**Telomeres: Their Structure and Maintenance**

Radmila Capkova Frydrychova and James M. Mason

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51356

### **1. Introduction**

Telomeres are essential nucleoprotein structures at the ends of eukaryotic chromosomes. They play several essential roles preserving genome stability and function, including distinguish‐ ing chromosome ends from DNA double stranded breaks (DSBs) and maintenance of chromo‐ some length. Due to the inability of conventional DNA polymerases to replicate the very end of a chromosome, sometimes known as the end replication problem, chromosome ends short‐ en with every round of DNA replication. In the absence of special telomere maintenance mechanisms this telomere shortening leads to replicative senescence and apoptosis. Several telomere maintenance mechanisms have been identified; these are reflected in several known types of telomeres. In most eukaryotes telomeres comprise a tandem array of a short, 5-8 bp, well conserved repeat unit, and telomere length is maintained by telomerase, a specialized reverse transcriptase that carries its own RNA template and adds telomeric sequences onto chromosome ends [1]. Nevertheless, in some organisms the array of short telomeric se‐ quence motifs has been replaced with less conventional sequences, such as satellite sequen‐ ces or transposable elements. The telomeres of such organisms are maintained through homologous recombination or through transposition of the mobile elements [2,3]. These dif‐ ferent telomere types present distinct difficulties for chromosome end protection. Telomeres maintained by telomerase are protected by a proteinaceous telomere cap, termed shelterin, that recognizes chromosome ends in a DNA sequence specific manner, while telomeres with long terminal repeat units are protected by a cap, termed terminin, that binds to chromo‐ some ends independently of DNA sequence.

### **2. The structure of telomeric DNA: "usual" and "unusual" telomeres**

The most common telomere structure found across the whole eukaryotic tree is a simple telomeric repeat of the form (TxAyGz)n generated by telomerase. For example, the sequence in

properly cited.

© 2013 Capkova Frydrychova and Mason; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 Capkova Frydrychova and Mason; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

unikonts generally, including animals, fungi and amoebozoa, is T2AG3, while in most plants and green algae it is T3AG3. Within these broad generalizations, however, there are excep‐ tions. Some species seem to have lost the canonical telomeric motif altogether. We will men‐ tion a few examples here, then describe one of these examples in more detail.

#### **2.1. Chromalveolata**

The terminal sequence motif seems to be quite variable among the Chromalveolates, while still adhering to the consensus telomeric motif (Figure 1). Apicomplexa species use three different motifs [4-6], and ciliates use two [4,7]. Dinoflagellates use T3AG3 [8], similar to plants and green algae, while diatoms use T2AG3 [9], similar to unikonts. Photosynthetic species in the Chromalveolates are derived from the engulfment of a red alga. The resulting nucleo‐ morphs retain the algal linear chromosomes and telomeres that are very different. The cryp‐ tomonad, *Guillardia theta*, for example, uses T3AG3 in its nucleus and (AG)7A2G6A in its nucleomorph [11,12].

**Figure 1.** Diagram showing five major eukaryotic supergroups and representative telomeric motifs. These groups are shown to have diverged from a single latest common ancestor, because the evolutionary relationships are not known. Trees connecting the major taxa within these supergroups are shown, but the branch lengths are arbitrary. Represen‐ tative telomeric motifs are shown for the major subtaxa. In some cases, two or three representative motifs are known for one of these taxa, as shown. Exceptions to these representations are discussed in the text. The figure was modified from [10] with permission.

It seems likely that the telomere binding proteins in these organisms are either different in the two intracellular bodies, or do not bind in a DNA sequence specific manner.

#### **2.2. Plantae**

unikonts generally, including animals, fungi and amoebozoa, is T2AG3, while in most plants and green algae it is T3AG3. Within these broad generalizations, however, there are excep‐ tions. Some species seem to have lost the canonical telomeric motif altogether. We will men‐

The terminal sequence motif seems to be quite variable among the Chromalveolates, while still adhering to the consensus telomeric motif (Figure 1). Apicomplexa species use three different motifs [4-6], and ciliates use two [4,7]. Dinoflagellates use T3AG3 [8], similar to plants and green algae, while diatoms use T2AG3 [9], similar to unikonts. Photosynthetic species in the Chromalveolates are derived from the engulfment of a red alga. The resulting nucleo‐ morphs retain the algal linear chromosomes and telomeres that are very different. The cryp‐ tomonad, *Guillardia theta*, for example, uses T3AG3 in its nucleus and (AG)7A2G6A in its

**Figure 1.** Diagram showing five major eukaryotic supergroups and representative telomeric motifs. These groups are shown to have diverged from a single latest common ancestor, because the evolutionary relationships are not known. Trees connecting the major taxa within these supergroups are shown, but the branch lengths are arbitrary. Represen‐ tative telomeric motifs are shown for the major subtaxa. In some cases, two or three representative motifs are known for one of these taxa, as shown. Exceptions to these representations are discussed in the text. The figure was modified

tion a few examples here, then describe one of these examples in more detail.

**2.1. Chromalveolata**

424 The Mechanisms of DNA Replication

nucleomorph [11,12].

from [10] with permission.

Among the Plantae, land plants and green algae mostly use T3AG3 as a telomeric motif, while the red algae have a very different sequence at their chromosome ends. The red alga *Cyanidioschyzon merolae*, for example, uses A2TG6 [13]. While telomeres in most green algae conform to the telomeric motif of this kingdom, the order Chlamydomonadales includes species that carry the telomeric motifs T4AG3, T3AG3 and T2AG3, apparently independently of phylogeny as determined by the 18S rDNA sequence [14]. Some species of this order lack all three of these telomeric motifs and carry unknown DNA sequences at their chromosome ends. It is possible that the 18S rDNA sequence does not represent an accurate reflection of phylogeny or the telomeric motif is quite variable in this order. In either case, it seems that sequence specific binding by telomeric proteins may have eased in this order.

Similarly, while most land plants retain the canonical T3AG3 telomeric motif, telomeres in a few orders differ from this structure. Within the monocot order Asparagales some species of Alliaceae have switched to the sequence T2AG3, and others appear to have lost the canonical telomeric sequence completely. It has been proposed that the telomeres of these latter Allia‐ ceae species are maintained through transposition of mobile elements or through homolo‐ gous recombination between the satellite sequences [15,16]. In the eudicot order Solanales the canonical telomeric motif as well as telomerase are absent from several genera of the family Solanaceae [17-20]. The actual telomeric sequence and compensation mechanism in this group of plants, however, remain unknown.

#### **2.3. Unikonta**

The T2AG3 telomeric sequence is found widely among the unikonts (Figure 1). While this is generally true within the fungi, representatives of two classes, Schizosaccharomycetes and Saccharomycetes, use variable, degenerate telomeric sequences that may result from replica‐ tion infidelity or slippage [12]. In *Saccharomyces cerevisiae*, for example, the repeat motif is TG1-3.

Similarly, T2AG3 is found widely at chromosome ends among metazoans. The animal phy‐ lum Arthropoda, however, uses the sequence T2AG2 at telomeres, and its sister phylum Tardigrada lacks both of these telomeric motifs [21]. Insects are the largest class of arthro‐ pods, and even here individual insect taxa may have different forms of the canonical se‐ quence or even unrelated telomeric sequences. Insects seem to have lost the canonical arthropod telomeric motif several times [22,23]. In some cases, such as the coleopteran superfamily Tenebrionoidea, the arthropod repeat has been replaced by a similar motif, in this case TCAG2 [24], while in many other instances the new telomeric DNA sequence has not been identified.

Insects of the orders Diptera, Mecoptera and Siphonaptera (superorder Antliophora) do not carry a canonical telomeric DNA sequence at their chromosome ends [23,25]; nor do they have a telomerase gene [26], indicating that telomerase was lost some 260-280 Mya. Even so, Diptera is one of the most successful insect orders, with some 152,000 species [27]. This suggests that telomerase and the canonical telomeric DNA sequences generated by telomer‐ ase, *per se*, are not critical for evolutionary survival. It is possible telomerase is expenda‐ ble, as long as the telomere capping complex is compatible with whatever terminal DNA sequence is present on chromosome ends. When the primary pathway for telomere replica‐ tion is defective, an alternative backup mechanism can restore telomere function. It was documented in yeast. Yeast mutants lacking telomerase showed the progressive telomere loss and, although the majority of the cells died, a minor subpopulation survived via homol‐ ogous recombination [28].

Long satellite sequences have been reported in nematoceran species. Chromosome tips of several *Chironomus* species (infraorder Culicomorpha) consist of large, 50-200 kb, blocks of complex, tandemly repeated sequences that have been classified into subfamilies based on sequence similarities. Different telomeres display different sets of subfamilies, and the distri‐ bution of subfamilies differs between individuals within a species. The variation of the satel‐ lite sequences supports the proposal that telomeres in *Chironomus* are elongated by a gene conversion mechanism involving these long blocks of complex repeat units [29-32]. A simi‐ lar situation has been found in *Anopheles gambiae* (infraorder Culicomorpha) using a plasmid fortuitously inserted into the complex telomeric sequences at the tip of chromosome *2*L. The telomere carrying the plasmid was found to engage in frequent recombination events that resulted in extension of the terminal array [33,34]. Recently, a similar case was reported in *Rhynchosciara americana* (infraorder Bibionomorpha). Tandem arrays of short repeats, 16 and 22 bp in length, were found to extend to chromosome ends [35]. Although telomere elonga‐ tion could not be assayed in this case, it seems likely that the mechanism is similar to that seen in other nematoceran species. In many respects, these complex arrays resemble subtelo‐ meric sequences [36], suggesting a possible mechanism for telomere formation upon the loss of telomerase.

Telomere structures have only been examined in a single brachyceran genus, *Drosophila* (in‐ fraorder Muscomorpha). Telomeric DNA sequences consist of long arrays of non-long ter‐ minal repeat (LTR) retrotransposons and are thus very different from those found in Nematocera. These telomeric transposons resemble long interspersed elements (LINEs) found in mammals, but have some differences that may reflect their telomere-specific 'life‐ style.' Three families of telomeric elements have been described in *Drosophila melanogaster* (subgenus Sophophora), *HeT-A*, *TART* and *TAHRE* [2]; in all cases these elements are attach‐ ed to the chromosome by their 3' oligo(A) tails. Many of the elements are truncated at the 5' end, possibly due to the end replication problem. *HeT-A* transposons are about 6 kb in length and make up about 80-90% of the elements found at chromosome ends. They are atypical LINE-like elements in three respects: the 3' untranslated region (UTR) comprises about 3 kb or half the length of the element; the transcriptional promoter is at the 3' end of the element to prevent loss when the element is present at the chromosome terminus with its 5' end exposed to incomplete DNA replication of linear DNA; and an open reading frame (ORF) coding for a reverse transcriptase is absent. *TART* elements are about 10 kb in length and make up about 10-20% of the telomeric retrotransposons. They are also unusual ele‐ ments, but in some ways that differ from *HeT-A*: they also have an unusually long 3' UTR; they have a relatively strong antisense promoter of unknown function and a pair of perfect long non-terminal repeats that may be important for replication [37,38]; they make a reverse transcriptase, but the encoded Gag-like protein is unable to target telomeres in the absence of the *HeT-A* Gag [39]. *TAHRE* elements closely resemble *HeT-A*, except they encode their own reverse transcriptase. Thus, while *TAHRE* seems to be the only one of the three ele‐ ments capable of independent transposition, it is by far the least abundant, comprising only 1% of the telomeric retrotransposons.

*HeT-A* and *TART* elements have also been found in *Drosophila virilis* (subgenus Drosophila) Although there is little sequence homology across species, the two types of retrotransposons can be recognized by their telomeric locations and unusual structures, as described above [40,41]. Given the difficulty in finding homology between evolutionarily related telomeric elements within the *Drosophila* genus, finding similar elements in other brachyceran species based on homology alone is unlikely. Thus, it is not known when these targeted transposi‐ tions took over the role of telomere maintenance from homologous recombination.

Human telomeres have been shown to form a large terminal loop dependent on the pres‐ ence of a 3' G strand overhang at the telomeric end. This 3' end is tucked back into the double-stranded DNA as a loop, termed a t-loop [42]. Similar t-loops may also be formed in yeast [43].

### **3. Proteins associated with telomeres**

suggests that telomerase and the canonical telomeric DNA sequences generated by telomer‐ ase, *per se*, are not critical for evolutionary survival. It is possible telomerase is expenda‐ ble, as long as the telomere capping complex is compatible with whatever terminal DNA sequence is present on chromosome ends. When the primary pathway for telomere replica‐ tion is defective, an alternative backup mechanism can restore telomere function. It was documented in yeast. Yeast mutants lacking telomerase showed the progressive telomere loss and, although the majority of the cells died, a minor subpopulation survived via homol‐

Long satellite sequences have been reported in nematoceran species. Chromosome tips of several *Chironomus* species (infraorder Culicomorpha) consist of large, 50-200 kb, blocks of complex, tandemly repeated sequences that have been classified into subfamilies based on sequence similarities. Different telomeres display different sets of subfamilies, and the distri‐ bution of subfamilies differs between individuals within a species. The variation of the satel‐ lite sequences supports the proposal that telomeres in *Chironomus* are elongated by a gene conversion mechanism involving these long blocks of complex repeat units [29-32]. A simi‐ lar situation has been found in *Anopheles gambiae* (infraorder Culicomorpha) using a plasmid fortuitously inserted into the complex telomeric sequences at the tip of chromosome *2*L. The telomere carrying the plasmid was found to engage in frequent recombination events that resulted in extension of the terminal array [33,34]. Recently, a similar case was reported in *Rhynchosciara americana* (infraorder Bibionomorpha). Tandem arrays of short repeats, 16 and 22 bp in length, were found to extend to chromosome ends [35]. Although telomere elonga‐ tion could not be assayed in this case, it seems likely that the mechanism is similar to that seen in other nematoceran species. In many respects, these complex arrays resemble subtelo‐ meric sequences [36], suggesting a possible mechanism for telomere formation upon the loss

Telomere structures have only been examined in a single brachyceran genus, *Drosophila* (in‐ fraorder Muscomorpha). Telomeric DNA sequences consist of long arrays of non-long ter‐ minal repeat (LTR) retrotransposons and are thus very different from those found in Nematocera. These telomeric transposons resemble long interspersed elements (LINEs) found in mammals, but have some differences that may reflect their telomere-specific 'life‐ style.' Three families of telomeric elements have been described in *Drosophila melanogaster* (subgenus Sophophora), *HeT-A*, *TART* and *TAHRE* [2]; in all cases these elements are attach‐ ed to the chromosome by their 3' oligo(A) tails. Many of the elements are truncated at the 5' end, possibly due to the end replication problem. *HeT-A* transposons are about 6 kb in length and make up about 80-90% of the elements found at chromosome ends. They are atypical LINE-like elements in three respects: the 3' untranslated region (UTR) comprises about 3 kb or half the length of the element; the transcriptional promoter is at the 3' end of the element to prevent loss when the element is present at the chromosome terminus with its 5' end exposed to incomplete DNA replication of linear DNA; and an open reading frame (ORF) coding for a reverse transcriptase is absent. *TART* elements are about 10 kb in length and make up about 10-20% of the telomeric retrotransposons. They are also unusual ele‐ ments, but in some ways that differ from *HeT-A*: they also have an unusually long 3' UTR; they have a relatively strong antisense promoter of unknown function and a pair of perfect

ogous recombination [28].

426 The Mechanisms of DNA Replication

of telomerase.

The telomere cap, a multiprotein structure at chromosome end ensuring stability and integ‐ rity of the genome, was revealed by early cytological observations of chromosomal rear‐ rangements after exposure to ionizing radiation [44]. The telomere cap allows cells to distinguish their natural chromosome ends from DSBs, thus protecting the chromosome ter‐ mini from inadvertent DNA damage response (DDR) activities. Defects in the cap, or DSBs elsewhere in the genome, lead to activation of cell cycle checkpoints followed by DDR mech‐ anisms. A consequence of inappropriate DSB repair are end-to-end fusions of chromosomes, i.e. formation of ring chromosomes or dicentric linear chromosomes, followed by chromo‐ some breakage, which results in genomic instability and loss of cellular viability [45,46]. Al‐ though, in this context, telomeres perform the same essential function across phyla, cap proteins of diverse organisms are less conserved that one might expect. Even within a single taxonomic class, such as mammals, telomeric proteins display less conservation than other chromosomal proteins [47]. In mammals the telomere-specific cap complex has been termed 'shelterin' (Figure 2). The six-protein complex is formed by double-stranded TTAGG repeatbinding proteins TRF1 and TRF2, which recruit TIN2 and TPP1. The latter proteins make a bridge between the TRF proteins and G-overhang-binding protein, POT1. The sixth protein is the TRF2-interacting protein RAP1 [46,48,49]. A characteristic of shelterin proteins is spe‐ cific and exclusive association with telomeric DNA, where they are permanently present throughout the cell cycle and serve as platform for a transient and dynamic recruitment of a number of telomere-associated factors, referred to as non-shelterin telomeric proteins. These non-shelterin proteins are required for telomere protection and replication but also have nu‐ merous nontelomeric functions. Examples include DDR proteins that are commonly in‐ volved in DSB repair through nonhomologous end joining (NHEJ) or homologous recombination (HR), such as ATM, ATR and Ku70/80, which associate with TRF1 and TRF2, and the MRN complex, composed of the MRE11, RAD50 and NBS1 (MRN) proteins, which associates with TRF2 [50-55]. Another protein associated with TRF2 is Apollo, an exonu‐ clease important for recreating the 3' overhang [51,56]. The binding of shelterin proteins and formation of a functional cap require a terminal DNA array of specific sequence and of satis‐ factory length.

Analysis of deleterious events at shelterin-free telomeres revealed six pathways for end pro‐ tection [57]. The primery protection by shelterin is against classical NHEJ and unwanted ac‐ tivation of ATM and ATR signaling. Additionally, shelterin provides a defense against alternate NHEJ, HR and 5' end resection. Another protective layer is achieved through the Ku70/80 heterodimer or 53BP1. 53BP1 minimizes resection but only at telomeres eliciting a DNA damage signal. Ku70/80 blocks alternate NHEJ and HR at telomeres independent of a DNA damage signal [57].

**Figure 2.** A. The telomere forms a t-loop structure characterized by invasion of the 3' overhang into a double stranded telomeric DNA. B. Six proteins, TRF1, TRF2, TPP1, POT1, TIN2, and RAP1 form a dedicated telomere-protection protein complex in humans [48,49,58].

Telomeres in *Saccharomyces cerevisiae* are protected by two separate protein complexes. One is the Rap1/Rif1/Rif2 complex, which localizes to double-stranded telomeric DNA. The other is the Cdc13/Stn1/Ten1 (CST) complex, which is targeted to the single-stranded G-overhangs through sequence-specific binding of Cdc13. Defects in the CST complex result in degrada‐ tion of the C-stand and activation of DDR mechanisms [47]. As with shelterin, CST interacts with numerous proteins required for telomere function. Some evolutionary conservation in the protein composition of the cap is expected, for instance similarities to CST and shelterin are observed in telomeric proteins of numerous organisms. This is documented for mamma‐ lian CST, which, although not involved in telomere capping, facilitates telomere replication and, if impaired, leads to catastrophic telomeric defects [59]. Another example is Ver, a com‐ ponent of the *Drosophila* cap with weak structural similarities to Stn1 [60,61].

A multiprotein capping complex in *Drosophila*, termed 'terminin,' is an analog of mammali‐ an shelterin [61]. One major difference between these two complexes is that terminin does not bind to a specific telomeric DNA sequence. Rather limited information is available about the structure and function of four known terminin proteins, HOAP, Moi, Ver, and HipHop. As with shelterin, terminin proteins localize specifically to telomeres and appear to function only at telomeres. HOAP is encoded by the *cav* gene [62]; Moi is a HOAP-binding protein encoded by *moi* [63,64]; Ver is structurally homologous to STN1 and is encoded by *ver* [60]; and HipHop is a HP1-HOAP interacting protein [65]. Assembly of the terminin complex re‐ quires strict dependencies. For example, the binding of HOAP and HipHoP to telomeres is interdependent, loss of one protein reduces binding of the other [65]; HOAP is required for Ver and Moi localization [61]. The terminin complex seems to occupy a broad region cover‐ ing a more than 10 kb from the chromosome termini [65]. As with shelterin proteins, defects in terminin proteins lead to frequent telomeric fusions.

merous nontelomeric functions. Examples include DDR proteins that are commonly in‐ volved in DSB repair through nonhomologous end joining (NHEJ) or homologous recombination (HR), such as ATM, ATR and Ku70/80, which associate with TRF1 and TRF2, and the MRN complex, composed of the MRE11, RAD50 and NBS1 (MRN) proteins, which associates with TRF2 [50-55]. Another protein associated with TRF2 is Apollo, an exonu‐ clease important for recreating the 3' overhang [51,56]. The binding of shelterin proteins and formation of a functional cap require a terminal DNA array of specific sequence and of satis‐

Analysis of deleterious events at shelterin-free telomeres revealed six pathways for end pro‐ tection [57]. The primery protection by shelterin is against classical NHEJ and unwanted ac‐ tivation of ATM and ATR signaling. Additionally, shelterin provides a defense against alternate NHEJ, HR and 5' end resection. Another protective layer is achieved through the Ku70/80 heterodimer or 53BP1. 53BP1 minimizes resection but only at telomeres eliciting a DNA damage signal. Ku70/80 blocks alternate NHEJ and HR at telomeres independent of a

**Figure 2.** A. The telomere forms a t-loop structure characterized by invasion of the 3' overhang into a double stranded telomeric DNA. B. Six proteins, TRF1, TRF2, TPP1, POT1, TIN2, and RAP1 form a dedicated telomere-protection protein

Telomeres in *Saccharomyces cerevisiae* are protected by two separate protein complexes. One is the Rap1/Rif1/Rif2 complex, which localizes to double-stranded telomeric DNA. The other is the Cdc13/Stn1/Ten1 (CST) complex, which is targeted to the single-stranded G-overhangs through sequence-specific binding of Cdc13. Defects in the CST complex result in degrada‐ tion of the C-stand and activation of DDR mechanisms [47]. As with shelterin, CST interacts with numerous proteins required for telomere function. Some evolutionary conservation in the protein composition of the cap is expected, for instance similarities to CST and shelterin are observed in telomeric proteins of numerous organisms. This is documented for mamma‐ lian CST, which, although not involved in telomere capping, facilitates telomere replication and, if impaired, leads to catastrophic telomeric defects [59]. Another example is Ver, a com‐

A multiprotein capping complex in *Drosophila*, termed 'terminin,' is an analog of mammali‐ an shelterin [61]. One major difference between these two complexes is that terminin does

ponent of the *Drosophila* cap with weak structural similarities to Stn1 [60,61].

factory length.

428 The Mechanisms of DNA Replication

DNA damage signal [57].

complex in humans [48,49,58].

As there is no specific telomeric DNA sequence in Drosophila, terminin binding to telomer‐ ic DNA is sequence-independent, which makes a substantial difference between mammali‐ an and Drosophila telomeres. In contrast to mammals, the complete loss of a Drosophila telomere does not definitely mean inescapable damage to genome stability and cell death, because under the right circumstances the telomere cap can be formed *de novo* as on any broken chromosome end and perform there the same protective functions as the regular telomere. This demonstrates that the telomeric retrotransposons, although important for telomere elon‐ gation, are not required as an unique assembly platform for cap formation [2,66,67].

Similar to shelterin, terminin presents a docking site for binding of additional proteins, called non-terminin capping proteins. Although not exclusively located at telomeres and having some telomere-unrelated functions, these proteins are required for the capping func‐ tion and, in many cases, facilitate terminin assembly. There are several known non-terminin proteins; most of them were identified because their mutants display frequent telomeric fu‐ sions [61]. The best characterized is HP1a that is encoded by *Su(var)205*. The presence of HP1 at telomeres is required for HOAP binding, which reveals the importance of HP1 for terminin assembly. As in mammals, other non-terminin proteins are DNA repair factors: the *Drosophila* homologs of the ATM kinase and proteins of the MRN complex. Defects in the MRN complex lead to reduction of HOAP and HP1 at telomeres and frequent telomeric fu‐ sions. Through its effects on the binding of HOAP and possibly other terminin components, the MRN complex seems to be essential for the terminin formation [61,68,69]. ATM prevents telomeric fusions, and defects in this protein partially affect HP1/HOAP localization [70-72]. Woc is a zinc-finger protein preventing telomeric fusions, but acting independently of HP1, HOAP, and RAD50 [73]. UbcD1 is an E2 ubiquitin conjugating enzyme. It has been suggest‐ ed that UbcD1-mediated ubiquitination of telomeric proteins is an essential post-translation‐ al modification ensuring their proper function [61,74]. In contrast to non-terminin and nonshelterin proteins that are largely conserved, a comparison between shelterin and terminin reveals no obvious homology in protein composition. Loss of conservation between shelter‐ in and terminin proteins may correspond the evolutionary stage when a Antliophoran an‐ cestor lost telomerase-based telomere elongation and had to evolve a sequence-independent protection of chromosome ends and acquire a new elongation system.

A highly condensed chromatin structure is a common characteristic of telomeres from yeast to man. Usually telomeres are heterochromatic, and the heterochromatic properties are thought to play an important role in telomeric function [75-77]. Telomeric chromatin is the source of telomeric position effect (TPE), a silencing of transgenes inserted into telomeres or their vicinity [78]. Besides the cap region, *Drosophila* telomeres contain two distinct chromatin domains: a subtelomeric region of repetitive DNA, termed TAS (telo‐ mere associated sequence), exhibiting features that resemble heterochromatin, and a termi‐ nal array of retrotransposons with euchromatic characteristics [79]. The *Drosophila* TAS region is, in contrast to retrotransposon array, the source of TPE [79,80]. Although organ‐ ized into a heterochromatic structure, the vertebrate TTAGGG sequence remains unmethy‐ lated due to the lack of a appropriate cytosine substrate. The subtelomeric region is, in contrast, heavily methylated by DNA methyltranferases DNMT1, DNMT3a and DNMT3b [81]. Both in vertebrates and *Drosophila*, telomeric and subtelomeric regions are enriched in histone H3 methylated at lysine 9 (H3K9me), mediated by a H3K9-specific histone meth‐ yltransferase and HP1.

#### **4. Telomeric replication and its difficulties**

Based on DNA and protein composition, telomeres are typical heterochromatin, so their rep‐ lication should correspond with a common paradigm of late heterochromatin replication. Based on early microscopic studies, it is generally accepted that DNA replication at early stages of S phase is associated with expressed genes, whereas repressed tissue-specific genes or heterochromatic regions are replicated during the late stages of replication [82-84]. The late replication seems to be common, but definitely is not universal [85]. Replication of hu‐ man telomeres takes place throughout S phase, and specific telomeres tend to replicate at defined stages, some replicating early and others late [86]. The pattern of replication timing seems to be conserved between homologous chromosomes and does not vary between cells of different individuals. Although no correlation was found with telomere length or telo‐ merase activity, a strong association was observed with nuclear localization. Late-replicating telomeres show a preferential association with the nuclear periphery, while early-replicating telomeres are preferentially located near the nuclear center [86]. A different situation was found in budding yeast, *Saccharomyces cerevisiae*, where early telomere replication correlates with short telomeric length and telomerase activity [87,88]. In fission yeast, *Schizosaccharo‐ myces pombe*, telomere replication corresponds to S/G2 phase [85,89].

Because of the repetitive nature of telomeric DNA, telomeres present a significant prob‐ lem for their replication. Spontaneous replication fork regression in telomeric DNA *in vi‐ tro* was determined to be 41% higher than seen in non-repeated DNA [90]. The obstacles during replication may lead to formation of cruciform intermediates, resulting in unwant‐ ed recombination events, amplifications or deletions [90,91]. Most of the telomere is repli‐ cated by a standard replication fork, however, to achieve efficient telomere replication a number of additional steps are needed. The process requires cooperation between stand‐ ard replication factors and telomeric proteins, DDR proteins and numerous additional fac‐ tors [47]. Examples of additional proteins are RecQ-type helicases that are present at replication forks in addition to standard helicases and are shown to unwind structures similar to chickenfoot intermediates [90,92]. Cooperation of replication factors with shelter‐ in proteins is also documented. TRF1 mutants showed a reduction in replication efficien‐ cy, suggesting that TRF1 promotes efficient replication of telomeric DNA by preventing fork stalling [93]. Similarly, Taz1, a TRF1 homolog in fission yeast, has been shown to prevent fork stalling [94]. Another example is mammalian CTC1; deletion of CTC1 results in increased loss of leading C-strand telomeres, dramatic telomere loss and accumulation of excessive single-stranded telomeric DNA [95].

A highly condensed chromatin structure is a common characteristic of telomeres from yeast to man. Usually telomeres are heterochromatic, and the heterochromatic properties are thought to play an important role in telomeric function [75-77]. Telomeric chromatin is the source of telomeric position effect (TPE), a silencing of transgenes inserted into telomeres or their vicinity [78]. Besides the cap region, *Drosophila* telomeres contain two distinct chromatin domains: a subtelomeric region of repetitive DNA, termed TAS (telo‐ mere associated sequence), exhibiting features that resemble heterochromatin, and a termi‐ nal array of retrotransposons with euchromatic characteristics [79]. The *Drosophila* TAS region is, in contrast to retrotransposon array, the source of TPE [79,80]. Although organ‐ ized into a heterochromatic structure, the vertebrate TTAGGG sequence remains unmethy‐ lated due to the lack of a appropriate cytosine substrate. The subtelomeric region is, in contrast, heavily methylated by DNA methyltranferases DNMT1, DNMT3a and DNMT3b [81]. Both in vertebrates and *Drosophila*, telomeric and subtelomeric regions are enriched in histone H3 methylated at lysine 9 (H3K9me), mediated by a H3K9-specific histone meth‐

Based on DNA and protein composition, telomeres are typical heterochromatin, so their rep‐ lication should correspond with a common paradigm of late heterochromatin replication. Based on early microscopic studies, it is generally accepted that DNA replication at early stages of S phase is associated with expressed genes, whereas repressed tissue-specific genes or heterochromatic regions are replicated during the late stages of replication [82-84]. The late replication seems to be common, but definitely is not universal [85]. Replication of hu‐ man telomeres takes place throughout S phase, and specific telomeres tend to replicate at defined stages, some replicating early and others late [86]. The pattern of replication timing seems to be conserved between homologous chromosomes and does not vary between cells of different individuals. Although no correlation was found with telomere length or telo‐ merase activity, a strong association was observed with nuclear localization. Late-replicating telomeres show a preferential association with the nuclear periphery, while early-replicating telomeres are preferentially located near the nuclear center [86]. A different situation was found in budding yeast, *Saccharomyces cerevisiae*, where early telomere replication correlates with short telomeric length and telomerase activity [87,88]. In fission yeast, *Schizosaccharo‐*

Because of the repetitive nature of telomeric DNA, telomeres present a significant prob‐ lem for their replication. Spontaneous replication fork regression in telomeric DNA *in vi‐ tro* was determined to be 41% higher than seen in non-repeated DNA [90]. The obstacles during replication may lead to formation of cruciform intermediates, resulting in unwant‐ ed recombination events, amplifications or deletions [90,91]. Most of the telomere is repli‐ cated by a standard replication fork, however, to achieve efficient telomere replication a number of additional steps are needed. The process requires cooperation between stand‐ ard replication factors and telomeric proteins, DDR proteins and numerous additional fac‐

yltransferase and HP1.

430 The Mechanisms of DNA Replication

**4. Telomeric replication and its difficulties**

*myces pombe*, telomere replication corresponds to S/G2 phase [85,89].

In yeast, the replication of telomeres is initiated in subtelomeric regions, and the replication fork moves towards the chromosome termini [96]. In mammalian cells, the origin of telo‐ meric replication and direction is ambiguous.

After the replication fork reaches the chromosome terminus, the lagging strand gains a 3' overhang due to the removal of the primer for the terminal Okazaki fragment. At the same time C-strand specific resection occurs by nucleases Exo1 and/or Dna2 to produce a G-over‐ hang on the leading strand [89]. If active, telomerase elongates the G-overhangs by addition of new telomeric repeats. Telomerase action is followed by complementary C-strand synthe‐ sis by DNA pol α. The process is terminated by additional processing to remove the RNA primer and to leave a 40-400 nucleotide G-overhang. The timing of the events differs be‐ tween species. In human cells, telomere replication occurs at the same time as telomerasemediated extension, and fill-in synthesis of C- strand is delayed until S/G2. Budding yeast shows tight coupling between G-strand extension and C-strand synthesis [89,91].

### **5. The mechanisms of telomeric elongation and their regulation**

Telomerase is a ribonucleoprotein reverse transcriptase that utilizes its protein subunit (TERT in mammals, Est2p in *S. cerevisiae*) to elongate the 3' end of telomeric DNA using an internal RNA subunit (TR) as a template [97-99]. Telomerase activity is related to cell prolif‐ eration status: it is high in actively cycling cultures and low in quiescent differentiated cells [100]. Telomerase is not detected in human mature sperm or unfertilized eggs, but after fer‐ tilization telomerase is rapidly activated. A dramatic increase is observed in blastocysts, but during later stages of gestation telomerase activity declines. In the 16-week fetus Wright [101] showed high levels of telomerase in liver and intestine; detectable activity in lung, skin, muscle, adrenal glands, and kidney; and very weak or no activity in brain, bone or pla‐ cental extracts. Most somatic cells in adults show no telomerase activity, as enzyme activity is limited to specific types of proliferative cells, such as embryonic, stem and epithelial cells, the germline, or cells of the hematopoetic system [102,103]. Telomerase activity is highly regulated. Reactivation of telomerase is associated with tumor development, and converse‐ ly, insufficient telomerase activity is linked to stem cell diseases, such as dyskeratosis conge‐ nita and aplastic anemia [104-106].

Telomerase is regulated through genetic, epigenetic and environmental factors: TERT and TR transcription, posttranscriptional and posttranslational modifications of TERT, and telo‐ merase recruitment and processivity [104]. TERT promoter activity has been studied exten‐ sively, and numerous transcription factors have been found to interact with TERT. TERT transcription is, for instance, activated by the oncogene c-Myc and suppressed by the tumorsuppressor WT1 (Wilm's tumor suppresor). Misregulation of TERT through the c-Myc or WT1 pathways is associated with telomerase reactivation in cancer cells [107,108]. Although transcription of TERT is the major determinant of telomerase activity, TERT transcript levels do not always correlate with enzyme activity. Posttranslational phosphorylation may regu‐ late telomerase activity, as may telomerase degradation through ubiquitination, as the halflife of telomerase activity was approximately 24 hours [109]. In human cells the POT1-TPP1 complex was found to be a key regulator of telomerase processivity [110-113].

Little is known about the regulation of telomere length in *Drosophila*, where two modes of telomere elongation have been described: transposition of telomeric elements and gene con‐ version. The process of telomeric transposition is composed of several steps: 1. transcription of the telomeric elements, 2. export of retroelement transcripts from the nucleus to the cyto‐ plasm, 3. translation, 4. nuclear re-import of the retroelement transcripts together the retro‐ element proteins, 5. recognition of chromosome ends, and 6. target-primed reverse transcription, which attaches the 3' oligo(A) tails of the elements to chromosome termini [2]. Transposition of these elements to chromosome termini does not depend on a specific DNA sequence at the target site and together with the loss of telomeric DNA results in tandem arrays of mixed complete and 5' truncated elements [2]. The regulation of telomere elonga‐ tion may be on the level of retroelement transcription and/or accessibility of the chromo‐ some ends for new retroelement attachments. A variety of proteins have been identified to play a role in *Drosophila* telomere capping, however, only a few proteins are known to func‐ tion in telomere elongation. HP1 was found to have a dual role in telomere protection and telomere length control. Compared to wild-type, heterozygotous *Su(var)205* mutants dis‐ played much longer telomeres associated with a dramatic increases in retroelement tran‐ scription and transposition [114-116]. The regulation of retroelement transcription by HP1 was observed along the terminal retrotransposon array, thus this HP1 function is not limited to the telomere cap [117]. No, or only minor, changes were observed in telomere length or retroelement transcription in mutants of genes involved in telomere capping, such as *cav*, *moi*, *ver* or *atm* [61,117], which may indicate that terminin does not control telomere length. Another gene regulating telomere length is *prod*. Although *prod* mutants showed elevated levels of *HeT-A* transcripts, no change in telomere length was observed, suggesting that ele‐ vated retroelement transcription is not sufficient for telomere length growth [118]. Similar data were observed for members of rasiRNA (repeat-associated small interfering RNA) pathway *aub* (*aubergine*) and *Spn-E.* Their mutants displayed higher *HeT-A* transcript levels [119], albeit without any significant increase in telomere length (our unpublished data). In parallel with telomerase activity, transcription of telomeric elements is observed only in pro‐ liferating cells, such as embryonic cells, cells of imaginal discs, testis and ovaries [120,121].

Telomere length is maintained through an interplay between telomere maintenance mecha‐ nisms and telomere shortening events. Based on human research it has been proposed that telomerase activity and telomere length are modulated by different endogenous and exoge‐ nous factors, such as emotional or physical stress, health conditions and aging [102]. How‐ ever, the prime factor in telomeric shortening may well be oxidative stress. Due to a high content of guanines, telomeres are particularly vulnerable to oxidative damage, and the im‐ pact of oxidative stress on telomeric length has been proposed to be much larger than the end-replication problem [122]. Endogenous oxidative stress is associated with several cellu‐ lar processes, such as the mitochondrial OXPHOS system and inflammation. Mitochondrial dysfunction-induced reactive oxygen species and hyperoxia *in vitro* lead to accelerated telo‐ mere shortening and reduced proliferative lifespan of cultured somatic cells [123]. Thus, short telomeric length in humans appears to be linked to the limited proliferative capacity of normal somatic cells, and it is likely that telomeric shortening is one of the key events relat‐ ed to cellular senescence and organismal aging. As telomeres shorten with age, telomere length is considered as a biomarker of aging and a forecaster of longevity [102].

### **6. Conclusion**

Telomerase is regulated through genetic, epigenetic and environmental factors: TERT and TR transcription, posttranscriptional and posttranslational modifications of TERT, and telo‐ merase recruitment and processivity [104]. TERT promoter activity has been studied exten‐ sively, and numerous transcription factors have been found to interact with TERT. TERT transcription is, for instance, activated by the oncogene c-Myc and suppressed by the tumorsuppressor WT1 (Wilm's tumor suppresor). Misregulation of TERT through the c-Myc or WT1 pathways is associated with telomerase reactivation in cancer cells [107,108]. Although transcription of TERT is the major determinant of telomerase activity, TERT transcript levels do not always correlate with enzyme activity. Posttranslational phosphorylation may regu‐ late telomerase activity, as may telomerase degradation through ubiquitination, as the halflife of telomerase activity was approximately 24 hours [109]. In human cells the POT1-TPP1

Little is known about the regulation of telomere length in *Drosophila*, where two modes of telomere elongation have been described: transposition of telomeric elements and gene con‐ version. The process of telomeric transposition is composed of several steps: 1. transcription of the telomeric elements, 2. export of retroelement transcripts from the nucleus to the cyto‐ plasm, 3. translation, 4. nuclear re-import of the retroelement transcripts together the retro‐ element proteins, 5. recognition of chromosome ends, and 6. target-primed reverse transcription, which attaches the 3' oligo(A) tails of the elements to chromosome termini [2]. Transposition of these elements to chromosome termini does not depend on a specific DNA sequence at the target site and together with the loss of telomeric DNA results in tandem arrays of mixed complete and 5' truncated elements [2]. The regulation of telomere elonga‐ tion may be on the level of retroelement transcription and/or accessibility of the chromo‐ some ends for new retroelement attachments. A variety of proteins have been identified to play a role in *Drosophila* telomere capping, however, only a few proteins are known to func‐ tion in telomere elongation. HP1 was found to have a dual role in telomere protection and telomere length control. Compared to wild-type, heterozygotous *Su(var)205* mutants dis‐ played much longer telomeres associated with a dramatic increases in retroelement tran‐ scription and transposition [114-116]. The regulation of retroelement transcription by HP1 was observed along the terminal retrotransposon array, thus this HP1 function is not limited to the telomere cap [117]. No, or only minor, changes were observed in telomere length or retroelement transcription in mutants of genes involved in telomere capping, such as *cav*, *moi*, *ver* or *atm* [61,117], which may indicate that terminin does not control telomere length. Another gene regulating telomere length is *prod*. Although *prod* mutants showed elevated levels of *HeT-A* transcripts, no change in telomere length was observed, suggesting that ele‐ vated retroelement transcription is not sufficient for telomere length growth [118]. Similar data were observed for members of rasiRNA (repeat-associated small interfering RNA) pathway *aub* (*aubergine*) and *Spn-E.* Their mutants displayed higher *HeT-A* transcript levels [119], albeit without any significant increase in telomere length (our unpublished data). In parallel with telomerase activity, transcription of telomeric elements is observed only in pro‐ liferating cells, such as embryonic cells, cells of imaginal discs, testis and ovaries [120,121].

complex was found to be a key regulator of telomerase processivity [110-113].

432 The Mechanisms of DNA Replication

The ends of all linear chromosomes face the same difficulties regardless of their struc‐ tures. Chromosome ends are not replicated completely by the standard replication machi‐ nery, resulting in loss of sequence and a 3' overhang on half of the replication products. Early eukaryotes may have solved the end replication problem by co-opting a reverse tran‐ scriptase that had arisen in a retrotransposable element [124] and using it to generate ar‐ rays of a simple repeat unit. They then solved the end protection problem by engineering long 3' overhangs on all termini, which could then loop around and tuck into the double stranded telomeric region and coating the terminal arrays with proteins that recognize the product of the reverse transcriptase. This combination of telomere maintenance by telomer‐ ase and protection by CST/shelterin served eukaryotes well and has been amazingly sta‐ ble for more than a billion years.

Depending on how strictly shelterin recognizes the telomeric motif, the sequence may be conserved over long expanses of time, as in unikonts. If shelterin is less strict in recognizing the telomerase-generated motif, this sequence may have more latitude to vary, as in chro‐ malveolates. If the protective telomere cap completely loses its ability to recognize the telo‐ meric sequence, telomerase and the canonical telomeric motif may be lost. Many eukaryotes, including yeast and humans use unequal homologous recombination/gene conversion as a backup telomere maintenance system. It appears that in some species of plants and animals telomerase has been lost, and gene conversion has taken over as the primary mechanism to maintain chromosome length, with the eventual loss of telomeric motif. Chromosome length maintenance and end protection must be maintained through all of this. The evolution of new telomere structures, therefore, requires a delicate interplay between these two func‐ tions, as well as other telomeric functions that may be less well understood, such as hetero‐ chromatin formation and meiotic chromosome pairing.

### **Acknowledgements**

The authors were supported in part by the Intramural Research Program, NIEHS, U. S. Na‐ tional Institutes of Health and by grant no. GAČR P501/10/1215 (Grant Agency of the Czech Republic, Prague).

### **Author details**

Radmila Capkova Frydrychova1\* and James M. Mason2\*

\*Address all correspondence to: radmila.frydrychova@hotmail.com

1 Institute of Entomology, Czech Republic

2 National Institute of Environmental Health Sciences, USA

#### **References**


[9] Bowler, C., Allen, A. E., Badger, J. H., Grimwood, J., Jabbari, K., Kuo, A., et al. (2008). The Phaeodactylum genome reveals the evolutionary history of diatom genomes. *Na‐ ture*, 456(7219), 239-244.

**Acknowledgements**

434 The Mechanisms of DNA Replication

Republic, Prague).

**Author details**

**References**

274(2), 92-97.

Radmila Capkova Frydrychova1\* and James M. Mason2\*

2 National Institute of Environmental Health Sciences, USA

1 Institute of Entomology, Czech Republic

\*Address all correspondence to: radmila.frydrychova@hotmail.com

exception providing new insights. *Bioessays*, 30(1), 25-37.

mere elongation. *Cell Mol Life Sci*, 60(11), 2325-2333.

somes. *Exp Cell Res*, 318(12), 1456-1460.

*Acids Res*, 23(11), 1942-1947.

The authors were supported in part by the Intramural Research Program, NIEHS, U. S. Na‐ tional Institutes of Health and by grant no. GAČR P501/10/1215 (Grant Agency of the Czech

[1] Greider, C.W., & Blackburn, E. H. (1996). Telomeres, telomerase and cancer. *Sci Am*,

[2] Mason, J. M., Frydrychova, R. C., & Biessmann, H. (2008). Drosophila telomeres: an

[3] Biessmann, H., & Mason, J. M. (2003). Telomerase-independent mechanisms of telo‐

[4] Zakian, V. A. (2012). Telomeres: The beginnings and ends of eukaryotic chromo‐

[5] Sohanpal, B. K., Morzaria, S. P., Gobright, E. I., & Bishop, R. P. (1995). Characterisa‐ tion of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome. *Nucleic*

[6] Liu, C., Schroeder, A. A., Kapur, V., & Abrahamsen, M. S. (1998). Telomeric sequen‐

[7] Klobutcher, L. A., Swanton, M. T., Donini, P., & Prescott, D. M. (1981). All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and

[8] Alverca, E., Cuadrado, A., Jouve, N., Franca, S., & Moreno Diaz., de la Espina. (2007). Telomeric DNA localization on dinoflagellate chromosomes: structural and evolu‐

ces of Cryptosporidium parvum. *Mol Biochem Parasitol*, 94(2), 291-296.

an unusual 3' terminus. *Proc Natl Acad Sci U S A*, 78(5), 3015-3019.

tionary implications. *Cytogenet Genome Res*, 116(3), 224-231.


[38] Sheen, F. M., & Levis, R. W. (1994). Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. *Proc Natl Acad Sci U S A*, 91(26), 12510-12514.

[24] Mravinac, B., Mestrovic, N., Cavrak, V. V., & Plohl, M. (2011). TCAGG, an alternative

[25] Mason, J. M., & Biessmann, H. (1995). The unusual telomeres of Drosophila. *Trends*

[26] Sasaki, T., & Fujiwara, H. (2000). Detection and distribution patterns of telomerase

[27] Wiegmann, B. M., Trautwein, M. D., Winkler, I. S., Barr, N. B., Kim, J. W., Lambkin, C., et al. (2011). Episodic radiations in the fly tree of life. *Proc Natl Acad Sci U S A*,

[28] Lundblad, V., & Blackburn, E. H. (1993). An alternative pathway for yeast telomere

[29] Nielsen, L., & Edstrom, J. E. (1993). Complex telomere-associated repeat units in members of the genus Chironomus evolve from sequences similar to simple telomer‐

[30] Nielsen, L., Schmidt, E. R., & Edstrom, J. E. (1990). Subrepeats result from regional DNA sequence conservation in tandem repeats in Chironomus telomeres. *J Mol Biol*,

[31] Cohn, M., & Edstrom, J. E. (1992). Chromosome ends in Chironomus pallidivittatus contain different subfamilies of telomere-associated repeats. *Chromosoma*, 101(10),

[32] Biessmann, H., Walter, M. F., & Mason, J. M. (1997). Drosophila telomere elongation.

[33] Biessmann, H., Kobeski, F., Walter, M. F., Kasravi, A., & Roth, C. W. (1998). DNA or‐ ganization and length polymorphism at the 2L telomeric region of Anopheles gam‐

[34] Roth, C. W., Kobeski, F., Walter, M. F., & Biessmann, H. (1997). Chromosome end elongation by recombination in the mosquito Anopheles gambiae. *Mol Cell Biol*, 17(9),

[35] Madalena, C. R., Amabis, J. M., & Gorab, E. (2010). Unusually short tandem repeats appear to reach chromosome ends of Rhynchosciara americana (Diptera: Sciaridae).

[36] Pryde, F. E., Gorham, H. C., & Louis, E. J. (1997). Chromosome ends: all the same un‐

[37] George, J. A., Traverse, K. L., DeBaryshe, P. G., Kelley, K. J., & Pardue, M. L. (2010). Evolution of diverse mechanisms for protecting chromosome ends by Drosophila TART telomere retrotransposons. *Proc Natl Acad Sci U S A*, 107(49), 21052-21057.

telomeric sequence in insects. *Chromosoma*, 120(4), 367-376.

activity in insects. *Eur J Biochem*, 267(10), 3025-3031.

maintenance rescues est1- senescence. *Cell.*, 73(2), 347-360.

ic repeats. *Mol Cell Biol*, 13(3), 1583-1589.

*Genet*, 11(2), 58-62.

436 The Mechanisms of DNA Replication

108(14), 5690-5695.

216(3), 577-584.

*Ciba Found Symp*, 211, 53-67.

biae. *Insect Mol Biol*, 7(1), 83-93.

*Chromosoma*, 119(6), 613-623.

der their caps. *Curr Opin Genet Dev*, 7(6), 822-828.

634-640.

5176-5183.


[68] Ciapponi, L., Cenci, G., & Gatti, M. (2006, Jul). The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability. *Genetics*, 173(3), 1447-1454.

[53] Celli, G. B., Denchi, E. L., & de, L. T. (2006). Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination. *Nat Cell*

[54] Denchi, E. L., & de, L. T. (2007). Protection of telomeres through independent control

[55] Lamarche, B. J., Orazio, N. I., & Weitzman, M. D. (2010). The MRN complex in dou‐ ble-strand break repair and telomere maintenance. *FEBS Lett*, 584(17), 3682-3695. [56] Wu, P., van Rooney, O. M., & de , S. L. T. (2010). Apollo contributes to G overhang

[57] Sfeir, A., & de , L. T. (2012). Removal of shelterin reveals the telomere end-protection

[59] Gu, P, Min, J. N., Wang, Y, Huang, C, Peng, T, & Chai, W. (2012). CTC1 deletion re‐ sults in defective telomere replication, leading to catastrophic telomere loss and stem

[60] Raffa, G. D., Raimondo, D., Sorino, C., Cugusi, S., Cenci, G., Cacchione, S., et al. (2010). Verrocchio, a Drosophila OB fold-containing protein, is a component of the

[61] Raffa, G. D., Ciapponi, L., Cenci, G., & Gatti, M. (2011). Terminin: a protein complex that mediates epigenetic maintenance of Drosophila telomeres. *Nucleus*, 2(5), 383-391.

[62] Cenci, G., Siriaco, G., Raffa, G. D., Kellum, R., & Gatti, M. (2003, Jan). The Drosophila

[63] Raffa, G. D., Siriaco, G, Cugusi, S, Ciapponi, L, Cenci, G, & Wojcik, E. (2009). The Drosophila modigliani (moi) gene encodes a HOAP-interacting protein required for

[64] Komonyi, O., Schauer, T., Papai, G., Deak, P., & Boros, I. M. (2009). A product of the bicistronic Drosophila melanogaster gene CG31241, which also encodes a trimethyl‐ guanosine synthase, plays a role in telomere protection. *J Cell Sci*, Mar 15, 122(Pt 6),

[65] Gao, G., Walser, J. C., Beaucher, M. L., Morciano, P., Wesolowska, N., Chen, J., et al. HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a se‐

[66] Biessmann, H., Kasravi, B., Bui, T., Fujiwara, G., Champion, L. E., & Mason, J. M. (1994, Apr). Comparison of two active HeT-A retroposons of Drosophila melanogast‐

[67] Capkova, F. R., Biessmann, H., & Mason, J. M. (2008). Regulation of telomere length

HOAP protein is required for telomere capping. *Nat Cell Biol*, 5(1), 82-84.

maintenance and protects leading-end telomeres. *Mol Cell*, 39(4), 606-617.

[58] Greider, C. W. (1999). Telomeres do D-loop-T-loop. *Cell*, 97(4), 419-422.

terminin telomere-capping complex. *Genes Dev*, 24(15), 1596-1601.

telomere protection. *Proc Natl Acad Sci U S A*, 106(7), 2271-2276.

quence-independent manner. *EMBO J*, 29(4), 819-829.

in Drosophila. *Cytogenet Genome Res*, 122(3-4), 356-364.

er. *Chromosoma*, 103(2), 90-98.

of ATM and ATR by TRF2 and POT1. *Nature*, 448(7157), 1068-1071.

*Biol*, 8(8), 885-890.

438 The Mechanisms of DNA Replication

769-774.

problem. *Science*, 336(6081), 593-597.

cell exhaustion. *EMBO J*, 31(10), 2309-2321.


[96] Raghuraman, M. K., Winzeler, E. A., Collingwood, D., Hunt, S., Wodicka, L., Con‐ way, A., et al. (2001). Replication dynamics of the yeast genome. Science Oct 5 , 294(5540), 115-121.

[82] Su, T. T. Heterochromatin replication: better late than ever. *Curr Biol*, 20(23), R 1018-

[83] Leach, T. J., Chotkowski, H. L., Wotring, M. G., Dilwith, R. L., & Glaser, R. L. (2000, Sep). Replication of heterochromatin and structure of polytene chromosomes. *Mol*

[84] Gilbert, D. M., Takebayashi, S. I., Ryba, T., Lu, J., Pope, B. D. , Wilson, K. A., et al. (2010). Space and time in the nucleus: developmental control of replication timing

[85] Kim, S. M., Dubey, D. D., & Huberman, J. A. Early-replicating heterochromatin.

[86] Arnoult, N., Schluth-Bolard, C., Letessier, A., Drascovic, I., Bouarich-Bourimi, R., Campisi, J., et al. (2010, Apr). Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear locali‐

[87] Bianchi, A., & Shore, D. Molecular biology Refined view of the ends. *Science*,

[88] Bianchi, A., & Shore, D. Early replication of short telomeres in budding yeast. *Cell*,

[89] Stewart, J. A., Chaiken, M. F., Wang, F., & Price, C. M. (2012). Maintaining the end: roles of telomere proteins in end-protection, telomere replication and length regula‐

[90] Fouche, N., Ozgur, S., Roy, D., & Griffith, J. D. (2006). Replication fork regression in

[91] Sampathi, S., & Chai, W. (2011, Jan). Telomere replication: poised but puzzling. *J Cell*

[92] Yang, Q., Zhang, R., Wang, X. W., Spillare, E. A., Linke, S. P., Subramanian, D., et al. (2002). The processing of Holliday junctions by BLM and WRN helicases is regulated

[93] Sfeir, A., Kosiyatrakul, S. T., Hockemeyer, D., Mac Rae, S. L., Karlseder, J., Schildk‐ raut, C. L., et al. (2009). Mammalian telomeres resemble fragile sites and require

[94] Miller, K. M., Rog, O., & Cooper, J. P. (2006). Semi-conservative DNA replication

[95] Gu, P, Min, J. N., Wang, Y, Huang, C, Peng, T, Chai, W, et al. CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell

through telomeres requires Taz1. NatureApr 6 , 440(7085), 824-828.

and chromosome architecture. *Cold Spring Harb Symp Quant Biol*, 75, 143-153.

R1020.

440 The Mechanisms of DNA Replication

*Cell Biol*, 20(17), 6308-16.

*Genes Dev*, 17(3), 330-335.

320(5881), 1301-1302.

128(6), 1051-1062.

*Mol Med*, 15(1), 3-13.

zation. *PLoS Genet*, 6(4), e1000920.

tion. Mutat Res Feb 1 , 730(1-2), 12-19.

repetitive DNAs. *Nucleic Acids Res*, 34(20), 6044-6050.

by. J Biol Chem Aug 30 , 277(35), 31980-31987.

exhaustion. *EMBO J*, 31(10), 2309-2321.

TRF1 for efficient replication. CellJul 10 , 138(1), 90-103.


[124] Belfort, M., Curcio, M. J., & Lue, N. F. (2011). Telomerase and retrotransposons: re‐ verse transcriptases that shaped genomes. Proc Natl Acad Sci U S A Dec 20 , 108(51), 20304-20310.

[111] Wang, F., Podell, E. R., Zaug, A. J., Yang, Y., Baciu, P., Cech, T. R., et al. (2007). The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature Feb 1 ,

[112] Zaug, A. J., Podell, E. R., Nandakumar, J., & Cech, T. R. (2010). Functional interaction between telomere protein TPP1 and telomerase. Genes Dev Mar 15 , 24(6), 613-22.

[113] Zaug, A. J., Podell, E. R., & Cech, T. R. (2005). Human POT1 disrupts telomeric Gquadruplexes allowing telomerase extension in vitro. Proc Natl Acad Sci U S A Aug

[114] Savitsky, M., Kravchuk, O., Melnikova, L., & Georgiev, P. (2002, May). Heterochro‐ matin protein 1 is involved in control of telomere elongation in Drosophila mela‐

[115] Perrini, B., Piacentini, L., Fanti, L., Altieri, F., Chichiarelli, S., & Berloco, M. (2004). HP1 controls telomere capping, telomere elongation, and telomere silencing by two

[116] Frydrychova, R. C., Biessmann, H., Konev, A. Y., Golubovsky, M. D., Johnson, J., Archer, T. K., et al. (2007, Jul). Transcriptional activity of the telomeric retrotranspo‐ son HeT-A in Drosophila melanogaster is stimulated as a consequence of subtermi‐ nal deficiencies at homologous and nonhomologous telomeres. *Mol Cell Biol*, 27(13),

[117] Frydrychova, R. C., Mason, J. M., & Archer, T. K. (2008, Sep). HP1 is distributed with‐ in distinct chromatin domains at Drosophila telomeres. *Genetics*, 180(1), 121-131. [118] Torok, T., Benitez, C., Takacs, S., & Biessmann, H. (2007, Apr). The protein encoded by the gene proliferation disrupter (prod) is associated with the telomeric retrotrans‐

[119] Savitsky, M., Kwon, D., Georgiev, P., Kalmykova, A., & Gvozdev, V. (2006). Telo‐ mere elongation is under the control of the RNAi-based mechanism in the Drosophi‐

[120] George, J. A., & Pardue, M. L. (2003, Feb). The promoter of the heterochromatic Dro‐ sophila telomeric retrotransposon, HeT-A, is active when moved into euchromatic

[121] Walter, M. F., & Biessmann, H. (2004, May). Expression of the telomeric retrotranspo‐ son HeT-A in Drosophila melanogaster is correlated with cell proliferation. *Dev*

[122] Oikawa, S., Tada-Oikawa, S., & Kawanishi, S. (2001). Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening. Biochemis‐

[123] Barja, G. (2002). Rate of generation of oxidative stress-related damage and animal

longevity. Free Radic Biol Med Nov 1 , 33(9), 1167-1172.

poson array in Drosophila melanogaster. *Chromosoma*, 116(2), 185-195.

different mechanisms in Drosophila. Mol Cell Aug 13 , 15(3), 467-476.

445(7127), 506-510.

442 The Mechanisms of DNA Replication

2 , 102(31), 10864-10869.

4991-5001.

nogaster. *Mol Cell Biol*, 22(9), 3204-3218.

la germline. Genes Dev Feb 1 , 20(3), 345-354.

locations. *Genetics*, 163(2), 625-635.

*Genes Evol*, 214(5), 211-9.

try Apr 17 , 40(15), 4763-8.

### **Chapter 18**

## **Telomere Shortening Mechanisms**

### Andrey Grach

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55244

### **1. Introduction**

Telomeres are the terminal regions of the linear chromosomes of eukaryotes, which are composed of telomeric DNA and associated specific telomeric proteins. In most kinds of organisms, telomeric DNA is presented by a large number of repetitive, strictly defined short nucleotide sequences, such as: TTAGGG (in vertebrates), TTTAGGG (in the majority of terrestrial plants) and TTGGGG (in the ciliated infusoria Tetrahymena), etc. Although telomeric proteins differ among different groups of organisms they perform similar functions, which mainly consist of telomere length regulation and their protection against degradation (Grach, 2009). For a long time, it was considered that telomeres did not code RNA molecules and thus proteins. Subsequently, it was found that RNA is still transcribed from telomeres but that it did not encode any proteins. Further studies showed that this RNA plays an important role in telomere length regulation and chromatin reorganisation during both development and cell differentiation (Azzalin et al., 2007). In spite of the fact that telomeres do not code proteins, they also perform very important functions, the main role of which is to maintain the stability and functionality of the cellular genome. Among these are the protection of chromosomes from fusion with each other (Blackburn, 2001), participation in mitotic and meiotic chromosome segregation (Conrad et al., 1997; Dynek and Smith, 2004; Kirk et al., 1997), the stabilisation of broken chromosome ends (Pennaneach et al., 2006), their attachment to the nuclear envelope (Hediger et al., 2002; Podgornaya et al., 2000), influencing gene expression (Baur et al., 2001; Pedram et al., 2006), counting the quantity of cell divisions (Allsopp et al., 1992; Kurenova & Mason, 1997; Olovnikov, 1973), and also an original buffer function (Olovnikov, 1973). The latter consists of the protection of the mRNA coding regions of chromosomes from the end replication problem. The end replication problem consists of the impossibility of the full reproduction of the previous length of linear DNA ends on the leading telomeres during of the S-phase of the cell division cycle. It is caused by peculiarities in their structure and the functioning of the DNA replication machinery. As a result the telomeric regions of chromo‐

© 2013 Grach; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Grach; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

somes in daughter cells are shortened by several tens of nucleotides at each cell division (Lingner et al., 1995). In addition to the end replication problem, the telomere repair problem can also play a role in telomere length shortening. This problem in turn can be divided into the end repair problem and the shelterin-mediated telomere repair problem. The end repair problem includes the incomplete repair of DNA ends and direct damage-mediated telomere shortening and it can occur at the extreme ends of chromosomes. The incomplete repair of DNA ends consists in the inability of a repair system to complete the repair of damage if it occurred at the extreme ends of telomeres, since repair proteins cannot work correctly on the brink of a template and, as a result, they will also be shortened. Direct damage-mediated telomere shortening is based on the fact that, in some cases, the repair of damage at the extreme ends of chromosomes cannot even begin, in contrast to the incomplete repair of DNA ends at which the repair process begins but is not fully completed. It can generally be invoked by the fact that the breaks occurring on the extreme ends of chromosomes lead to the complete separation of the terminal DNA section and as a result repair system proteins are not able to even partially restore such damage. The consequence of this - and also of the subsequent actions of nucleases, which restore previous telomere ends' configuration - is the DNA ends shortening again. The shelterin-mediated telomere repair problem consists of the inability of the proteins involved in DNA damage response to detect and repair the damage occurring at telomeres due to the fact that the telomeric proteins in combination with telomeric DNA form a special structure on the telomeres called a telomeric loop (t-loop) that directly blocks DNA damage response proteins, as well as they block various DNA repair pathways themselves that is especially actual for the uncapped telomere condition when t-loop is not yet formed. This ultimately leads to the accumulation of damage and telomere shortening, and occurs along the entire length of telomeres where there is a telosome organisation and not just at their extreme ends as is the case with the end repair problem.

Telomere shortening is closely related to the replicative potential of cells and their lifespan. Thus, in accordance with A. M. Olovnikov's telomere theory of aging, when the telomere length approaches a certain critical level the cells stop dividing and begin ageing and are exposed to apoptosis upon reaching that level (Olovnikov, 1973). This fact has been confirmed experimentally in a number of studies (Allsopp et al., 1992; Allsopp et al., 1995; Aubert & Lansdorp, 2008). Besides playing a key role in aging, telomeres are also have great significance for carcinogenesis, as some cells with shortened telomeres acquire mechanisms to bypass the aging program and gain (among other characteristics) the ability to maintain telomere length and hence to "unlimited" quantity of divisions (Desmaze et al., 2003; Londoño-Vallejo, 2008; Stewart & Weinberg, 2006). The ability to elongate telomeres in vertebrates can be realised by means of two known mechanisms. The first and the most widespread mechanism among tumours provides for the use of a special enzyme called telomerase. It is a ribonucleoprotein enzyme consisting of a catalytic subunit, a telomerase RNA molecule and several additional components. Joining in with the ends of telomeres, its catalytic subunit uses reverse transcrip‐ tion of RNA, which is a part of telomerase to elongate a G-rich chain of telomeric DNA, which corresponds to the 3'-end regions of the DNA. Further, a C-rich chain corresponding to 5'-end DNA is synthesised on a template of a significantly elongated G-rich chain by a regular DNA polymerase reaction. As a result, the telomere ends gain the same structure as they had prior to the telomerase action but they become much longer in this case (Blackburn & Collins, 2011; Dong et al., 2005; Testorelli, 2003). The second mechanism, which is found in a minority of neoplasm types, is accomplished by recombination-mediated telomere replication and belongs to the alternative lengthening of telomeres mechanisms (ALT) (Grach, 2011a; Grach, 2011b; Henson et al., 2002; Muntoni & Reddel, 2005; Stewart, 2005). Besides the elongation of telomeres in tumour cells, telomerase also has a high activity in stem and germ cells, thereby providing them a high proliferative capacity (Meeker & Coffey, 1997). Meanwhile, its activity is low or absent in normal somatic cells, making their replicative capacity strictly limited (Rhyu, 1995). As for ALT, it is usually repressed in normal cells by telomeric proteins and certain other factors (Grach, 2011b).

In recent years, the study of telomeres has becomes increasingly popular among scientists who are engaged both with different branches of molecular biology as well as with the most distant problems in the whole of modern medicine. Such heightened interest in their study is first of all caused by the fact that telomeres perform very important functions in the maintenance of eukaryotic cell genome normal functionality. Besides this, telomeres define the replicative capacity of cells and play a key role in their aging and transformation processes which make these end structures an even more important subject for research. All of the above mentioned roles of telomeres depend upon their shortening and, therefore, telomere shortening mecha‐ nisms are among the key aspects of telomere biology, because the loss of chromosome functions, cell aging and degeneration are associated with the telomeric regions of chromo‐ somes' length shortening. In this respect, the study of these mechanisms as well as the factors involved in their protection and elongation are of primary importance as long as our cumu‐ lative knowledge can help in the future in the struggle against aging, tumours and many other diseases, the treatment of which requires a high replicative capacity in the cells. Based on the great significance of the telomere shortening process, the nub of various telomere-shortening mechanisms will be considered in detail in this chapter, namely the end replication and telomere repair problems.

### **2. The end replication problem**

somes in daughter cells are shortened by several tens of nucleotides at each cell division (Lingner et al., 1995). In addition to the end replication problem, the telomere repair problem can also play a role in telomere length shortening. This problem in turn can be divided into the end repair problem and the shelterin-mediated telomere repair problem. The end repair problem includes the incomplete repair of DNA ends and direct damage-mediated telomere shortening and it can occur at the extreme ends of chromosomes. The incomplete repair of DNA ends consists in the inability of a repair system to complete the repair of damage if it occurred at the extreme ends of telomeres, since repair proteins cannot work correctly on the brink of a template and, as a result, they will also be shortened. Direct damage-mediated telomere shortening is based on the fact that, in some cases, the repair of damage at the extreme ends of chromosomes cannot even begin, in contrast to the incomplete repair of DNA ends at which the repair process begins but is not fully completed. It can generally be invoked by the fact that the breaks occurring on the extreme ends of chromosomes lead to the complete separation of the terminal DNA section and as a result repair system proteins are not able to even partially restore such damage. The consequence of this - and also of the subsequent actions of nucleases, which restore previous telomere ends' configuration - is the DNA ends shortening again. The shelterin-mediated telomere repair problem consists of the inability of the proteins involved in DNA damage response to detect and repair the damage occurring at telomeres due to the fact that the telomeric proteins in combination with telomeric DNA form a special structure on the telomeres called a telomeric loop (t-loop) that directly blocks DNA damage response proteins, as well as they block various DNA repair pathways themselves that is especially actual for the uncapped telomere condition when t-loop is not yet formed. This ultimately leads to the accumulation of damage and telomere shortening, and occurs along the entire length of telomeres where there is a telosome organisation and not just at their

Telomere shortening is closely related to the replicative potential of cells and their lifespan. Thus, in accordance with A. M. Olovnikov's telomere theory of aging, when the telomere length approaches a certain critical level the cells stop dividing and begin ageing and are exposed to apoptosis upon reaching that level (Olovnikov, 1973). This fact has been confirmed experimentally in a number of studies (Allsopp et al., 1992; Allsopp et al., 1995; Aubert & Lansdorp, 2008). Besides playing a key role in aging, telomeres are also have great significance for carcinogenesis, as some cells with shortened telomeres acquire mechanisms to bypass the aging program and gain (among other characteristics) the ability to maintain telomere length and hence to "unlimited" quantity of divisions (Desmaze et al., 2003; Londoño-Vallejo, 2008; Stewart & Weinberg, 2006). The ability to elongate telomeres in vertebrates can be realised by means of two known mechanisms. The first and the most widespread mechanism among tumours provides for the use of a special enzyme called telomerase. It is a ribonucleoprotein enzyme consisting of a catalytic subunit, a telomerase RNA molecule and several additional components. Joining in with the ends of telomeres, its catalytic subunit uses reverse transcrip‐ tion of RNA, which is a part of telomerase to elongate a G-rich chain of telomeric DNA, which corresponds to the 3'-end regions of the DNA. Further, a C-rich chain corresponding to 5'-end DNA is synthesised on a template of a significantly elongated G-rich chain by a regular DNA polymerase reaction. As a result, the telomere ends gain the same structure as they had prior

extreme ends as is the case with the end repair problem.

446 The Mechanisms of DNA Replication

#### **2.1. Early views of the end replication problem**

#### *2.1.1. The end replication problem as a cause of telomere length shortening, which determines the replicative potential of cells*

For the first time, the problem of eukaryotic linear chromosome ends' replication was proposed and described in detail in the form of the theory of marginotomy by A. M. Olovnikov in Russian in 1971 (Olovnikov, 1971). One year later, in 1972, the problem was also described by J. D. Watson, independently of Olovnikov (Watson, 1972). In 1973, the problem proposed by Olovnikov was represented in its English version (Olovnikov, 1973). At the heart of this problem, as was suggested, lies the inability of the usual DNA replication system to fully complete the replication of linear DNA ends in the process of cell division. As a result of this, it was assumed that the telomeric regions of chromosomes are shortened by roughly tens of base pairs (bps) at each cell doubling (Olovnikov, 1973). This state of affairs should explain why normal somatic cells, having divided a number of times, stop their further reproduction, start ageing and undergo apoptosis (i.e. the causes of the Hayflick cell division limit) (Hayflick, 1965). It was therefore suggested that in all of the non-transformed somatic cells of the organism, the telomere replication mechanism is absent and as a result of which they are gradually shortened on their division. When telomeres shorten to the definite minimal length needed for their normal functioning, the cells stop their division, age and then die. In other words, it was suggested that the telomere shortening process is a kind of "counter" which determines the replicative potential of cells (Olovnikov, 1973). These suppositions have been confirmed experimentally in several studies (Allsopp et al., 1992; Allsopp et al., 1995; Levy, et al., 1992). Thus, in one such experiment it was found that cells with shortened telomeres could perform far fewer divisions than cells with long telomeres (Allsopp et al., 1992).

#### *2.1.2. The old theoretical model of the end replication problem*

We now consider the actual mechanisms of the incomplete replication of the ends of linear DNA. As is known, every human chromosome consists of two anti-parallel DNA strands, which together form a single linear double-stranded DNA molecule with two ends. When the end replication problem was described for the first time, it was still considered that according to the generally accepted DNA model its ends would also have a double-stranded structure. Proceeding from this understanding of DNA, has been formulated the old theoretical model of the end replication problem, which was based on two possible mechanisms by means of which DNA ends could not be completely replicated.

The first mechanism suggests that DNA polymerase implementing DNA synthesis only in the 5' → 3' direction should have besides the catalytic centre also the DNA binding site, which should be located in front of catalytic one and be responsible for attachment of enzyme to a parent DNA strand. As such, and during DNA replication, when a DNA polymerase ap‐ proaches the very end of the template by moving in front of the DNA binding site it cannot continue synthesis and so dissociates from the DNA because it will have nothing more to bind to. As a result, the end portion of a template which is equal in length to a DNA binding centre cannot be replicated, since a DNA polymerase cannot simply bring its catalytic centre to the last nucleotides of a parent strand without being dissociated from the DNA. Thus, after an incomplete replication process of such a kind, the 3'-end of a new DNA strand should become shortened by several nucleotides when compared with the parental one (Olovnikov, 1973).

The second mechanism of the incomplete replication of DNA ends was based on the fact that a DNA polymerase is not able to begin new DNA synthesis itself but is capable only of elongating already existing oligonucleotides. Therefore, at the very beginning of replication, primase synthesises a short RNA primer of around 9-12 nucleotides long, which is subse‐ quently elongated by a leading strand DNA polymerase. When the DNA polymerase has already synthesised a long enough DNA strand, the RNA primer is removed by RNase after that the gap is filled by the polymerase and the ends between the DNA fragments are connected by a ligase. The DNA end regions there do not form an exception and are replicated according to the same principle. However, the problem arises with the RNA primer, which is attached to the 3'-end of the DNA and defines the 5'-end of a new strand. The end gap arising after removal of the RNA primer cannot be filled later by the DNA polymerase, as there is no free 3'-end, which it could elongate. As a result, such incomplete end replication mechanism leads, this time, to the shortening of the 5'-end of a new DNA strand (Olovnikov, 1973).

it was assumed that the telomeric regions of chromosomes are shortened by roughly tens of base pairs (bps) at each cell doubling (Olovnikov, 1973). This state of affairs should explain why normal somatic cells, having divided a number of times, stop their further reproduction, start ageing and undergo apoptosis (i.e. the causes of the Hayflick cell division limit) (Hayflick, 1965). It was therefore suggested that in all of the non-transformed somatic cells of the organism, the telomere replication mechanism is absent and as a result of which they are gradually shortened on their division. When telomeres shorten to the definite minimal length needed for their normal functioning, the cells stop their division, age and then die. In other words, it was suggested that the telomere shortening process is a kind of "counter" which determines the replicative potential of cells (Olovnikov, 1973). These suppositions have been confirmed experimentally in several studies (Allsopp et al., 1992; Allsopp et al., 1995; Levy, et al., 1992). Thus, in one such experiment it was found that cells with shortened telomeres could

perform far fewer divisions than cells with long telomeres (Allsopp et al., 1992).

We now consider the actual mechanisms of the incomplete replication of the ends of linear DNA. As is known, every human chromosome consists of two anti-parallel DNA strands, which together form a single linear double-stranded DNA molecule with two ends. When the end replication problem was described for the first time, it was still considered that according to the generally accepted DNA model its ends would also have a double-stranded structure. Proceeding from this understanding of DNA, has been formulated the old theoretical model of the end replication problem, which was based on two possible mechanisms by means of

The first mechanism suggests that DNA polymerase implementing DNA synthesis only in the 5' → 3' direction should have besides the catalytic centre also the DNA binding site, which should be located in front of catalytic one and be responsible for attachment of enzyme to a parent DNA strand. As such, and during DNA replication, when a DNA polymerase ap‐ proaches the very end of the template by moving in front of the DNA binding site it cannot continue synthesis and so dissociates from the DNA because it will have nothing more to bind to. As a result, the end portion of a template which is equal in length to a DNA binding centre cannot be replicated, since a DNA polymerase cannot simply bring its catalytic centre to the last nucleotides of a parent strand without being dissociated from the DNA. Thus, after an incomplete replication process of such a kind, the 3'-end of a new DNA strand should become shortened by several nucleotides when compared with the parental one (Olovnikov, 1973).

The second mechanism of the incomplete replication of DNA ends was based on the fact that a DNA polymerase is not able to begin new DNA synthesis itself but is capable only of elongating already existing oligonucleotides. Therefore, at the very beginning of replication, primase synthesises a short RNA primer of around 9-12 nucleotides long, which is subse‐ quently elongated by a leading strand DNA polymerase. When the DNA polymerase has already synthesised a long enough DNA strand, the RNA primer is removed by RNase after that the gap is filled by the polymerase and the ends between the DNA fragments are connected by a ligase. The DNA end regions there do not form an exception and are replicated according

*2.1.2. The old theoretical model of the end replication problem*

448 The Mechanisms of DNA Replication

which DNA ends could not be completely replicated.

Thus, according to the old theoretical model of the end replication problem, the 5' overhang at one end and the 3' overhang at the other can be formed at both daughter DNAs arising due to the peculiarities in the functioning of the DNA polymerase system. Such single-stranded protrusions should be cut by nucleases further in order to achieve double-stranded DNA ends' structure - which, as it was supposed earlier, they had initially. Consequently, the daughter chromosome telomere ends upon completion of the replication process will have the same configuration as they had before doubling. However, at the same time they will be shortened by a certain number of nucleotides (Olovnikov, 1973). The old theoretical model of the end replication problem is depicted in Figure 1.

#### *2.1.3. Experimental confirmation of the old theoretical model of the end replication problem*

As is shown in Fig. 1, the old theoretical model of the end replication problem assumes that incomplete replication can result from two mechanisms at both DNA ends - both on leading and lagging strands, resulting in each daughter chromosome being shortened at each of it's telomeres simultaneously. Further experimental verification of these circumstances has demonstrated quite different results for the leading strand and has completely confirmed the suppositions concerning the lagging DNA strand. In the course of one piece of research into the end replication problem using the artificially-created linear DNA replication system of the SV40 virus *in vitro*, it has been clearly determined that leading strand is synthesised entirely up to the very 5'-end of the template (Ohki et al., 2001). The explanation for this is that the DNA helicase unwinds its parent DNA to the very end and thus allows the completion of the synthesis of a new DNA strand. This discovery could abruptly undermine the possibility of the existence of the first proposed incomplete DNA replication mechanism, which assumes that due to the peculiarities of the functioning of DNA polymerase, the leading DNA strand synthesis cannot be fully completed up to the very end of the template. However, it is perhaps too early to judge this.

The results of another study of the end replication problem have shown the absolutely opposite situation. In the course of experiments looking into G-rich and C-rich single-stranded DNA in human fibroblasts, researchers unexpectedly revealed that the 5'-end of the DNA leading strand template is not replicated completely in the proliferating cells. Therefore, the 5' overhangs appears in these cells predominantly during S-phase. This information provides grounds to suppose that the replication fork can terminate before reaching the chromosome's end. The authors of this study explain this in such a way that if the last RNA primer of the lagging strand is to be created as closely as possible to the 3'-end of the template then, in this case, the complete synthesis of the leading strand up to the very end of the 5'-end of a template is possible. If priming occurs more centromerically, then incomplete DNA leading strand replication and related to it, enhanced telomere shortening may be observed (Cimino-Reale et al., 2003).

Similar results have also been observed in one more study, although it was not carried out with nuclear DNA but rather with the linear mitochondrial DNA end regions of the yeasts (in humans, mitochondrial DNA is ring-shaped). It was found during this work that the DNA polymerase stops at a distance of approximately 110 nucleotides from the 5'-end of a template and does not continue further leading strand synthesis, thereby again leaving the 5'-overhangs. However, the authors of the research could not explain why this happens (Nosek et al., 1995).

Consequently, the results of the researches just reviewed are quite conflicting. Unlike the first investigation described, where DNA leading strand synthesis continues up to the very end of the 5'-end of the parent DNA, creating "blunt" end, in the second and in the third studies we observed the incomplete replication of the leading strand with the creation of a "sharp" end of the DNA molecule. Moreover, none of the studies describe the possibility that complete or incomplete replication of the parent DNA 5'-ends was due to the peculiarities in the function of DNA polymerase as had been suggested by Olovnikov in its first incomplete DNA ends replication mechanism. Instead they consider quite other reasons for - in one case of complete leading strand synthesis to the very end of a template, and in another instance of incomplete leading strand replication.

Therefore, this data cannot fully support or refute the possibility of the inability of DNA polymerase to complete the replication of the 5'-end through a failure to bring its catalytic site to the last nucleotides of a template. It is also very important to note that the above mentioned studies, which describe the incomplete synthesis of the DNA leading strand with the creation of a 5' overhang, in practice are almost unique in their kind. The prevailing majority of studies show that leading strand is synthesised completely up to the very end of a template (Chai et al., 2006; Lingner et al., 1995, Wright et al., 1997).

It was also shown in the experimental research reviewed by us initially that lagging strand synthesis stops within the area located at a distance of approximately 500 bps from the end of a parent strand leaving 3' overhangs there (Ohki et al., 2001). This in its turn fully supports assumptions concerning existence of the second mechanism of incomplete DNA replication described above. As too long extension of incomplete replication was found here, in this work the authors have reviewed somewhat in a new way this mechanism, performed on the lagging strand. It is known that the length of RNA primers range from 9 to 12 nucleotides, which has been described in most of the studies that we have analysed (Griep, 1995; Hao & Tan, 2002; Sfeir et al., 2005). In some cases, primers of 20-30 nucleotides in length are also mentioned (Bouche et al., 1978; Dai et al., 2009). Nonetheless, the length of an incomplete replication of a DNA lagging strand is much longer, and has been discovered to be as long as 500 nucleotides. Therefore, the authors of this research propose the following mechanisms for the incomplete replication of the DNA lagging strand. It should be noted that the first mechanism completely corresponds to that proposed in the old theoretical model of the end replication problem for DNA. As has already been noted, it is based on the removal of the end RNA primer and the further failure to fill the resulting gap with deoxyribonucleotides. As lagging strand's incom‐ plete replication reached approximately 500 nucleotides and the RNA primer length ranges

replication and related to it, enhanced telomere shortening may be observed (Cimino-Reale et

Similar results have also been observed in one more study, although it was not carried out with nuclear DNA but rather with the linear mitochondrial DNA end regions of the yeasts (in humans, mitochondrial DNA is ring-shaped). It was found during this work that the DNA polymerase stops at a distance of approximately 110 nucleotides from the 5'-end of a template and does not continue further leading strand synthesis, thereby again leaving the 5'-overhangs. However, the authors of the research could not explain why this happens (Nosek et al., 1995). Consequently, the results of the researches just reviewed are quite conflicting. Unlike the first investigation described, where DNA leading strand synthesis continues up to the very end of the 5'-end of the parent DNA, creating "blunt" end, in the second and in the third studies we observed the incomplete replication of the leading strand with the creation of a "sharp" end of the DNA molecule. Moreover, none of the studies describe the possibility that complete or incomplete replication of the parent DNA 5'-ends was due to the peculiarities in the function of DNA polymerase as had been suggested by Olovnikov in its first incomplete DNA ends replication mechanism. Instead they consider quite other reasons for - in one case of complete leading strand synthesis to the very end of a template, and in another instance of incomplete

Therefore, this data cannot fully support or refute the possibility of the inability of DNA polymerase to complete the replication of the 5'-end through a failure to bring its catalytic site to the last nucleotides of a template. It is also very important to note that the above mentioned studies, which describe the incomplete synthesis of the DNA leading strand with the creation of a 5' overhang, in practice are almost unique in their kind. The prevailing majority of studies show that leading strand is synthesised completely up to the very end of a template (Chai et

It was also shown in the experimental research reviewed by us initially that lagging strand synthesis stops within the area located at a distance of approximately 500 bps from the end of a parent strand leaving 3' overhangs there (Ohki et al., 2001). This in its turn fully supports assumptions concerning existence of the second mechanism of incomplete DNA replication described above. As too long extension of incomplete replication was found here, in this work the authors have reviewed somewhat in a new way this mechanism, performed on the lagging strand. It is known that the length of RNA primers range from 9 to 12 nucleotides, which has been described in most of the studies that we have analysed (Griep, 1995; Hao & Tan, 2002; Sfeir et al., 2005). In some cases, primers of 20-30 nucleotides in length are also mentioned (Bouche et al., 1978; Dai et al., 2009). Nonetheless, the length of an incomplete replication of a DNA lagging strand is much longer, and has been discovered to be as long as 500 nucleotides. Therefore, the authors of this research propose the following mechanisms for the incomplete replication of the DNA lagging strand. It should be noted that the first mechanism completely corresponds to that proposed in the old theoretical model of the end replication problem for DNA. As has already been noted, it is based on the removal of the end RNA primer and the further failure to fill the resulting gap with deoxyribonucleotides. As lagging strand's incom‐ plete replication reached approximately 500 nucleotides and the RNA primer length ranges

al., 2003).

450 The Mechanisms of DNA Replication

leading strand replication.

al., 2006; Lingner et al., 1995, Wright et al., 1997).

**Figure 1.** Schematic representation of the old theoretical model of the end replication problem, described by A. M. Olovnikov in 1971.

from 9 to 12 nucleotides, the authors of the work considered it very unlikely that this mecha‐ nism was the only one for the shortening of the 5'-end of daughter DNA. The second assumed mechanism consists of the inability of the DNA polymerase α-primase to begin lagging strand synthesis at the very end of a linear DNA molecule that can be the main cause of the end replication problem of the parent DNA's 3'-end (Ohki et al., 2001). It is also interesting that the length of Okazaki fragments, which represent the short DNA fragments with RNA primers at the 5'-end and are the key feature of the lagging strand, can range from between 100 to 500 nucleotides (Burgers, 2009; Mackenney et al., 1997; Zheng & Shen, 2011). Therefore, if such a fragment cannot be formed at the very end of the parent strand then the DNA daughter strand after the replication process will appear to be shortened in its length. Besides this research, many other studies showing similar results with incomplete lagging strand synthesis are also known.

Thus, most of the conditions of the old theoretical model of the end replication problem initially proposed by Olovnikov were confirmed absolutely in the course of the experiments carried out. In particular, it has been confirmed that telomeres are shortened at every cell division and that, specifically, these circumstances define the replicative potential of the cells and appear to be the cause of their aging and subsequent programmed cell death. Nevertheless, it was a failure to acquire any information absolutely confirming that the specifically incomplete replication of the DNA strands and subsequent cleavage of the resulting single-stranded overhangs of the molecule by nucleases results in telomeres' ends shortening, as was supposed by the old theoretical model. Unfortunately, we also failed to find any experimental data which fully confirms the first mechanism for the incomplete replication of DNA ends, assuming that DNA polymerase is not able to completely copy the 5'-end of the DNA leading strand template since it is incapable of bringing its catalytic site to the last nucleotides of a parent strand. At the same time, the results of the studies show that the 5'-end of a template remains in some cases not completely replicated, but other reasons for this, which are not directly related to DNA polymerase are specified in these cases. Therefore, the assumption regarding incomplete replication of the DNA leading strand is basically confirmed, but it is still unclear whether DNA polymerase directly plays a key role here or whether some other factors are involved (such as the ones that have been mentioned by the authors of the studies already discussed). Given all this, the assumption concerning the second mechanism of the incomplete replication of DNA ends is completely confirmed. Thus, the 3'-ends of the parent DNA, as was confirmed by the results of the experiments and initially stated in the theory, cannot be completely replicated during the lagging strand synthesis. As long as the extension of the incomplete replication of a lagging strand was much longer than the RNA primer length, it was supposed that the reason for the incomplete formation of a lagging strand along with the end primer removal might be due to the inability to prime and create the whole Okazaki fragment at the very DNA end. Given that the incomplete synthesis of the DNA leading strand is described only in some studies, but in the overwhelming majority of works it is shown that leading strand synthesis is performed completely up to the very end of a template, and that the 3'-end of the DNA template cannot for sure be replicated completely, the old theoretical model of end replication problem was also named a problem of incomplete lagging strand synthesis.

#### **2.2. Modern views of the end replication problem**

#### *2.2.1. The establishment of the fact that telomere ends have a single-stranded structure*

In the early 1980s, the data began to appear suggesting that both ends of each chromosome need not necessarily have a double-stranded structure but that they have a single-stranded structure (i.e. they are represented by 3'-overhangs). In 1981, it was noted for the first time that the ends of the linear minichromosomes, which are present in macronuclei of such ciliates as *Oxytricha*, *Stylonychia* and *Euplotes*, possess G-rich 3'-overhangs between 12 and 16 nucleotides long (Klobutcher et al., 1981). Later on, in 1989, similar results were also acquired for the linear extrachromosomal ribosomal DNA of ciliate *Tetrahymena* and - evolutionarily distant from it – the slim mould *Didymium* (Henderson & Blackburn, 1989). Later, in 1993, it was found that the telomeres of the yeast *Saccharomyces cerevisiae* also gain 3'-end overhangs in the late S phase of the cell cycle and which differ a little in their dimensions from the ones which were described in previous works, being formed by more than 30 nucleotides (Wellinger et al., 1993). Unlike the above-mentioned organisms, which have a constant G-overhang length, the telomeric overhangs of higher eukaryotes display variability, even among the different cells studied in one group. As has been demonstrated by the results of a great number of studies, human telomeres possess very heterogeneous 3' overhangs, ranging from very short ones 35 nucleo‐ tides long and even less, to very long ones with an extension of 500 nucleotides or more. Furthermore, such varying in their dimensions G-overhangs are observed in all types of examined cells including the telomerase-positive transformed cells, telomerase-negative normal mitotic cells and post-mitotic cells (Cimino-Reale et al., 2001; Makarov et al., 1997; McElligott & Wellinger, 1997; Stewart et al., 2003; Wright et al., 1997). All these observations allow the supposition that G-overhangs are a general feature of eukaryotic chromosome telomeres.

#### *2.2.2. A new theoretical model of the end replication problem*

after the replication process will appear to be shortened in its length. Besides this research, many other studies showing similar results with incomplete lagging strand synthesis are also

Thus, most of the conditions of the old theoretical model of the end replication problem initially proposed by Olovnikov were confirmed absolutely in the course of the experiments carried out. In particular, it has been confirmed that telomeres are shortened at every cell division and that, specifically, these circumstances define the replicative potential of the cells and appear to be the cause of their aging and subsequent programmed cell death. Nevertheless, it was a failure to acquire any information absolutely confirming that the specifically incomplete replication of the DNA strands and subsequent cleavage of the resulting single-stranded overhangs of the molecule by nucleases results in telomeres' ends shortening, as was supposed by the old theoretical model. Unfortunately, we also failed to find any experimental data which fully confirms the first mechanism for the incomplete replication of DNA ends, assuming that DNA polymerase is not able to completely copy the 5'-end of the DNA leading strand template since it is incapable of bringing its catalytic site to the last nucleotides of a parent strand. At the same time, the results of the studies show that the 5'-end of a template remains in some cases not completely replicated, but other reasons for this, which are not directly related to DNA polymerase are specified in these cases. Therefore, the assumption regarding incomplete replication of the DNA leading strand is basically confirmed, but it is still unclear whether DNA polymerase directly plays a key role here or whether some other factors are involved (such as the ones that have been mentioned by the authors of the studies already discussed). Given all this, the assumption concerning the second mechanism of the incomplete replication of DNA ends is completely confirmed. Thus, the 3'-ends of the parent DNA, as was confirmed by the results of the experiments and initially stated in the theory, cannot be completely replicated during the lagging strand synthesis. As long as the extension of the incomplete replication of a lagging strand was much longer than the RNA primer length, it was supposed that the reason for the incomplete formation of a lagging strand along with the end primer removal might be due to the inability to prime and create the whole Okazaki fragment at the very DNA end. Given that the incomplete synthesis of the DNA leading strand is described only in some studies, but in the overwhelming majority of works it is shown that leading strand synthesis is performed completely up to the very end of a template, and that the 3'-end of the DNA template cannot for sure be replicated completely, the old theoretical model of end replication problem was also named a problem of incomplete lagging strand synthesis.

**2.2. Modern views of the end replication problem**

*2.2.1. The establishment of the fact that telomere ends have a single-stranded structure*

In the early 1980s, the data began to appear suggesting that both ends of each chromosome need not necessarily have a double-stranded structure but that they have a single-stranded structure (i.e. they are represented by 3'-overhangs). In 1981, it was noted for the first time that the ends of the linear minichromosomes, which are present in macronuclei of such ciliates as *Oxytricha*, *Stylonychia* and *Euplotes*, possess G-rich 3'-overhangs between 12 and 16 nucleotides

known.

452 The Mechanisms of DNA Replication

Based on numerous experimental observations showing that telomere ends' structure is not double-stranded but single-stranded, J. Lingner et al. have shown that this situation consid‐ erably changes established views as to the end replication problem. In particular, they demonstrated that the second mechanism of incomplete DNA replication, based on last RNA primer removal, no longer necessarily appears to be a problem for DNA replication machinery and the cause of telomere shortening. As primer cutting all the same leads to the creation of a 3' overhang, which also existed prior to replication and which is a normal structural feature of chromosome ends, so no genetic informational loss occurs in this case. In this respect, the incomplete synthesis of the lagging strand up to the end of a template can be considered now to be the mechanism of normal single-stranded 3' overhang telomere ends' structural forma‐ tion. At the same time, a problem arises with the leading strand synthesis. This is caused by the fact that in the course of replication on such telomeres, daughter chromosomes lose the 3' overhang which was present in the parent chromosome and in the absence of telomerase this will accordingly result in their shortening. Moreover, if it is not restored by this enzyme to its previous state then, and only in this case, in the next round of replication might be observed the problem of incomplete lagging strand synthesis and already associated to it DNA short‐ ening (Lingner et al., 1995) (Fig. 2). Thus, the result of replication with the new theoretical model proposed by Lingner et al. is a formation of two daughter DNAs which have one "sharp" end with a 3' overhang forming due to lagging strand synthesis and the other "blunt" end (or a "sharp" one with a 5' overhang if we take into consideration the possibility of performing the first mechanism of the old model of the end replication problem proposed by Olovnikov) forming on the leading strand. In contrast to the earlier proposed theoretical model of the end replication problem where overhangs should be cut, now single-stranded 3'-end protrusions remain intact, forming the natural eukaryotic chromosomes ends. Given all this, if according to a new theoretical model leading strand synthesis results in the loss of 3' overhangs and the formation of "blunt" DNA ends, but the results of many experiments show that both chromo‐ some ends have G-overhangs, and given that incomplete lagging strand replication assumes its formation only on one end, then there should also be a mechanism creating such an overhang on leading telomeres (Fig. 2).

Lingner et al. have also proposed two possible mechanisms for previous 3' overhang formation which also guarantee that DNA shortening, due to a problem of incomplete lagging strand synthesis, can never occur. The first mechanism presupposes that after the DNA replication process the end of the newly synthesised leading strand in the "blunt" DNA end can be elongated by telomerase and as a result a "sharp" DNA end, with a previous 3' overhang, will be restored. The possible caveat of this variant is that the DNA molecule with the "blunt" end acts here as a substrate for telomerase but telomerase is able to elongate only single-stranded ends rather than double-stranded ends as was found earlier *in vitro*. Nevertheless, the possibility that telomerase access could be provided in this case by helicases, nucleases or proteins binding single-stranded DNA has been considered. The second mechanism assumes that telomerase acts before the replication process, elongating the 3' overhang. It creates a template for the gap-filling synthesis of the complementary C-strand. As a result of the elongation of the 5'-end by conventional DNA replication machinery and the subsequent removal of the RNA primer, a telomere end region acquires a 3' overhang structure again but becomes much longer. Now, when the replication process approaches its end, the overhang on the leading strand is also lost but the genetic material no longer decreases, since before replication the parent strand of the leading strand was elongated. Further, the so-formed DNA "blunt" end and, in particular, its 5'-end region are exposed to processing by nucleases resulting in the formation of a "sharp" end with a previous 3' overhang that existed prior to the elongation by the telomerase (Lingner et al., 1995).

#### *2.2.3. Experimental evidence for the new theoretical model of the end replication problem*

These mechanisms describe the different possibilities for the creation of a previous 3' over‐ hang and opposition to telomere shortening due to an inability during leading DNA strand synthesis to create such a single-stranded protrusion. However, both of these mechanisms are based on the possibility of telomerase action. At the same time telomerase activity is either very low in most normal human somatic cells or else is not observed at all (Rhyu, 1995). In spite of this, 3' overhangs are observed at both chromosome ends in normal cells (Makarov et al., 1997). There are also the results of focused experimental studies, showing that the removal of the genes coding for telomerase components does not affect the G-overhang structure considerably and this in turn also shows that the formation of such overhangs occurs irrespective of telomerase activity (Dionn & Wellinger, 1996; Hemann & Greider, 1999; Yuan et al., 1999). Besides, it was found during another study that these overhangs are exposed to cell cycle-regulated changes independent of telomerase activity (Dai et al., 2010). At the same time, telomerase in the cells where it is present is capable of elongating the 3' overhang after it is formed and thus make it like in the previous parental telomere. On this basis, it might be supposed in principle that previous 3' overhang in cells where there is no telomerase activity cannot be restored, but at the same time in its place a new overhang, by means of a special mechanism which will be reviewed later, is formed which results in the telomere shortening. It is should also be mentioned that if it were forever restored to a previous state with the telomerase participation that was as‐ sumed in the above described mechanisms, it would lead to telomeres not be shortened during the course of cell doubling. This is equivalent to the acquisition of the unlimited replication potential which is observed mainly in the transformed cells. At the same time, the second mechanism reviewed presupposes that after preliminary elongation by polymerases, a DNA "blunt" end formed during replication due to the impossibility of creating a 3' overhang through leading strand synthesis is exposed to treatment by nucleases which process its C-rich strand and thus create an overhang of a specific length. Recent studies suggest that such postreplication treatment of a parent strand by nucleases, independently of whether there is telomerase in the cells or not, seems likely to be the main mechanism of 3' overhang forma‐ tion in the leading telomeres (Lenain et al., 2006; van Overbeek & de Lange, 2006; Wu et al., 2010). If the parent 3' overhang before replication were to be elongated by telomerase, then the nucleases activity would further lead to previous 3' overhang formation, i.e. telomere end length does not decrease upon that and even increases, and if not, then these enzymes will create a new 3' overhang resulting in the shortening of the telomere's length. It is important to note that there are studies, showing that telomerase elongates the 3' overhangs of the leading daughter telomeres (Chai et al., 2006). Therefore, the first of the above reviewed mechanisms can be considered more realistic for previous 3' overhang restoration, especially taking into account that the DNA's "blunt" end after replication is necessarily exposed to the nuclease's influence and only after this does it become accessible for telomerase. It is also important to note that if in a case of accomplishment of the first mechanism of the old model of the end replication problem a DNA " sharp" end with a 5' overhang will be formed, the telomere ends shortening in that case would be even greater, as the incomplete synthesis of the DNA leading strand up to the end of a template and - related to this - excessive post-replication processing will take place. The latter is caused by the situation that nucleases now, in order to create a 3' over‐ hang, will not only remove a certain number of C-strand nucleotides as a part of the doublestranded DNA, but also its single-stranded overhang. The schematic representation of the new theoretical model of the end replication problem is presented in Fig. 2.

to a new theoretical model leading strand synthesis results in the loss of 3' overhangs and the formation of "blunt" DNA ends, but the results of many experiments show that both chromo‐ some ends have G-overhangs, and given that incomplete lagging strand replication assumes its formation only on one end, then there should also be a mechanism creating such an

Lingner et al. have also proposed two possible mechanisms for previous 3' overhang formation which also guarantee that DNA shortening, due to a problem of incomplete lagging strand synthesis, can never occur. The first mechanism presupposes that after the DNA replication process the end of the newly synthesised leading strand in the "blunt" DNA end can be elongated by telomerase and as a result a "sharp" DNA end, with a previous 3' overhang, will be restored. The possible caveat of this variant is that the DNA molecule with the "blunt" end acts here as a substrate for telomerase but telomerase is able to elongate only single-stranded ends rather than double-stranded ends as was found earlier *in vitro*. Nevertheless, the possibility that telomerase access could be provided in this case by helicases, nucleases or proteins binding single-stranded DNA has been considered. The second mechanism assumes that telomerase acts before the replication process, elongating the 3' overhang. It creates a template for the gap-filling synthesis of the complementary C-strand. As a result of the elongation of the 5'-end by conventional DNA replication machinery and the subsequent removal of the RNA primer, a telomere end region acquires a 3' overhang structure again but becomes much longer. Now, when the replication process approaches its end, the overhang on the leading strand is also lost but the genetic material no longer decreases, since before replication the parent strand of the leading strand was elongated. Further, the so-formed DNA "blunt" end and, in particular, its 5'-end region are exposed to processing by nucleases resulting in the formation of a "sharp" end with a previous 3' overhang that existed prior to the elongation

*2.2.3. Experimental evidence for the new theoretical model of the end replication problem*

These mechanisms describe the different possibilities for the creation of a previous 3' over‐ hang and opposition to telomere shortening due to an inability during leading DNA strand synthesis to create such a single-stranded protrusion. However, both of these mechanisms are based on the possibility of telomerase action. At the same time telomerase activity is either very low in most normal human somatic cells or else is not observed at all (Rhyu, 1995). In spite of this, 3' overhangs are observed at both chromosome ends in normal cells (Makarov et al., 1997). There are also the results of focused experimental studies, showing that the removal of the genes coding for telomerase components does not affect the G-overhang structure considerably and this in turn also shows that the formation of such overhangs occurs irrespective of telomerase activity (Dionn & Wellinger, 1996; Hemann & Greider, 1999; Yuan et al., 1999). Besides, it was found during another study that these overhangs are exposed to cell cycle-regulated changes independent of telomerase activity (Dai et al., 2010). At the same time, telomerase in the cells where it is present is capable of elongating the 3' overhang after it is formed and thus make it like in the previous parental telomere. On this basis, it might be supposed in principle that previous 3' overhang in cells where there is no telomerase activity cannot be restored, but at the same time in its place a new overhang, by means of a special mechanism which will be reviewed

overhang on leading telomeres (Fig. 2).

454 The Mechanisms of DNA Replication

by the telomerase (Lingner et al., 1995).

It is clearly shown in Fig. 2 that under the new theoretical model of the end replication problem the incomplete DNA lagging strand synthesis, as a consequence of the impossibility of creating an Okazaki fragment and the removal of an end RNA primer, no longer leads to the daughter telomere's shortening but appears instead to be a kind of mechanism of their normal 3' overhanging structures' renewal. At the same time, during the synthesis of the leading strand, the DNA replication machinery is not able to recreate such an overhang on other chromosome ends as for its synthesis there is simply no template. Therefore, the leading telomeres of daughter chromosomes with respect to a parent chromosome lose their 3' overhang, which can be the cause of their further shortening. Experimental evidence for the claim that it is 3' overhang loss, which really leads to telomere shortening is derived from one study where it was found that the length of this overhang completely agrees with the chromosome end regions' shortening rate (Huffman et al., 2000). Nevertheless, there is also the data from another study showing that the G-overhang length does not correlate with the telomere-shortening rate (Keys et al., 2004). The authors of the research suggest that besides the 3' overhang loss in the course of DNA replication; the telomere-shortening rate is also influenced by damage from oxygen free radicals (Keys et al., 2004). As a result of such replication, there occurs the

**Figure 2.** New theoretical model of the end replication problem.

formation of DNA daughter molecules that have one "sharp" end with a 3' overhang and the other "blunt" end. Taking into account that both chromosome ends have G-overhangs the "blunt" end on the leading telomere is further exposed to treatment by nucleases which cut its C-strand and thereby create the new 3' overhang, the length of which, will determine the rate of telomere shortening due to the end replication problem in the next replication cycle. The processing of the 5'-end of a parent strand of DNA can occur with the participation of such factors as the MRN protein complex (MRE11-RAD50-NBS1) as well as EXO1 and Apollo nucleases (Dewar & Lydall, 2010; Larrivee et al., 2004; Lenain et al., 2006; Maringele & Lydall, 2002; Tran et al., 2004; van Overbeek & de Lange, 2006; Wu et al., 2010; Zubko et al., 2004). Given this, the likely leading role is assigned to the Apollo nuclease, as RNA interference mediated repression of the gene encoding Apollo nuclease, leads to the loss of 3' overhangs, subsequent cell cycle arrest and programmed death (van Overbeek & de Lange, 2006). The dominant role of such overhangs on the ends of chromosomes, as has long been established in the course of experiments, consists of the formation of special structures called telomeric loops (t-loops, see below), which protect DNA ends from being recognised as double-stranded breaks by the repair system proteins and other enzymatic influences (Grach, 2009; Griffith et al., 1999; Stansel et al., 2001). This is why it is so important that the leading telomere form a new 3' overhang, even taking into account some DNA parent strand shortening. It should also be noted that if we assume - hypothetically - the possibility that chromosomes could function normally if a 3' overhang was present on only one DNA end, at the same time if in this case the parent 5'-end was never cut by nucleases, it would lead to the impossibility of telomere shortening in a considerable number of primary cells and their immortalisation without telomerase. Fortunately, it is not possible because then there would be a high probability of such cells transforming. As is shown in Fig. 2, telomere shortening as a result of one round of DNA replication happens due to the impossibility of recreating a 3' overhang on a leading daughter telomere and a subsequent undercut of a 5'-end by nucleases in creating such a new overhang. If, after these events, a telomerase does not become active and does not elongate this new 3' overhang, thereby providing the possibility of recreating a previous overhang, then the shortened 3'-ends, having passed to the next round of replication will lead to a situation such that now, on their template, even shorter 5'-ends will be created as a result of incomplete lagging strand synthesis. Therefore, it is possible to say that in such cases telomere shortening can be performed by means of incomplete lagging strand synthesis, which, it should be especially emphasised, is possible only after the previous loss of the 3' overhang or, as some have noted, after incomplete DNA leading strand synthesis, and so cannot proceed on the initial chromosomes. It is interesting to notice here that in certain scientific works, which describe the new model of the end replication problem, the 3' overhang loss is designated as a problem of incomplete leading strand synthesis. This description - as it appears to us - does not fully correspond to the actuality because, in this case, a template is replicated to the very 5'-end, unlike the variant proposed by the first mechanism of the old model of the end replication problem, where its incomplete replication with C-overhang formation can be carried out. Therefore, with regard to a single-stranded template, the leading strand synthesis here is performed completely, however the truth is that in relation to the parent doublestranded DNA with a 3' overhang on both sides it does not do so completely. Thus, irrespective of these descriptions, but in the case of absence of telomerase activity, it seems to be possible that the following chain of events under the new theoretical model of the end replication problem lead to a daughter telomeres' shortening: a 3' overhang loss on the DNA daughter strand, the processing of the 5'-end of the DNA parent strand within one round of replication, and then the incomplete replication of a shortened 3'-end of the previous DNA daughter strand in the next one.

In order to understand in more detail how incomplete DNA lagging strand synthesis and 3' overhang loss on the leading telomere is accomplished under the new theoretical model of the end replication problem, let us examine the structure of replication forks on both chromosome ends, as presented by Fig. 3.

formation of DNA daughter molecules that have one "sharp" end with a 3' overhang and the other "blunt" end. Taking into account that both chromosome ends have G-overhangs the "blunt" end on the leading telomere is further exposed to treatment by nucleases which cut its C-strand and thereby create the new 3' overhang, the length of which, will determine the rate of telomere shortening due to the end replication problem in the next replication cycle. The processing of the 5'-end of a parent strand of DNA can occur with the participation of such factors as the MRN protein complex (MRE11-RAD50-NBS1) as well as EXO1 and Apollo nucleases (Dewar & Lydall, 2010; Larrivee et al., 2004; Lenain et al., 2006; Maringele & Lydall, 2002; Tran et al., 2004; van Overbeek & de Lange, 2006; Wu et al., 2010; Zubko et al., 2004). Given this, the likely leading role is assigned to the Apollo nuclease, as RNA interference

**Figure 2.** New theoretical model of the end replication problem.

456 The Mechanisms of DNA Replication

Fig. 3 shows two replication bubbles on eukaryotic chromosome ends, each of which consists of a pair of replication forks moving in opposite directions. As is known, in most cases the initiation of replication is accomplished from a non-telomeric origin (Gilson & Geli, 2007). Later on, one fork of the replication bubble moves towards a centromere and another one towards a telomeric end. Here it is seen that helicase unwinds a double-stranded DNA molecule up to the very ends. It allows for DNA polymerases to finish leading strand synthesis completely, to the very end of a template. The double-stranded ends on the leading telomeres are formed upon that. As is clear from the figure, in such cases, the previous 3' overhang, which earlier was on the parent DNA ends, cannot be reproduced, in principle, in daughter molecules during leading strand formation as there is nothing for it to be synthesised on and, consequently, it is lost, resulting in telomere shortening. While the leading strand concerning the parent strand is synthesised completely, the lagging strand synthesis cannot be completed up to the end of the template. Earlier, it was thought that the removal of the end RNA primer is responsible for it. However, today many researchers are inclined to consider that this is not the only reason and it is also probably significantly complemented with the impossibility of creating the last Okazaki fragment. This situation is also well represented by Fig. 3. As is known, first of all the leading strand is synthesised in the motion of the unwinding of the parent DNA, and later on, after the DNA polymerase has synthesised a certain leading strand extension, it moves to a lagging strand and elongates it, thereby catching up with the first one. When such synthesis of both strands reaches the last point of unwinding - which can correspond to the 5'-end of the parent DNA - there remains a long stretch of single-stranded DNA in the form of a 3' overhang beyond its limits. Upon this, there is no more space for synthesising the leading strand in order that later on a DNA polymerase can move and fill such an overhang with a lagging strand. In this connection, the Okazaki fragment on the 3' overhang is not created and it remains nonreplicated, and after the last primer removal its length increases a little more. However, as an overhang - which occurred prior to replication - is created anyway, the telomere's shortening does not happen in this case.

**Figure 3.** Telomeric replication forks.

Thereby, almost all conceptions of the new theoretical model of the end replication problem have been proved to be true in the course of the studies. It has been proved experimentally that 3' overhang loss in particular leads to telomere shortening. Besides it has also been confirmed that a new overhang is formed due to leading telomere processing by nucleases. At the same time, the assumptions as to the point at which a telomerase itself directly restores a previous overhang were not confirmed. For all this, according to new views of the end replication problem and which have been confirmed by a number of studies, from now on it should be construed not as the lagging strand synthesis problem but rather as the DNA leading strand synthesis problem.

#### **2.3. The conclusion of this section**

Fig. 3 shows two replication bubbles on eukaryotic chromosome ends, each of which consists of a pair of replication forks moving in opposite directions. As is known, in most cases the initiation of replication is accomplished from a non-telomeric origin (Gilson & Geli, 2007). Later on, one fork of the replication bubble moves towards a centromere and another one towards a telomeric end. Here it is seen that helicase unwinds a double-stranded DNA molecule up to the very ends. It allows for DNA polymerases to finish leading strand synthesis completely, to the very end of a template. The double-stranded ends on the leading telomeres are formed upon that. As is clear from the figure, in such cases, the previous 3' overhang, which earlier was on the parent DNA ends, cannot be reproduced, in principle, in daughter molecules during leading strand formation as there is nothing for it to be synthesised on and, consequently, it is lost, resulting in telomere shortening. While the leading strand concerning the parent strand is synthesised completely, the lagging strand synthesis cannot be completed up to the end of the template. Earlier, it was thought that the removal of the end RNA primer is responsible for it. However, today many researchers are inclined to consider that this is not the only reason and it is also probably significantly complemented with the impossibility of creating the last Okazaki fragment. This situation is also well represented by Fig. 3. As is known, first of all the leading strand is synthesised in the motion of the unwinding of the parent DNA, and later on, after the DNA polymerase has synthesised a certain leading strand extension, it moves to a lagging strand and elongates it, thereby catching up with the first one. When such synthesis of both strands reaches the last point of unwinding - which can correspond to the 5'-end of the parent DNA - there remains a long stretch of single-stranded DNA in the form of a 3' overhang beyond its limits. Upon this, there is no more space for synthesising the leading strand in order that later on a DNA polymerase can move and fill such an overhang with a lagging strand. In this connection, the Okazaki fragment on the 3' overhang is not created and it remains nonreplicated, and after the last primer removal its length increases a little more. However, as an overhang - which occurred prior to replication - is created anyway, the telomere's shortening

Thereby, almost all conceptions of the new theoretical model of the end replication problem have been proved to be true in the course of the studies. It has been proved experimentally

does not happen in this case.

458 The Mechanisms of DNA Replication

**Figure 3.** Telomeric replication forks.

Thus, summarising all of the aforesaid, the views of the end replication problem as the cause of telomere shortening have changed over a period of several decades. Initially, when everyone considered that the structure of chromosome ends was double-stranded, it was supposed that telomeres were shortened mainly due to incomplete DNA lagging strand synthesis, which leads to the formation of 3' overhangs in support of which there is much experimental data. Furthermore, some studies have demonstrated the possibility of the accomplishment of incomplete leading strand synthesis up to the very end of a template with 5' overhangs forming, but the truth is that their number is small. Such overhangs further should be undercut, which would lead to the single-step shortening of chromosomes from both ends. As most of these works nevertheless provide evidence in favour of the idea that the replication of 5'-ends was carried out completely, it was later considered that telomeres were shortened only due to the problem of incomplete lagging strand synthesis. Here, it would seem that if the telomere shortening mechanism acts only from one end of a chromosome then the its other end would never decrease in length. Actually, this is not precisely true. The matter is that, if we were to monitor two strands of any initially parent DNA then one of them - after a certain number of divisions and in case of the absence of telomerase - will be really shortened from one end and the other one would be from the opposite end. If we continuously monitor some formed daughter strands, then in the subsequent generations of the cells there will also appear chromosomes which are shortened at their own two ends. After the establishment of the important circumstance that the structure of telomere ends is not double-stranded but 3' overhanging single-stranded, the problem of incomplete lagging strand synthesis already actually ceased to be treated as being the problem, since it no longer led to telomere shortening now, and only restored a previous configuration of their ends, which is important for normal chromosome functioning. At the same time, the existence of 3' overhangs on the chromosomes' ends creates a significant problem for leading strand synthesis. It is caused by the fact that in the course of replication, the 3' overhang which is present in the parent DNA on two ends cannot be renewed in the daughter DNAs during leading strand formation because of the absence of a template for its synthesis; in this connection it will be absent at one end in one daughter molecule and on another end of another one. Such a 3' overhang loss, the further processing of the 5'-ends of leading telomeres resulting in the formation of new G-overhangs and also the subsequent incomplete lagging strand synthesis in the next generation on a template of an already shortened 3'-end, actually lead to telomere shortening. In this connec‐ tion the end replication problem is inverted from the lagging strand to the leading strand. However, this does not mean that leading strand is necessarily synthesised incompletely up to the very end of a template, and it can be reproduced fully in this case. Thus, all of the observations described above have shaped our current thinking about telomere shortening during cell division.

### **3. The telomere repair problem**

#### **3.1. Early ideas of the telomere repair problem — The incomplete repair of double-stranded DNA ends**

The problem of the incomplete repair of the very ends of DNA was also described, first, by A. M. Olovnikov as early as 1995. As with the old model of the end replication problem it was based on the idea that telomere ends have a double-stranded structure. The two suggested mechanisms of incomplete DNA ends' repair that are actually similar to the mechanisms of incomplete replication described in the previous section were distinguished. The essence of the first one concluded that if a single-stranded break (SSB) or "nick" occurred close to a 3' end of a DNA strand at a distance of just several nucleotides, then this damage could not in principle be repaired. It was presumably connected with the following situation. The short end oligonucleotide created by the nick could not remain hybridized to the rest of the DNA molecule resulting in the formation of a gap with a protruding 5'-end. Later on, a repair DNA polymerase should attach to a DNA molecule and, while moving along the undamaged C-rich strand, synthesizes the lost 3'-end region on its template. However, it could not be performed in this case as DNA polymerase again, as well as with respect to the replication of the very 5' end, not able to bring its catalytic site to the last nucleotides of a template in order to reproduce them on a complementary strand. Therefore, the DNA molecule remains shortened at the 3' end afterwards. The second suggested mechanism provided for the impossibility of damage repair if a nick happened near the 5'-end of the DNA strand. A gap formed after the separation of a DNA fragment that was too short to remain hybridized to the template, could not be filled in for another reason in this case. The chain which was subject to repair and shortened due to the single-stranded break has no 3'-end or primer to which a DNA polymerase can add nucleotides in the course of repair synthesis and, therefore, should also remain non-elongated. On this basis, in both cases of the incomplete repair of DNA ends, single-stranded overhangs are formed which should be cut further by nucleases that would subsequently lead to telomere shortening (Olovnikov, 1995a; Olovnikov, 1995b; Olovnikov, 1995c).

#### **3.2. The end repair problem — The incomplete repair of DNA ends and direct damagemediated telomere shortening**

The discovery that telomere ends had a single-stranded 3' overhanging structure, besides changing views on the end replication problem also considerably changed conceptions of the repair of chromosome ends. Before considering in detail exactly what these changes are characterised by, it is necessary to clearly define what should be understood by an incomplete DNA ends' repair. It is suggested by A. M. Olovnikov that it can proceed in two cases. In the first case, damage resulting in the breaking off of a single-stranded DNA fragment happens near the very end of the 3'-end of a double helix, and further DNA polymerase is not able to completely synthesise the lost region insofar as by moving along the opposite undamaged strand it cannot bring its catalytic site to the last nucleotides of the template. The second case assumes that damage with the separation of a DNA fragment happens near the 5'-end of a double-stranded molecule, and as a result DNA polymerase cannot restore the lost part again, since there is no available 3'-end as a primer to elongate. It is known that to begin polynucleo‐ tide synthesis at primer absence, as already noted, it is not able. Moreover there appears that the gap is so short that primase cannot even create an RNA primer. It is thus meant that in the first case a DNA polymerase binds to a DNA molecule and synthesizes several nucleotides, but the truth is that the last bases, which should lie opposite the very edge of the template, do not form (i.e. the damage repair process starts but does not come to its completion), and that in the second case the DNA polymerase, due to a RNA primer absence, cannot attach to a DNA double-stranded molecule and even to begin damage repair process. Based on these differen‐ ces, specifically as to whether the repair process can start but does not come to its end or whether it does not start at all, we propose to look at the problem of incomplete DNA ends' repair and related to it telomere shortening somewhat in a new way. In particular, it is proposed that, under the problem of incomplete repair to consider that, the repair of damage at the end of double-stranded DNA starts but cannot finish completely because of the inability of most repair system proteins to correctly function at the very edge of a template that leads to chromosome ends shortening. It is necessary to emphasise that when noting that repair cannot be finished it is meant not only that DNA polymerase is unable to copy a template completely up to the very end in the course of repair DNA synthesis, but also that other enzymes can begin and even accomplish some stage of the repair process, however that later, due to certain reasons, repair cannot continue and so it finishes prematurely. As such, in the first case described, it is possible to say that what is actually accomplished is the incomplete repair of DNA ends. In addition, it is also proposed that if the repair of damage at a DNA end cannot start at all, again owing to the inability of repair proteins to work correctly at the edge of a template - and it will lead to chromosome telomere regions shortening, then this situation further should be designated as direct damage-mediated telomere shortening (DDMTS). The second described case can be related to this. Thus, we define two possible variants by means of which telomere shortening can be performed in a case where damage occurs at the very ends of a double-stranded DNA molecule, namely incomplete DNA ends' repair and direct damage-mediated telomere shortening.

to the very end of a template, and it can be reproduced fully in this case. Thus, all of the observations described above have shaped our current thinking about telomere shortening

**3.1. Early ideas of the telomere repair problem — The incomplete repair of double-stranded**

The problem of the incomplete repair of the very ends of DNA was also described, first, by A. M. Olovnikov as early as 1995. As with the old model of the end replication problem it was based on the idea that telomere ends have a double-stranded structure. The two suggested mechanisms of incomplete DNA ends' repair that are actually similar to the mechanisms of incomplete replication described in the previous section were distinguished. The essence of the first one concluded that if a single-stranded break (SSB) or "nick" occurred close to a 3' end of a DNA strand at a distance of just several nucleotides, then this damage could not in principle be repaired. It was presumably connected with the following situation. The short end oligonucleotide created by the nick could not remain hybridized to the rest of the DNA molecule resulting in the formation of a gap with a protruding 5'-end. Later on, a repair DNA polymerase should attach to a DNA molecule and, while moving along the undamaged C-rich strand, synthesizes the lost 3'-end region on its template. However, it could not be performed in this case as DNA polymerase again, as well as with respect to the replication of the very 5' end, not able to bring its catalytic site to the last nucleotides of a template in order to reproduce them on a complementary strand. Therefore, the DNA molecule remains shortened at the 3' end afterwards. The second suggested mechanism provided for the impossibility of damage repair if a nick happened near the 5'-end of the DNA strand. A gap formed after the separation of a DNA fragment that was too short to remain hybridized to the template, could not be filled in for another reason in this case. The chain which was subject to repair and shortened due to the single-stranded break has no 3'-end or primer to which a DNA polymerase can add nucleotides in the course of repair synthesis and, therefore, should also remain non-elongated. On this basis, in both cases of the incomplete repair of DNA ends, single-stranded overhangs are formed which should be cut further by nucleases that would subsequently lead to telomere

shortening (Olovnikov, 1995a; Olovnikov, 1995b; Olovnikov, 1995c).

**mediated telomere shortening**

**3.2. The end repair problem — The incomplete repair of DNA ends and direct damage-**

The discovery that telomere ends had a single-stranded 3' overhanging structure, besides changing views on the end replication problem also considerably changed conceptions of the repair of chromosome ends. Before considering in detail exactly what these changes are characterised by, it is necessary to clearly define what should be understood by an incomplete DNA ends' repair. It is suggested by A. M. Olovnikov that it can proceed in two cases. In the first case, damage resulting in the breaking off of a single-stranded DNA fragment happens

during cell division.

460 The Mechanisms of DNA Replication

**DNA ends**

**3. The telomere repair problem**

Based on these new conceptions, let us consider once again Olovnikov's theoretical model of incomplete DNA ends' repair. As was mentioned, since it is described in the first variant that a DNA polymerase attaches to a DNA molecule, reproduces several nucleotides but subse‐ quently cannot finish repair synthesis to the very end of a template, then in this case there occurs incomplete DNA repair. In the second variant, the enzyme cannot even attach itself to a template to begin repair - that was designated as DDMTS. It is necessary to note here that if, in the case of the first variant, the gap is very short then the DNA polymerase - even if it attaches itself to a template - will also not be able to begin repair because it will place its anchor region directly onto the very end of an undamaged DNA strand and, as a result, it will be immediately separated from the DNA molecule. This situation can already be regarded as DDMTS. At the same time, if with the second variant the gap will be long enough for an RNA primer to be formed, and then it is possible that there will be two variants, in both cases of which there will now occur the incomplete repair of the DNA ends. If the gap will be long enough to fit just an RNA primer, then in this case it might be supposed that when a DNA polymerase attaches itself, it will not be able to synthesise nucleotides as well, but as far as primase synthesizes the primer, then it is possible to consider that repair has started and that one of its stages has finished, but also that another one is not able to begin. In the future, such a primer is removed by RNase and a gap of the same length as it was before the repair arises. In the case where the length of a gap is such that in spite of the RNA primer several nucleotides are able to fit there, then the DNA polymerase synthesises them. However, after primer removal there will be a gap anyway but which the truth is that will be smaller than before the repair. As a result, such situation should also be viewed as incomplete repair. Ultimately, it is important to note that at replicative and cell senescence stages it is known that repair systems act poorly. Therefore, if a long enough gap appears at one of the DNA ends in senescent cells, then it will probably not be even partially repaired, and as a result DDMTS will take place. Thus, if such gaps are repaired in young cells, even if incompletely, then in old ones they will be not be repaired. It is also necessary to emphasise that at one time is apparently possible to separate only terminal single-stranded DNA fragment that was less than nine nucleotides in length, since it is widely known that RNA primers 9-12 nucleotides in size are strongly hybridized to a template. For that matter, when it was mentioned that a gap can arise is longer than primer itself or else the same, it can proceed only according to several steps, i.e. a successive separation of several fragments 8 nucleotides in size or less. Thus, if a break occurs, e.g. at a distance of 9 or more nucleotides, then such a terminal oligonucleotide will not only be able to hang on a template but will also be reunited with the remaining proximal part by DNA ligase. In summary, it is also necessary to add that since it was experimentally discovered that 5'-end copying by a DNA polymerase in the course of replication is, in most cases, accomplished completely, then in such a case only the second variant of incomplete repair based on RNA primer removal so described could feasibly be carried out.

#### **3.3. Modern views of the end repair problem**

Now let us consider what exactly are characterized by the changing of conceptions of repair at the ends of chromosomes, if the telomeres have single-stranded 3' overhanging structure. They are characterised by the following circumstances. First, given such telomere ends organisation, the problem of incomplete repair can arise, as it seems to be possible, only if a single-stranded break occurs at a distance of up to approximately eight nucleotides towards the centromere from a place where the 3' overhang begins and the 5'-end of complementary strand is situated (Fig. 4). In such a case, if a DNA polymerase even manages to copy a template up to the very 5'-end in the course of the repair synthesis of the lost single-stranded DNA fragment, the previous 3' overhang would still not be able to renew, and so it can be seen that in such a situation repair has begun but cannot be finished, insofar as the damage could not be fully repaired. If we take into account that a DNA polymerase might not be able to copy the last nucleotides of a template, then in such a case if a break with a subsequent separation of a DNA fragment occurs at a very short distance (e.g. of one nucleotide) from the above mentioned place, then repair will not begin and it will be designated as DDMTS. Secondly, if a break occurs somewhere at the 3' overhang or near its base (i.e. at a region where the opposite C-rich strand ends), then the distal part of the overhang or its entirely will separate from the DNA molecule and will be lost, as a result of which DDMTS will be observed - as far as repair in that case cannot even begin in principle due to single-stranded DNA fragment loss and the absence of a template for synthesizing the new one (Figure 5). With both variants, the new overhang will form in the future through 5'-end processing by the nucleases. Thirdly, an incomplete repair problem of the 5'-end, which should arise due to RNA primer removal on telomeres with "blunt" ends, is no longer a problem, and hence the reason for telomere shortening in instances with "sharp" ends, because as is the case with lagging strand synthesis at DNA replication, its cutting out leads only to the formation of the previous 3' overhanging configuration of telomere ends (Figure 6). Thus, single-stranded end breaks, at the 3' over‐ hanging telomere structure, can lead to incomplete telomere repair and further telomere shortening only if they will occur on a G-rich strand at a distance of several nucleotides in front of a place where a complementary C-strand ends. If the breaks affect the 3' overhang itself, then this will lead to DDMTS. Finally, the breaks of a C-rich strand occurring near the very 5' ends will repair completely.

same time, if with the second variant the gap will be long enough for an RNA primer to be formed, and then it is possible that there will be two variants, in both cases of which there will now occur the incomplete repair of the DNA ends. If the gap will be long enough to fit just an RNA primer, then in this case it might be supposed that when a DNA polymerase attaches itself, it will not be able to synthesise nucleotides as well, but as far as primase synthesizes the primer, then it is possible to consider that repair has started and that one of its stages has finished, but also that another one is not able to begin. In the future, such a primer is removed by RNase and a gap of the same length as it was before the repair arises. In the case where the length of a gap is such that in spite of the RNA primer several nucleotides are able to fit there, then the DNA polymerase synthesises them. However, after primer removal there will be a gap anyway but which the truth is that will be smaller than before the repair. As a result, such situation should also be viewed as incomplete repair. Ultimately, it is important to note that at replicative and cell senescence stages it is known that repair systems act poorly. Therefore, if a long enough gap appears at one of the DNA ends in senescent cells, then it will probably not be even partially repaired, and as a result DDMTS will take place. Thus, if such gaps are repaired in young cells, even if incompletely, then in old ones they will be not be repaired. It is also necessary to emphasise that at one time is apparently possible to separate only terminal single-stranded DNA fragment that was less than nine nucleotides in length, since it is widely known that RNA primers 9-12 nucleotides in size are strongly hybridized to a template. For that matter, when it was mentioned that a gap can arise is longer than primer itself or else the same, it can proceed only according to several steps, i.e. a successive separation of several fragments 8 nucleotides in size or less. Thus, if a break occurs, e.g. at a distance of 9 or more nucleotides, then such a terminal oligonucleotide will not only be able to hang on a template but will also be reunited with the remaining proximal part by DNA ligase. In summary, it is also necessary to add that since it was experimentally discovered that 5'-end copying by a DNA polymerase in the course of replication is, in most cases, accomplished completely, then in such a case only the second variant of incomplete repair based on RNA primer removal so

Now let us consider what exactly are characterized by the changing of conceptions of repair at the ends of chromosomes, if the telomeres have single-stranded 3' overhanging structure. They are characterised by the following circumstances. First, given such telomere ends organisation, the problem of incomplete repair can arise, as it seems to be possible, only if a single-stranded break occurs at a distance of up to approximately eight nucleotides towards the centromere from a place where the 3' overhang begins and the 5'-end of complementary strand is situated (Fig. 4). In such a case, if a DNA polymerase even manages to copy a template up to the very 5'-end in the course of the repair synthesis of the lost single-stranded DNA fragment, the previous 3' overhang would still not be able to renew, and so it can be seen that in such a situation repair has begun but cannot be finished, insofar as the damage could not be fully repaired. If we take into account that a DNA polymerase might not be able to copy the last nucleotides of a template, then in such a case if a break with a subsequent separation of a DNA fragment occurs at a very short distance (e.g. of one nucleotide) from the above

described could feasibly be carried out.

462 The Mechanisms of DNA Replication

**3.3. Modern views of the end repair problem**

As is known, apart from single-stranded breaks, there are also such basic types of DNA damage as various nucleotide modifications, double-stranded breaks and cross-links (Sancar et al., 2004). Various nucleotide modifications (of a single one, a pair or else several) arising at DNA ends in most cases cannot lead to single-stranded breaks of the molecule in themselves. Further, they are exposed to various repair pathways, such as a direct repair (DR), a base excision repair (BER) and a nucleotide excision repair (NER) (Sancar et al., 2004). Since a direct repair is accomplished without any breakage of the phosphodiester backbone it cannot, in principle, lead to telomere shortening and, therefore, in our case, is of no particular interest. This type of reactions includes the photoreactivation of ultraviolet-induced pyrimidine dimers by a DNA photolyase enzyme, the removal of the O6 -methyl group from O6 -methylguanine (O6 MeGua) in DNA by the DNA methyltransferase enzyme, and the repair of apurinic/ apyrimidinic sites through the direct insertion of bases by the insertase enzyme (Sancar et al., 2004). In addition, the repair of single-stranded DNA breaks by DNA ligase enzymes also belongs to this type but only if they do not arise at the very ends of DNA and do not lead to gap formation. Base excision repair consists of the cutting out of damaged nucleotide from a DNA strand by means of several reactions with the participation of DNA glycosylases, APendonuclease and phosphodiesterase, resulting in the formation of a very short gap (Fromme & Verdine, 2005; Krokan et al., 1997; Seeberg et al., 1995). This gap should be filled further by a DNA polymerase on a template of an undamaged complementary strand, after which the free ends are sealed by a ligase. If such a form of repair is carried out somewhere in the middle of the DNA molecule or near its 5'-end, then no problem will arise. However, if it proceeds at a distance of, say, 9 nucleotides from the place where the 3' overhang begins, i.e. the 9th nucleotide will be removed at that, then the end DNA fragment 8 nucleotides long up to the 5'-end of an opposite strand, together with the 3' overhang, would be lost. It will lead to gap formation, which can be filled further to form a "blunt" DNA end, but upon this, as well as in the case of a single-stranded break forming at a distance of up to 8 nucleotides and the subsequent formation of the same gap as discussed earlier, the previous 3' overhang it will not be able to restore itself and the telomere will shorten. Thus, base excision repair imposed on the end regions of a G-strand may be lead to an incomplete DNA repair. It is also interesting to discuss the situation where such a damaged nucleotide arises within the 3' overhang itself. BER system enzymes are apparently incapable of acting on a single-stranded DNA. As a result, such damage will not be repaired, and where further the base modification can similarly lead to DNA strand breaking and 3' overhang distal part loss, then this situation should be considered as DDMTS. However, if repair enzymes all the same could cut out a damaged nucleotide, which again will lead to the separation and loss of the 3' overhang terminal fragment, and so there will be an incomplete repair in this case. As for the nucleotide excision repair, it is very similar to BER but is accomplished by other enzymes, and in this case not only one damaged nucleotide is removed but up to thirty (de Laat et al., 1999; Reardon & Sancar, 2005; Sancar et al., 2004). With NER as well as with BER, if the gap is formed on the G-strand of telomeric DNA, in such a way that 8 or less nucleotides remain up to the end of a doublestranded DNA structure in the G-strand, then again a short end fragment of a G-strand together with the 3' overhang will be separated and lost and as a result an incomplete repair and related to it telomere shortening will be observed subsequently again. In order not to repeat this, it may be said that all other situations involving NER at the end of the telomere, including whether several nucleotides on the 3' overhang will be damaged, are similar to those that have been reviewed in relation to BER.

Double-stranded breaks (DSBs) of DNA can be repaired by three mechanisms: non-homolo‐ gous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologydirected repair (HDR) (Chu, 1997; Liang et al., 2008; Lieber et al., 2003). The first mechanism consists of the direct joining of broken ends by a specialised enzyme DNA ligase IV with the participation of the protein Ku and DNA-PK, which is carried out within G0/G1 and the early S phases of the cell cycle (Lieber et al., 2003). The second mechanism does not depend on these proteins and also differs from NHEJ in that this mechanism of DSBs repair uses 5-25 base pair microhomologous sequences to align the broken strands before joining, and it is carried out within the S phase of the cell cycle (Liang et al., 2008). The third mechanism is based on homologous recombination of a damaged chromosome with a sister chromatid or homologous undamaged chromosome, and therefore the damaged chromosome is repaired on their template that is carried out within the late S and G2 phases of the cell cycle (Chu, 1997). Nevertheless, NHEJ and HDR seem to be the main mechanisms for DSB's repair. If DSB occurs near the very end of a chromosome at a distance of only several nucleotides from the place where the 3' overhang begins, then such damage will probably not be repaired since the distal double-stranded fragment of DNA would be too short for the repair enzymes to bind to it, and, in the case of NHEJ and MMEJ, connect it to the rest of a molecule. Additionally, HDR also would be ineffective in repairing such damage since the homology tract would be again too short to effectively engage the enzymes that catalyze homologous recombination. As a result, the repair of such damage will not begin and there will be observed DDMTS. In addition, it is also necessary to note that if telomere damage occurs at a great enough distance from the very end of a DNA, but still within the telomere region organized by the telosome, it is unlikely that it could be repaired by homologous recombination. This is caused by the ability of telomeric proteins to block recombination events for the preventing of the elongation of chromosome telomere regions through ALT (Grach, 2011b).

**Figure 4.** Incomplete double-stranded DNA end repair.

subsequent formation of the same gap as discussed earlier, the previous 3' overhang it will not be able to restore itself and the telomere will shorten. Thus, base excision repair imposed on the end regions of a G-strand may be lead to an incomplete DNA repair. It is also interesting to discuss the situation where such a damaged nucleotide arises within the 3' overhang itself. BER system enzymes are apparently incapable of acting on a single-stranded DNA. As a result, such damage will not be repaired, and where further the base modification can similarly lead to DNA strand breaking and 3' overhang distal part loss, then this situation should be considered as DDMTS. However, if repair enzymes all the same could cut out a damaged nucleotide, which again will lead to the separation and loss of the 3' overhang terminal fragment, and so there will be an incomplete repair in this case. As for the nucleotide excision repair, it is very similar to BER but is accomplished by other enzymes, and in this case not only one damaged nucleotide is removed but up to thirty (de Laat et al., 1999; Reardon & Sancar, 2005; Sancar et al., 2004). With NER as well as with BER, if the gap is formed on the G-strand of telomeric DNA, in such a way that 8 or less nucleotides remain up to the end of a doublestranded DNA structure in the G-strand, then again a short end fragment of a G-strand together with the 3' overhang will be separated and lost and as a result an incomplete repair and related to it telomere shortening will be observed subsequently again. In order not to repeat this, it may be said that all other situations involving NER at the end of the telomere, including whether several nucleotides on the 3' overhang will be damaged, are similar to those that have

Double-stranded breaks (DSBs) of DNA can be repaired by three mechanisms: non-homolo‐ gous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and homologydirected repair (HDR) (Chu, 1997; Liang et al., 2008; Lieber et al., 2003). The first mechanism consists of the direct joining of broken ends by a specialised enzyme DNA ligase IV with the participation of the protein Ku and DNA-PK, which is carried out within G0/G1 and the early S phases of the cell cycle (Lieber et al., 2003). The second mechanism does not depend on these proteins and also differs from NHEJ in that this mechanism of DSBs repair uses 5-25 base pair microhomologous sequences to align the broken strands before joining, and it is carried out within the S phase of the cell cycle (Liang et al., 2008). The third mechanism is based on homologous recombination of a damaged chromosome with a sister chromatid or homologous undamaged chromosome, and therefore the damaged chromosome is repaired on their template that is carried out within the late S and G2 phases of the cell cycle (Chu, 1997). Nevertheless, NHEJ and HDR seem to be the main mechanisms for DSB's repair. If DSB occurs near the very end of a chromosome at a distance of only several nucleotides from the place where the 3' overhang begins, then such damage will probably not be repaired since the distal double-stranded fragment of DNA would be too short for the repair enzymes to bind to it, and, in the case of NHEJ and MMEJ, connect it to the rest of a molecule. Additionally, HDR also would be ineffective in repairing such damage since the homology tract would be again too short to effectively engage the enzymes that catalyze homologous recombination. As a result, the repair of such damage will not begin and there will be observed DDMTS. In addition, it is also necessary to note that if telomere damage occurs at a great enough distance from the very end of a DNA, but still within the telomere region organized by the telosome, it is unlikely that it could be repaired by homologous recombination. This is caused by the ability of

been reviewed in relation to BER.

464 The Mechanisms of DNA Replication

**Figure 5.** Direct damage-mediated telomere shortening.

**Figure 6.** Complete double-stranded DNA end repair.

DNA cross-links represent connections between the nucleotide bases by covalent bonds (normally they are bound by hydrogen bonds) which can be of two types: intrastrand and interstrand (Dronkert & Kanaar, 2001). The latter ones represent a very serious problem for DNA replication machinery insofar as before a DNA polymerase replicates a parent DNA, it is at first unwound by helicase through the breaking of the hydrogen bonds between two strands. Therefore, if two complementary nucleotides are covalently linked, helicase will not be able to separate them and this will lead to the stalling of the replication fork and potential DSB formation. Two mechanisms for the repair of such damage are known which can be carried out during different phases of a cell cycle. Both of these mechanisms begin identically by forming cuts on both sides of the cross-link on one DNA strand by NER system proteins, after which there occurs a twisting of the oligonucleotide carrying the damage and gap formation. Later, such a gap will be filled; one mechanism for this is by TLS (Translesion synthesis), which makes use of DNA polymerases that are able to replicate DNA despite template damage arising before them. This is followed by another round of NER during which the second DNA strand is cleaved, and adduct removal is carried out. The second gap that is formed can than be filled by a conventional DNA polymerase on a complementary strand template and the ends are connected by DNA ligase. In the case of the other mechanism, the filling of the first gap is carried out during the course of recombination on a homologous chromosome template within a G2 phase of a cell cycle, upon completion of which NER proceeds again. The subsequent stages of repair are the same as those for the first mechanism. If cross-links occur somewhere on the non-telomeric chromosome regions, then these two repair mechanisms can act without any problems arising. If, however, they arise too close to an end of a double-stranded DNA, in such a way that there are 8 or less nucleotides left towards the place where the 3' overhang begins on a G-strand during gap formation, then NER system activity will lead subsequently to such overhang loss and incomplete repair with telomere shortening. It could probably be restored to a previous state only in the case of the recombi‐ nation-mediated DNA synthesis, which on the very ends of chromosomes, is very tightly blocked, as has already been mentioned. Every other event here is similar to those that have been described in relation to BER and NER.

Thus, we have reviewed the possible cases of the occurrence of incomplete repair and of DDMTS for various types of the damage of chromosome ends with 3' overhangs, which in their turn, should lead to the telomere shortening. At the same, not all such possible cases (as well as variations of damage and mechanisms for their repair) have been analysed but only those that seem to be the most important. Also, it should be emphasized that every possible case of incomplete repair and DDMTS, which has been assumed, can arise on uncapped linear telomere ends. If telomere ends are in a capped condition (i.e. in the form of a t-loop) then already other such cases will probably be observed, which also will lead to their shortening. Nevertheless, it seems for us that if telomere ends are organised into t-loops then the cases of incomplete repair and DDMTS characterised by telomere shortening will occur much less frequently than with linear telomeres. It should be noted that experimental data fully and directly confirming the appearance of incomplete repair or DDMTS for different described variants of damage could not be found. At the same time, there are many studies providing general information, demonstrating that various kinds of damage can occur on telomeres, which are repaired much less efficiently than those which are formed on the non-telomeric chromosome regions, and that they lead to telomere shortening (Passos et al., 2007).

#### **3.4. The shelterin-mediated telomere repair problem**

**Figure 6.** Complete double-stranded DNA end repair.

466 The Mechanisms of DNA Replication

DNA cross-links represent connections between the nucleotide bases by covalent bonds (normally they are bound by hydrogen bonds) which can be of two types: intrastrand and interstrand (Dronkert & Kanaar, 2001). The latter ones represent a very serious problem for DNA replication machinery insofar as before a DNA polymerase replicates a parent DNA, it is at first unwound by helicase through the breaking of the hydrogen bonds between two strands. Therefore, if two complementary nucleotides are covalently linked, helicase will not be able to separate them and this will lead to the stalling of the replication fork and potential DSB formation. Two mechanisms for the repair of such damage are known which can be carried out during different phases of a cell cycle. Both of these mechanisms begin identically by forming cuts on both sides of the cross-link on one DNA strand by NER system proteins, after which there occurs a twisting of the oligonucleotide carrying the damage and gap formation. Later, such a gap will be filled; one mechanism for this is by TLS (Translesion synthesis), which makes use of DNA polymerases that are able to replicate DNA despite template damage arising before them. This is followed by another round of NER during which the second DNA strand is cleaved, and adduct removal is carried out. The second gap that is formed can than be filled by a conventional DNA polymerase on a complementary strand template and the ends are connected by DNA ligase. In the case of the other mechanism, the

Many investigations have been performed focussing on the influence of reactive oxygen species (ROS) on the occurrence of telomere damage and associated telomere shortening. In one of the earliest studies it was found that mild hyperoxia leads to accelerated telomere shortening and inhibits the proliferation of fibroblasts which, as it was supposed at that time, could happen due to the accumulation of single-stranded DNA breaks on chromosome end regions (von Zglinicki et al., 1995). In another study, it was found that oxidative stress really leads to the accumulation of single-stranded breaks on telomeres whereupon they actually become rapidly shortened (Petersen et al., 1998). In yet another study, it was found that ultraviolet radiation combined with riboflavin induces the formation of 8-oxo-7, 8-dihydro-2' deoxyguanosine (8-oxodG) in DNA fragments with telomere sequences that further leads to the appearance of breaks in the area of the central guanine of GGG sequences. It was also shown that under the influence of hydrogen dioxide (H2O2) together with Cu (II) on these fragments DNA damage also occurred, which included the formation of 8-oxodG at the GGG sequence in the telomere sequence (5'-TTAGGG-3'), and which also led to breaks. Therefore, it was concluded that the formation of 8-oxodG in a GGG telomere sequence triplet induced by oxidative stress could play an important role in the acceleration of telomere shortening (Kawanishi & Oikawa, 2004). Along with these studies, many others showing that ROS leads to telomeric DNA damage formation and to their subsequent shortening are known (Passos & Von Zglinicki, 2006; Richter & von Zglinicki, 2007; Saretzki et al., 1999; Tchirkov & Lansdorp, 2003; Toussaint et al., 2000; von Zglinicki, 2000; von Zglinicki et al., 2000; von Zglinicki, 2002). It is also possible that besides the occurrence of single-stranded breaks on telomeres under the influence of ROS, there could also be oxidative modifications of nucleotides, which should be subject to repair by BER and NER systems, as well as double-stranded breaks (Passos et al., 2007). ROS has special importance concerning telomere damage, because unlike other mutagenic factors such as ionizing radiation, ultraviolet emanation, different chemical agents etc, ROS are constantly formed by mitochondria in a human organism during its normal metabolic activity. This is what triggered a strong interest to their study. Based on this, it may be that these other damaging agents can affect an organism and damage its telomeres in only very rare cases, while ROS continuously damages chromosomes' end regions, leading to their shortening. This situation actually should explain the fact that under normal conditions in the course of a cell's division, the telomere shortening rate considerably exceeds that which is expected only as a result of 3' overhang loss under the end replication problem (Keys et al., 2004). There are also studies which demonstrate that ROS can directly damage mitochondria themselves, and their mitochondrial DNA (mtDNA) in particular, thereby leading to their dysfunction which in turn can lead to the more intensive production of free radicals and, as a consequence, can result in even more intensive telomere damage and their shortening (Liu et al., 2002; Passos et al., 2006; Passos et al., 2007).

As was already noted, the damage occurring on telomeres is repaired less efficiently than that which originates in other genome regions (Kruk et al., 1995; Petersen et al., 1998; von Zglinicki, 2002). The reasons for such deficiencies in telomere-specific repair have not yet been com‐ pletely established. At the same time, it is supposed that a basic role in the significant lessening of damage repair efficacy on telomeres belongs to the formation at their ends of the t-loops (capped telomere condition) (Passos et al., 2007). It was mentioned earlier that t-loops protect telomere ends from the activity of repair system proteins and another enzymatic influences (Grach, 2009; Griffith et al., 1999; Stansel et al., 2001). The example of the latter, incidentally, could be the telomerase attaching to the 3' overhang and its elongation. How does such repair suppression on telomeres by t-loops proceed? In order to answer this question let us first consider in detail what these t-loops represent and what actually characterises the response to DNA damage.

A t-loop represents a structure on eukaryotic chromosome ends which is formed at the bending back and subsequent insertion of a single-stranded telomeric DNA (3' overhang) into a doublestranded one (Grach, 2009). Upon this, the G-overhang forms a heteroduplex with the complementary C-strand region in double-stranded telomeric DNA, which is accompanied by the local untwisting of the latter and which leads to the formation of the so-called displacement loop (D-loop). The latter represents a triple-stranded structure which consists of a doublestranded DNA, formed by a 3' overhang and a C-strand of the previous double-stranded DNA, and single-stranded DNA, corresponding to the G-strand region of the previous doublestranded DNA (Fig. 7).

**Figure 7.** Structural organisation of a mammalian t-loop (Grach, 2009).

fragments DNA damage also occurred, which included the formation of 8-oxodG at the GGG sequence in the telomere sequence (5'-TTAGGG-3'), and which also led to breaks. Therefore, it was concluded that the formation of 8-oxodG in a GGG telomere sequence triplet induced by oxidative stress could play an important role in the acceleration of telomere shortening (Kawanishi & Oikawa, 2004). Along with these studies, many others showing that ROS leads to telomeric DNA damage formation and to their subsequent shortening are known (Passos & Von Zglinicki, 2006; Richter & von Zglinicki, 2007; Saretzki et al., 1999; Tchirkov & Lansdorp, 2003; Toussaint et al., 2000; von Zglinicki, 2000; von Zglinicki et al., 2000; von Zglinicki, 2002). It is also possible that besides the occurrence of single-stranded breaks on telomeres under the influence of ROS, there could also be oxidative modifications of nucleotides, which should be subject to repair by BER and NER systems, as well as double-stranded breaks (Passos et al., 2007). ROS has special importance concerning telomere damage, because unlike other mutagenic factors such as ionizing radiation, ultraviolet emanation, different chemical agents etc, ROS are constantly formed by mitochondria in a human organism during its normal metabolic activity. This is what triggered a strong interest to their study. Based on this, it may be that these other damaging agents can affect an organism and damage its telomeres in only very rare cases, while ROS continuously damages chromosomes' end regions, leading to their shortening. This situation actually should explain the fact that under normal conditions in the course of a cell's division, the telomere shortening rate considerably exceeds that which is expected only as a result of 3' overhang loss under the end replication problem (Keys et al., 2004). There are also studies which demonstrate that ROS can directly damage mitochondria themselves, and their mitochondrial DNA (mtDNA) in particular, thereby leading to their dysfunction which in turn can lead to the more intensive production of free radicals and, as a consequence, can result in even more intensive telomere damage and their shortening (Liu et

As was already noted, the damage occurring on telomeres is repaired less efficiently than that which originates in other genome regions (Kruk et al., 1995; Petersen et al., 1998; von Zglinicki, 2002). The reasons for such deficiencies in telomere-specific repair have not yet been com‐ pletely established. At the same time, it is supposed that a basic role in the significant lessening of damage repair efficacy on telomeres belongs to the formation at their ends of the t-loops (capped telomere condition) (Passos et al., 2007). It was mentioned earlier that t-loops protect telomere ends from the activity of repair system proteins and another enzymatic influences (Grach, 2009; Griffith et al., 1999; Stansel et al., 2001). The example of the latter, incidentally, could be the telomerase attaching to the 3' overhang and its elongation. How does such repair suppression on telomeres by t-loops proceed? In order to answer this question let us first consider in detail what these t-loops represent and what actually characterises the response to

A t-loop represents a structure on eukaryotic chromosome ends which is formed at the bending back and subsequent insertion of a single-stranded telomeric DNA (3' overhang) into a doublestranded one (Grach, 2009). Upon this, the G-overhang forms a heteroduplex with the complementary C-strand region in double-stranded telomeric DNA, which is accompanied by the local untwisting of the latter and which leads to the formation of the so-called displacement

al., 2002; Passos et al., 2006; Passos et al., 2007).

468 The Mechanisms of DNA Replication

DNA damage.

In t-loop formation, the primary role belongs to specific telomeric proteins, which are collec‐ tively referred to as the shelterin complex or telosome. Telomeric proteins differ slightly with different groups of organisms, but along with this they carry out similar functions. In mam‐ mals, shelterin includes six basic proteins, namely TRF1, TRF2, Rap1, TIN2, Pot1 and TPP1. These in turn can be divided into three groups: 1). double-stranded telomeric DNA binding proteins (TRF1 and TRF2); 2). single-stranded telomeric DNA binding proteins (Pot1); 3). proteins necessary for higher-order nucleoprotein complex formation (Rap1, TIN1 and TPP1) (Grach, 2009). The role of TRF1 function in t-loop formation it is to promote the bending back, twisting and linking of double-stranded telomeric DNA regions (Bianchi et al., 1997; Bianchi et al., 1999; Griffith et al., 1998; Griffith et al., 1999). Subsequently, TRF2 carries out the selfintroduction of single-stranded telomeric DNA into a double-stranded one (Greider et al., 1999; Griffith et al., 1999; Stansel et al., 2001). Pot1 binds to single-stranded regions of telomeric DNA, which are represented only by a G-strand, and stabilises them (Baumann & Cech, 2001; Bunch et al., 2005; Churikov et al., 2006). Moreover, if a telomere end turns up in an uncapped condition, then Pot1 will cover the 3' overhang. If it turns up in a capped condition, then this protein will bind a single-stranded G-strand region in a D-loop structure. The Rap1 protein interacts with telomeres through binding with TRF2 (Li et al., 2000). It is suggested that the main functions of Rap1 are connected with its ability to recruit various repair system proteins, including Mre11/Rad50/Nbs1, Ku70/80 and PARP-1, to the telomeres (O'Connor et al., 2004). TIN2's role consists of both the binding of TRF1 proteins among themselves (Kim et al., 1999) and the binding of TRF1 with TRF2 (Ye et al., 2004). TPP1 is also necessary for the binding of TRF1 and TRF2 sub-complexes. It assists in the stabilising of TRF1-TIN2-TRF2 interaction. Besides this, TPP1 also directly binds Pot1 and regulates its activity (O'Connor et al., 2006). Thus, TIN2 and TPP1 play a key role in the association of different telomeric proteins in a single functional complex, which participates in the formation of t-loops and the capping of telomere ends, and this provides telomeres protection from different kinds of enzymatic action.

The DNA damage response is characterised by the following circumstances. It is possible to distinguish such basic key points as DNA damage detection from repair itself. Earlier, we considered the most important repair mechanisms, and therefore we will analyse the damage detection pathways. As is known, there are two distinct DNA damage detection pathways, which can potentially be activated by a chromosome's natural ends, namely the ATM kinase pathway and the ATR kinase pathway (de Lange, 2010). The ATM kinase pathway is triggered in response to double-stranded DNA breaks (Lee & Paull, 2007). Upon this, the process by which the ATM kinase response is accomplished is still not completely clear (Lee & Paull, 2007). It is known that the Mre11/Rad50/Nbs1 complex (sensor proteins which take part in double-stranded breaks' detection) binds to DNA ends upon this and activates the ATM kinase in a combination with Tip60 HAT (Carson et al., 2003; de Lange, 2010; Williams et al., 2010). Later on, the ATM kinase phosphorylates some key proteins involved in the damage response to DNA double-strand breaks and initiates the activation of the DNA damage checkpoint, which pauses the cell cycle and allows time for a cell to repair damage before continuing its division (Lee & Paull, 2007). Upon this, Tip60 HAT, through histone acetylation, modulates repair proteins' loading and repair of double-stranded DNA breaks (Murr et al., 2006). In that case, if the damage is not resolved, the p53 protein is then activated, which triggers an apoptosis program (Polyak et al., 1997). The ATR kinase pathway is activated in response to the singlestranded DNA, and is based on the point that the abundant single-strand DNA binding protein RPA recognises and associates with single-stranded DNA, resulting in an RPA-ssDNA complex (Cimprich & Cortez, 2008; de Lange, 2010; Nam & Cortez, 2011). Further, the ATR kinase together with the ATRIP protein recognises such a single-stranded DNA coated with RPA and attached to the DNA's damage site (Cimprich & Cortez, 2008). At the same, the ATR kinase's recruitment to the RPA-ssDNA complex strongly depends on the ATRIP protein, which itself directly attaches to RPA-ssDNA, and thus binds this complex with the ATR (Zou & Elledge, 2003). The checkpoint clamp complex containing RAD9-HUS1-RAD1 (9-1-1) proteins, which take part in checkpoint activation, cell cycle arrest, and recruitment of specific DNA polymerases and other repair proteins to damaged DNA is also independently recruited to the DNA damage site by RAD17 protein (checkpoint clamp loader) (Bermudez et al., 2003; Sohn & Cho, 2009). Besides, the ATR activator TOPB1 is recruited to the DNA damage site (Choi et al., 2009). After these events, the activation of ATR by TOPBP1 and the phosphoryla‐ tion of downstream targets in a signal transduction cascade proceeds, which eventually leads to checkpoint activation, cell cycle arrest and subsequent damage repair (Cimprich & Cortez, 2008). Later on, all of the events are similar to those which were considered concerning the ATM kinase pathway, i.e. if damage is completely repaired then the cell cycle is resumed and the cell will continue its division, and if not then there will occur a trigger of the apoptosis program and subsequent cell death.

Now that we have considered what t-loops represent in themselves and what the DNA damage response is characterised by, it is possible to answer the question – how the repair of damage on telomeres is so strongly repressed. As such, it was proposed that shelterin hides the chromosome end from the ATM kinase pathway of DNA damage detection by remodelling telomeres into a closed structure – the t-loop. In a t-loop, Mre11/Rad50/Nbs1 is unlikely to recognise the telomere end as a double-stranded DNA end, which thus prevents ATM kinase activation, with subsequent cell cycle arrest and initiation of DNA damage repair (de Lange, 2009; de Lange, 2010; Griffith et al., 1999). This situation can be implemented as after the DNA replication, as well as after the double-stranded breaks occurred at telomeres. On the other hand, ATR signalling on telomeres is blocked by the shelterin Pot1 protein. It was noted earlier that, telomeres, on their own ends, contain a single-stranded DNA. This DNA at the uncapped condition of telomere ends is represented by 3' overhangs, and at the capped condition by a single-stranded region of a G-strand as a part of a D-loop. Furthermore, such single-stranded DNA can arise after a single-stranded damage and double-stranded breaks occurred at telomeres. It is potentially capable of activating the ATR kinase; however it was suggested that Pot1 binds a single-stranded telomere DNA and excludes the RPA protein from it. Later on, in the absence of this protein, such single-stranded DNA can no longer be distinguished by the ATR-ATRIP complex as damage, which prevents ATR kinase pathway activation and all subsequent events, including repair (de Lange, 2009; de Lange, 2010; Denchi & de Lange, 2007).

al., 2006). Thus, TIN2 and TPP1 play a key role in the association of different telomeric proteins in a single functional complex, which participates in the formation of t-loops and the capping of telomere ends, and this provides telomeres protection from different kinds of enzymatic

The DNA damage response is characterised by the following circumstances. It is possible to distinguish such basic key points as DNA damage detection from repair itself. Earlier, we considered the most important repair mechanisms, and therefore we will analyse the damage detection pathways. As is known, there are two distinct DNA damage detection pathways, which can potentially be activated by a chromosome's natural ends, namely the ATM kinase pathway and the ATR kinase pathway (de Lange, 2010). The ATM kinase pathway is triggered in response to double-stranded DNA breaks (Lee & Paull, 2007). Upon this, the process by which the ATM kinase response is accomplished is still not completely clear (Lee & Paull, 2007). It is known that the Mre11/Rad50/Nbs1 complex (sensor proteins which take part in double-stranded breaks' detection) binds to DNA ends upon this and activates the ATM kinase in a combination with Tip60 HAT (Carson et al., 2003; de Lange, 2010; Williams et al., 2010). Later on, the ATM kinase phosphorylates some key proteins involved in the damage response to DNA double-strand breaks and initiates the activation of the DNA damage checkpoint, which pauses the cell cycle and allows time for a cell to repair damage before continuing its division (Lee & Paull, 2007). Upon this, Tip60 HAT, through histone acetylation, modulates repair proteins' loading and repair of double-stranded DNA breaks (Murr et al., 2006). In that case, if the damage is not resolved, the p53 protein is then activated, which triggers an apoptosis program (Polyak et al., 1997). The ATR kinase pathway is activated in response to the singlestranded DNA, and is based on the point that the abundant single-strand DNA binding protein RPA recognises and associates with single-stranded DNA, resulting in an RPA-ssDNA complex (Cimprich & Cortez, 2008; de Lange, 2010; Nam & Cortez, 2011). Further, the ATR kinase together with the ATRIP protein recognises such a single-stranded DNA coated with RPA and attached to the DNA's damage site (Cimprich & Cortez, 2008). At the same, the ATR kinase's recruitment to the RPA-ssDNA complex strongly depends on the ATRIP protein, which itself directly attaches to RPA-ssDNA, and thus binds this complex with the ATR (Zou & Elledge, 2003). The checkpoint clamp complex containing RAD9-HUS1-RAD1 (9-1-1) proteins, which take part in checkpoint activation, cell cycle arrest, and recruitment of specific DNA polymerases and other repair proteins to damaged DNA is also independently recruited to the DNA damage site by RAD17 protein (checkpoint clamp loader) (Bermudez et al., 2003; Sohn & Cho, 2009). Besides, the ATR activator TOPB1 is recruited to the DNA damage site (Choi et al., 2009). After these events, the activation of ATR by TOPBP1 and the phosphoryla‐ tion of downstream targets in a signal transduction cascade proceeds, which eventually leads to checkpoint activation, cell cycle arrest and subsequent damage repair (Cimprich & Cortez, 2008). Later on, all of the events are similar to those which were considered concerning the ATM kinase pathway, i.e. if damage is completely repaired then the cell cycle is resumed and the cell will continue its division, and if not then there will occur a trigger of the apoptosis

action.

470 The Mechanisms of DNA Replication

program and subsequent cell death.

Along with the blocking of ATM and ATR kinase DNA damage detection pathways, shelterin can also block the DNA repair reactions by the direct blocking of repair proteins. It was mentioned earlier that the repair of double-stranded breaks can basically be performed by two pathways - NHEJ and HDR. These two pathways in turn are triggered in a manner similar to the ATM and ATR signalling pathways in various ways (de Lange, 2010). NHEJ first employs the ring-shaped Ku70/80 protein complex, which loads onto DNA ends and facilitates their further synapsis and ligation by DNA ligase IV. As such, there is the suggestion, that a t-loop in addition to the repression of the ATM signalling pathway, also - probably - effectively blocks Ku70/80 joining and thus could thwart NHEJ in its earliest steps (de Lange, 2009; de Lange, 2010; Palm & de Lange, 2008). Besides this, the possibility was discussed that additional mechanisms not involving the t-loop can be used for telomere ends' protection from NHEJ (de Lange, 2010). It is suggested that POT1 contributes in NHEJ repression, especially after DNA replication when the t-loop is not yet formed (de Lange, 2009). HDR is initiated when Rad51 (the protein playing one of the most important roles in homologous recombination, since it organizes the proteinaceous complex which is necessary for chromosomes pairing and subsequent DNA strands exchange (Babynin, 2007)) replaces RPA on a single-stranded DNA (de Lange, 2010). In this connection, for blocking such a pathway of DSB's repair at telomeres, it is enough simply to repress RPA binding. Such repression on the telomere ends of mammals is carried out with the help of the POT1 protein, the binding of which to a single-stranded telomeric DNA, as was already noted, excludes RPA (de Lange, 2010). Therefore, POT1 is probably capable not only of blocking the ATR signalling pathway, and NHEJ after DNA replication, but also HDR on telomeres (de Lange, 2010). Besides this, there is data suggesting that Ku70/80 is also capable of repressing HDR in the absence of POT1 (Celli et al., 2006). It is necessary to also note that there is experimental data showing that TRF2 overexpression weakens the repair of single-stranded breaks on telomeres, resulting in their accelerated shortening. This suggests the possibility that the repair of such damage on telomeres, as well as the other types of single-stranded damage, is again hindered by t-loops and shelterin, in the formation of which TRF2 participates. In this connection, at increased TRF2 production, the tloops are probably formed more intensively in this case, and damages are repaired less effectively, leading to accelerated telomere shortening (Richter et al., 2007).

Thus, shelterin can inhibit repair on telomeres by the repression of various pathways of DNA damage detection, as well as of their repair itself. T-loop formation, in particular, leads to the blocking of the access of Mre11/Rad50/Nbs1 and Ku70/80 to double-stranded DNA ends, which prevents the activation of ATM signalling pathway and NHEJ that in its turn protects DNA natural ends, but blocks the repair of double-stranded breaks at telomeres. Besides, it is possible that the t-loop sterically blocks the repair of single-stranded damage at the telomeres by its three-dimensional structure. POT1 binding to single-stranded telomeric DNA excludes RAP from it and, therefore, prevents the recognition of damage by ATR in a complex with ATRIP, and which in turn prevents ATR kinase pathway activation. POT1 also blocks the binding of the Rad51 protein to single-stranded telomeric DNA, which prevents repair through HDR. Besides this, it is supposed that POT1 - after DNA replication when the t-loop yet is not formed - takes part in NHEJ repression, which is carried out, apparently, by Ku70/80 hetero‐ dimer blocking. There is also data suggesting that telomeric proteins themselves directly repress the pathways of single-stranded damage repair as well.

All of these mechanisms for repair repression on telomeres would seem, at first sight, to be the enemies for them, since repair deficiency leads to the circumstance where damage, occurring at chromosomes' ends is badly repaired and this leads to their shortening and, ultimately leads to cell cycle arrest and apoptosis. Nevertheless, a certain amount of time is needed for this purpose, which in some cases can be a quite considerable. On the other hand, if telomeric proteins did not repress the DNA damage response at chromosomes' ends, it would result in apoptosis being triggered immediately rather than after telomeres had been shortened to a critical degree, which is caused by the following circumstances. If shelterin were be absent on distal telomere regions, or in other words if nucleosome organisation would be observed rather than telosome organisation, that probably would not distinguish them from other chromosome areas – it would lead to such a case whereby DNA natural ends would be recognised by the damage detection system as double-stranded breaks. In this connection, Mre11/Rad50/Nbs1 would activate subsequently the ATM kinase pathway. Besides this, it is known that MRN, attaching to double-stranded DNA ends and recognising them as DSBs, is also capable of performing the 5'-3' resection of such ends, thus creating 3' single-stranded tails or overhangs (Mimitou & Symington, 2009). With the absence of telosome and, in this case, of the POT1 protein in particular, this would now lead to the activation of the ATR kinase pathway. The activation of any of the DNA damage detection pathways would lead to cell cycle arrest and damage repair. The repair of such, let us say, false DSBs in the case of ATM signalling pathway would probably be carried out by the NHEJ way. Insofar as during NHEJ broken chromosome ends are directly joined, but in our hypothetical variant the natural ends of different chromo‐ somes would be recognized as broken ends, then NHEJ in this case could lead to various chromosomes' fusion with one another. This situation will result in genome instability, which leads to the initiation of the apoptosis program. The repair of damage by the ATR kinase pathway due to the presence of recombinogenic 3' single-stranded protrusions would possibly occur through HDR. In such a case, the fusion of different chromosomes and, later on, cell destruction through the initiation of the apoptosis program, will also take place. There is experimental data supporting all of this and showing that repression of the shelterin proteins leads to chromosomes' fusion and subsequent apoptosis. Besides this, such chromosome ends' vulnerability from homologous recombination could lead to continuous telomere elongation through the ALT which would essentially increase the probability of cell transformation. Thus, the repair suppression mechanisms on telomeres, although they lead to the accumulation of damage and shortening, at the same time protect chromosomes' ends from fusion among themselves and ALT activation. In the case of protection against chromosome fusion, it appreciably extends cells' lifespan. As for protection from ALT, thus shelterin prevents inappropriate telomere elongation and importantly the probability of cells' transformation. Therefore, the repair suppressing mechanisms on telomeres appear as friends for them.

weakens the repair of single-stranded breaks on telomeres, resulting in their accelerated shortening. This suggests the possibility that the repair of such damage on telomeres, as well as the other types of single-stranded damage, is again hindered by t-loops and shelterin, in the formation of which TRF2 participates. In this connection, at increased TRF2 production, the tloops are probably formed more intensively in this case, and damages are repaired less

Thus, shelterin can inhibit repair on telomeres by the repression of various pathways of DNA damage detection, as well as of their repair itself. T-loop formation, in particular, leads to the blocking of the access of Mre11/Rad50/Nbs1 and Ku70/80 to double-stranded DNA ends, which prevents the activation of ATM signalling pathway and NHEJ that in its turn protects DNA natural ends, but blocks the repair of double-stranded breaks at telomeres. Besides, it is possible that the t-loop sterically blocks the repair of single-stranded damage at the telomeres by its three-dimensional structure. POT1 binding to single-stranded telomeric DNA excludes RAP from it and, therefore, prevents the recognition of damage by ATR in a complex with ATRIP, and which in turn prevents ATR kinase pathway activation. POT1 also blocks the binding of the Rad51 protein to single-stranded telomeric DNA, which prevents repair through HDR. Besides this, it is supposed that POT1 - after DNA replication when the t-loop yet is not formed - takes part in NHEJ repression, which is carried out, apparently, by Ku70/80 hetero‐ dimer blocking. There is also data suggesting that telomeric proteins themselves directly

All of these mechanisms for repair repression on telomeres would seem, at first sight, to be the enemies for them, since repair deficiency leads to the circumstance where damage, occurring at chromosomes' ends is badly repaired and this leads to their shortening and, ultimately leads to cell cycle arrest and apoptosis. Nevertheless, a certain amount of time is needed for this purpose, which in some cases can be a quite considerable. On the other hand, if telomeric proteins did not repress the DNA damage response at chromosomes' ends, it would result in apoptosis being triggered immediately rather than after telomeres had been shortened to a critical degree, which is caused by the following circumstances. If shelterin were be absent on distal telomere regions, or in other words if nucleosome organisation would be observed rather than telosome organisation, that probably would not distinguish them from other chromosome areas – it would lead to such a case whereby DNA natural ends would be recognised by the damage detection system as double-stranded breaks. In this connection, Mre11/Rad50/Nbs1 would activate subsequently the ATM kinase pathway. Besides this, it is known that MRN, attaching to double-stranded DNA ends and recognising them as DSBs, is also capable of performing the 5'-3' resection of such ends, thus creating 3' single-stranded tails or overhangs (Mimitou & Symington, 2009). With the absence of telosome and, in this case, of the POT1 protein in particular, this would now lead to the activation of the ATR kinase pathway. The activation of any of the DNA damage detection pathways would lead to cell cycle arrest and damage repair. The repair of such, let us say, false DSBs in the case of ATM signalling pathway would probably be carried out by the NHEJ way. Insofar as during NHEJ broken chromosome ends are directly joined, but in our hypothetical variant the natural ends of different chromo‐ somes would be recognized as broken ends, then NHEJ in this case could lead to various

effectively, leading to accelerated telomere shortening (Richter et al., 2007).

472 The Mechanisms of DNA Replication

repress the pathways of single-stranded damage repair as well.

It follows from the discussion above that the DNA damage response on telomeres, as well as damage detection and their repair pathways is potentially detrimental. This view was held for a long period of time. However, opinions changed when it was discovered that the proteins involved in the DNA damage response were present on the functional telomeres of mammals and interact with shelterin components, as previously in some way mentioned in reference to telomeric Rap1 protein, which recruits various proteins of the repair system to telomeres (Boulton & Jackson, 1996; Francia et al., 2006; Nugent et al., 1998). Moreover, further experi‐ ments have shown that they are also involved in telomere maintenance. This discovery was absolutely unexpected and somewhat paradoxical, as earlier it was thought that these proteins were the enemies for telomeres. However, it has now been discovered that this is not exactly the case, which has radically changed established views. Moreover, from now on DNA damage response proteins should be considered at the same time as equally the enemies and friends of telomeres. For the first time, such a role for DNA damage response proteins was found in yeast, where the NHEJ factor Ku is required for the maintenance of telomeres (Boulton & Jackson, 1996; Nugent et al., 1998; Polotnianka et al., 1998). In particular, in one such study it was found that in cells lacking telomerase but with functional Ku, telomere shortening slightly decelerates, i.e. it is less than in cells with repressed Ku function (Nugent et al., 1998). Further studies have shown that in addition to Ku, other proteins, involved in various DNA damage detection and their repair pathways, widely interact with telomeric proteins (Francia et al., 2006; Hsu et al., 1999; Hsu et al., 2000; Lenain et al., 2006; Palm & de Lange, 2008; Tarsounas et al., 2004; van Overbeek & de Lange, 2006; Zhu et al., 2000). Such factors in the mammals were called "shelterin accessory factors". Although they are present on telomeres transiently, at the same time they are very important for the maintenance of their normal structural organisation and functioning (Palm & de Lange, 2008).

Thus we emphasise, once again, that the main cause of poor damage repair on telomeres in comparison with other chromosome regions is the formation by their ends of t-loops, a process in which shelterin directly participates. Shelterin protects telomeres from the influence of both different sensor proteins that carry out DNA damage detection and the repair proteins themselves. Although this in turn leads to an accumulation of damage on telomeres and their shortening, at the same time it protects chromosomes from joining with each other, which ensures the maintenance of the integrity and normal efficiency of the cell genome. While shelterin protects telomeres from the action of DNA damage response proteins, it effectively interacts with them, which is also very important for telomere maintenance. It is necessary to add to all this also that in our opinion the cause of an inefficient damage repair on telomeres - specifically on their very ends - and their subsequent shortening, besides formation by telomeric proteins of the t-loops as well as their direct inhibiting influence on the DNA repair proteins is the fact that DNA repair proteins themselves cannot act correctly on the very DNA molecule end, which was discussed in the beginning of the section and can lead to incomplete repair or DDMTS.

#### **3.5. The conclusion of this section**

In summary, Olovnikov first described the problem of repairing the damage occurring on telomeres in the early 1990s. As at that time, it was considered that telomere ends had a doublestranded structure and so the model of telomere repair problem was described according to that conception. At that time, this problem was named the incomplete DNA repair problem, under which was considered the possibility of whether damage (DNA single-stranded break) occurred at a distance of only several nucleotides from the very end of a DNA molecule, then it could not be completely repaired in the course of copying of the complementary DNA strand because of DNA polymerases' functional peculiarities. As a result of this, telomeres should be shortened. In addition, based on that model, as well as on the point that in certain cases repair can start but does not come to completion or else does not begin at all, we considered it necessary to propose – an incomplete DNA repair and damage-mediated telomere shortening, or DDMTS. Upon this, incomplete repair and DDMTS - which in several different ways can lead to telomere shortening - can be considered to be two variants of the end repair problem. Considering these proposed new conceptions, the old model of the end repair problem was revised. As the time, it was found that the structure of telomere ends is not double-stranded, as was supposed earlier, but 3' overhanging single-stranded, we considered the eventualities of incomplete repair and the occurrence of DDMTS on the very linear telomere ends in instances of single-stranded breaks and other types of damage in accordance with this model. Theoretically, it is expected that incomplete repair and DDMTS for different variants of damage should uniquely lead to telomere shortening. However, it is not known whether it can actually occur, as unfortunately we were unable to find the experimental data confirming these assumptions. At the same time, there is much general data showing that damage on telomeres occurs and that this damage is repaired less effectively than damage in other regions of the chromosome. It was demonstrated that researchers' main attention in this has been given to telomere damage by reactive oxygen species, which are constantly formed in mitochondria at normal cell vital activity. It should explain why the genuine telomere-shortening rate exceeds the one that is expected as being only as a result of end replication problem. The circumstances by which the damage on telomeres is repaired more poorly has been explained well by many researchers in terms of t-loop formation. Therefore, in order to demonstrate exactly how tloops repress repair, we considered what they represent in themselves and also what DNA damage response is characterised by. As a result, it was shown that t-loops, formed with the participation of the shelterin protein complex, as well as the telomeric proteins themselves, block various damage detection mechanisms and their repair pathways directly, and this actually causes telomere repair deficiency. This can be designated as the "shelterin-mediated telomere repair problem". It should be distinguished from the end repair problem, which can be carried out not along the whole telomere length but only on their ends, because of the inability of repair proteins to act on a template end. Thus, in principle, it is possible to identify two problems of repair on telomeres – the end repair problem, which is carried out near the very DNA molecule ends, and the shelterin-mediated telomere repair problem, which can affect all telomere regions where there is telosome organisation. At the same time, both of these problems can be referred to, in general, as the telomere repair problem.

#### **4. Conclusion**

different sensor proteins that carry out DNA damage detection and the repair proteins themselves. Although this in turn leads to an accumulation of damage on telomeres and their shortening, at the same time it protects chromosomes from joining with each other, which ensures the maintenance of the integrity and normal efficiency of the cell genome. While shelterin protects telomeres from the action of DNA damage response proteins, it effectively interacts with them, which is also very important for telomere maintenance. It is necessary to add to all this also that in our opinion the cause of an inefficient damage repair on telomeres - specifically on their very ends - and their subsequent shortening, besides formation by telomeric proteins of the t-loops as well as their direct inhibiting influence on the DNA repair proteins is the fact that DNA repair proteins themselves cannot act correctly on the very DNA molecule end, which was discussed in the beginning of the section and can lead to incomplete

In summary, Olovnikov first described the problem of repairing the damage occurring on telomeres in the early 1990s. As at that time, it was considered that telomere ends had a doublestranded structure and so the model of telomere repair problem was described according to that conception. At that time, this problem was named the incomplete DNA repair problem, under which was considered the possibility of whether damage (DNA single-stranded break) occurred at a distance of only several nucleotides from the very end of a DNA molecule, then it could not be completely repaired in the course of copying of the complementary DNA strand because of DNA polymerases' functional peculiarities. As a result of this, telomeres should be shortened. In addition, based on that model, as well as on the point that in certain cases repair can start but does not come to completion or else does not begin at all, we considered it necessary to propose – an incomplete DNA repair and damage-mediated telomere shortening, or DDMTS. Upon this, incomplete repair and DDMTS - which in several different ways can lead to telomere shortening - can be considered to be two variants of the end repair problem. Considering these proposed new conceptions, the old model of the end repair problem was revised. As the time, it was found that the structure of telomere ends is not double-stranded, as was supposed earlier, but 3' overhanging single-stranded, we considered the eventualities of incomplete repair and the occurrence of DDMTS on the very linear telomere ends in instances of single-stranded breaks and other types of damage in accordance with this model. Theoretically, it is expected that incomplete repair and DDMTS for different variants of damage should uniquely lead to telomere shortening. However, it is not known whether it can actually occur, as unfortunately we were unable to find the experimental data confirming these assumptions. At the same time, there is much general data showing that damage on telomeres occurs and that this damage is repaired less effectively than damage in other regions of the chromosome. It was demonstrated that researchers' main attention in this has been given to telomere damage by reactive oxygen species, which are constantly formed in mitochondria at normal cell vital activity. It should explain why the genuine telomere-shortening rate exceeds the one that is expected as being only as a result of end replication problem. The circumstances by which the damage on telomeres is repaired more poorly has been explained well by many researchers in terms of t-loop formation. Therefore, in order to demonstrate exactly how t-

repair or DDMTS.

474 The Mechanisms of DNA Replication

**3.5. The conclusion of this section**

In summarising all the data, it is necessary to emphasise that there exist two basic telomereshortening mechanisms – the end replication problem and the telomere repair problem. The end replication problem, which is based on the 3' overhang loss during the course of DNA leading strand synthesis while the genetic material is doubling, has been studied in depth. The study of this problem allowed for the discovery of the telomerase enzyme and finding of the connection between telomere shortening and ageing, as well as carcinogenesis and various degenerative diseases. At the same time, many aspects of the end replication problem are still not absolutely clear. Therefore, further detailed studies of this process are necessary. The problem of telomere repair has been studied much less. Thus, there is no experimental data fully confirming that the end repair problem - which includes incomplete repair and DDMTS - can really be carried out on telomere ends and so lead to their shortening. At the same time, there is general data showing that damage on telomeres is frequently formed and repaired much less efficiently than on other chromosome regions leading to telomeric shortening. The researchers' main focus has been given to the influence of ROS on telomere damage. Given that they are constantly formed in cells by mitochondria, this is quite justifiable. At the same time, it is also necessary to study other negative factors concerning telomere damage and their shortening. Faint damage repair on telomeres is explained mainly by the formation at their ends of the t-loops, which are created with the participation of the telomeric protein complex shelterin and block different proteins involved in DNA damage response in order to prevent chromosomes' fusion with each other. This situation was referred to as the shelterin-mediated telomere repair problem. In this connection, it is necessary to study in more detail the structure of the proteins included in shelterin and their functions as well as mechanisms for t-loop formation. It is also important to study the proteins, which take part in DNA damage detection and the repair process itself. The latter should be studied in relation to both DNA damage response and their role in telomere maintenance. Thus, telomere-shortening mechanisms remain quite poorly understood and require further research.

### **Author details**

Andrey Grach\*

Khmelnitsky Regional Hospital, Ukraine

### **References**


[10] Bianchi, A.; Stansel, R. M., Fairall, L., Griffith, J. D., Rhodes, D. & de Lange, T. (1999). TRF1 Binds a Bipartite Telomeric Site With Extreme Spatial Flexibility. *The EMBO Journal*, Vol.18, No.20, (October 1999), pp. 5735-5744.

**Author details**

476 The Mechanisms of DNA Replication

Andrey Grach\*

**References**

194-200.

182-193.

Khmelnitsky Regional Hospital, Ukraine

88, No.2, (April 2008), pp. 557-579.

100, No.4, (February 2003), pp. 1633-1638.

[1] Allsopp, R. C.; Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B., Greider, C. W. & Harley, C. B. (1992). Telomere Length Predicts Replicative Capacity of Human Fibroblasts. *Proceedings of the National Academy of Sciences of the United*

[2] Allsopp, R. C.; Chang, E., Kashefi-Aazam, M., Rogaev, E. I., Piatyszek, M. A., Shay, J. W. & Harley C. B. (1995). Telomere Shortening is Associated with Cell Division *in vi‐ tro* and *in vivo*. *Experimental Cell Research*, Vol.220, No.1, (September 1995), pp.

[3] Aubert, G. & Lansdorp, P. M. (2008). Telomeres and Aging. *Physiological Reviews*, Vol.

[4] Azzalin, C. M.; Reichenbach, P., Khoriauli, L., Giulotto, E. & Lingner J. (2007). Telo‐ meric Repeat Containing RNA and RNA Surveillance Factors at Mammalian Chro‐

[5] Babynin, E. V. (2007). Molecular Mechanism of Homologous Recombination in Meio‐ sis: Origin and Biological Significance. *Tsitologiia*, Vol.49, No.3, (March 2007), pp.

[6] Baumann, P. & Cech, T. R. (2001). Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans. *Science*, Vol.292, No.5519, (May 2001), pp. 1171-1175.

[7] Baur, J. A.; Zou, Y., Shay, J. W. & Wright, W. E. (2001). Telomere Position Effect in

[8] Bermudez, V. P.; Lindsey-Boltz, L. A., Cesare, A. J., Maniwa, Y., Griffith, J. D., Hur‐ witz, J. & Sancar, A. (2003). Loading of the Human 9-1-1 Checkpoint Complex onto DNA by the Checkpoint Clamp Loader hRad17-Replication Factor C Complex *in vi‐ tro*. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.

[9] Bianchi, A.; Smith, S., Chong, L., Elias, P. & de Lange, T. (1997). TRF1 is a Dimer and Bends Telomeric DNA. *The EMBO Journal*, Vol.16, No.7, (April 1997), pp. 1785-1794.

Human Cells. *Science*, Vol.292, No.5524, (June 2001), pp. 2075-2077.

mosome Ends. *Science*, Vol.318, No.5851, (November 2007), pp. 798-801.

*States of America*, Vol.89, No.21, (November 1992), pp. 10114-10118.


[36] Dong, C. K.; Masutomi, K. & Hahn, W. C. (2005). Telomerase: Regulation, Function and Transformation. *Critical Reviews in Oncology Hematology*, Vol.54, No.2, (May 2005), pp. 85-93.

[23] Cimino-Reale, G.; Pascale, E., Battiloro, E., Starace, G., Verna, R. & D'Ambrosio, E. (2001). The Length of Telomeric G-rich Strand 3'-Overhang Measured by Oligonu‐

cleotide Ligation Assay. *Nucleic Acids Research*, Vol.29, No.7, (April 2001), E35.

*try*, Vol.278, No.4, (January 2003), pp. 2136-2140.

*ence*, Vol.276, No.5316, (May 1997), pp. 1252-1255.

*Biochemistry*, Vol.106, No.5, (April 2009), pp. 798-811.

326, No.5955, (November 2009), pp. 948-952.

*ular Cell*, Vol.38, No.6, (June 2010), pp. 777-779.

*Letters*, Vol.584, No.17, (September 2010), pp. 3673-3674.

2788-2801.

478 The Mechanisms of DNA Replication

768-785.

2007), pp. 1068-1071.

(May 2003), pp. 173-182.

13902-13907.

[24] Cimino-Reale, G.; Pascale, E., Alvino, E., Starace, G. & D'Ambrosio, E. (2003). Long Telomeric C-Rich 5'-Tails in Human Replicating Cells. *The Journal of Biological Chemis‐*

[25] Cimprich, K. A. & Cortez, D. (2008). ATR: an Essential Regulator of Genome Integri‐ ty. *Nature Reviews Molecular Cell Biology*, Vol.9, No.8, (August 2008), pp. 616-627.

[26] Conrad, M. N.; Dominguez, A. M. & Dresser, M. E. (1997). Ndj1p, a Meiotic Telomere Protein Required for Normal Chromosome Synapsis and Segregation in Yeast. *Sci‐*

[27] Dai, H.; Liu, J., Malkas, L. H. & Hickey, R. J. (2009). Characterization of RNA Primers Synthesized by the Human Breast Cancer Cell DNA Synthesome. *Journal of Cellular*

[28] Dai, X.; Huang, C., Bhusari, A., Sampathi, S., Schubert, K. & Chai, W. (2010). Molecu‐ lar Steps of G-Overhang Generation at Human Telomeres and its Function in Chro‐ mosome End Protection. *The EMBO Journal*, Vol.29, No.16, (August 2010), pp.

[29] de Laat, L.; Jaspers, N. G. & Hoeijmakers, J. H. (1999). Molecular Mechanism of Nu‐ cleotide Excision Repair. *Genes and Development*, Vol.13, No.7, (April 1999), pp.

[30] de Lange, T. (2009). How Telomeres solve the End-Protection Problem. *Science*, Vol.

[31] de Lange, T. (2010). Telomere Biology and DNA Repair: Enemies with Benefits. *FEBS*

[32] Denchi, E. L. & de Lange, T. (2007). Protection of Telomeres through Independent Control of ATM and ATR by TRF2 and POT1. *Nature*, Vol.448, No.7157, (August

[33] Desmaze, C.; Soria, J. C., Freulet-Marrière, M. A., Mathieu, N. & Sabatier, L. (2003). Telomere-Driven Genomic Instability in Cancer Cells. *Cancer Letters*, Vol.194, No.2,

[34] Dewar, J. M. & Lydall, D. (2010). Telomere Replication: Mre11 Leads the Way. *Molec‐*

[35] Dionne, I. & Wellinger, R. J. (1996). Cell Cycle-Regulated Generation of Single-Stranded G-Rich DNA in the Absence of Telomerase. *Proceedings of the National Acad‐ emy of Sciences of the United States of America*, Vol.93, No.24, (November 1996), pp.


and an unusual 3' Terminus. *Proceedings of the National Academy of Sciences of the Unit‐ ed States of America*, Vol.78, No.5, (May 1981), pp. 3015-3019.

[63] Krokan, H. E.; Standal, R., & Slupphaug, G. (1997). DNA Glycosylases in the Base Ex‐ cision Repair of DNA. *Biochemical Journal*, Vol.325, No.Pt 1, (July 1997), pp. 1-16.

[50] Hayflick, L. (1965). The Limited *in vitro* Lifetime of Human Diploid Cell Strains. *Ex‐*

[51] Hediger, F.; Neumann, F. R., Van Houwe, G., Dubrana, K. & Gasser, S. M. (2002). Live Imaging of Telomeres: yKu and Sir Proteins Define Redundant Telomere-An‐ choring Pathways in Yeast. *Current Biology*, Vol.12, No.24, (December 2002), pp.

[52] Hemann, M. T. & Greider, C. W. (1999). G-Strand Overhangs on Telomeres in Telo‐ merase-Deficient Mouse Cells. *Nucleic Acids Research*, Vol.27, No.20, (October 1999),

[53] Henderson, E. R. & Blackburn, E. H. (1989). An Overhanging 3' Terminus is a Con‐ served Feature of Telomeres. *Molecular and Cellular Biology*, Vol.9, No.1, (January

[54] Henson, J. D.; Neumann, A. A., Yeager, T. R. & Reddel, R. R. (2002). Alternative Lengthening of Telomeres in Mammalian Cells. *Oncogene*, Vol.21, No.4, (January

[55] Hsu, H. L.; Gilley, D., Blackburn, E. H. & Chen, D. J. (1999). Ku is Associated with the Telomere in Mammals. *Proceedings of the National Academy of Sciences of the United*

[56] Hsu, H. L.; Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. C., Campisi, J., Kohwi-Shigematsu, T. & Chen, D. J. (2000). Ku Acts in a Unique Way at the Mammalian Telomere to Prevent End Joining. *Genes and Development*, Vol.14, No.

[57] Huffman, K. E.; Levene, S. D., Tesmer, V. M., Shay, J. W. & Wright, W. E. (2000). Te‐ lomere Shortening is Proportional to the Size of the G-Rich Telomeric 3'-Overhang.

*The Journal of Biological Chemistry*, Vol.275, No.26, (June 2000), pp. 19719-19722.

[58] Kawanishi, S. & Oikawa, S. (2004). Mechanism of Telomere Shortening by Oxidative Stress. *Annals of the New York Academy of Sciences*, Vol.1019, (June 2004), pp. 278-284.

[59] Keys, B.; Serra, V., Saretzki, G. & Von Zglinicki, T. (2004). Telomere Shortening In Human Fibroblasts is not Dependent on the Size of the Telomeric-3'-Overhang. *Aging*

[60] Kim, S. H.; Kaminker, P. & Campisi, J. (1999). TIN2, a New Regulator of Telomere Length in Human Cells. *Nature Genetics*, Vol.23, No.4, (December 1999), pp. 405-412.

[61] Kirk, K. E.; Harmon, B. P., Reichardt, I. K., Sedat, J. W. & Blackburn E. H. (1997). Block in Anaphase Chromosome Separation Caused by a Telomerase Template Mu‐

[62] Klobutcher, L. A.; Swanton, M. T., Donini, P. & Prescott, D. M. (1981). All Gene-Sized DNA Molecules in Four Species of Hypotrichs have the Same Terminal Sequence

tation. *Science*, Vol.275, No. 5305, (March 1997), pp. 1478-1481.

*States of America*, Vol.96, No.22, (October 1999), pp. 12454-12458.

*perimental Cell Research*, Vol.37, (March 1965), pp. 614-636.

2076-2089.

480 The Mechanisms of DNA Replication

pp. 3964-3969.

1989), pp. 345-348.

2002), pp. 598-610.

22, (November 2000), pp. 2807-2812.

*Cell*, Vol.3, No.3, (June 2004), pp. 103-109.


[87] O'Connor, M. S.; Safari, A., Liu, D., Qin, J. & Songyang, Z. (2004). The Human Rap1 Protein Complex and Modulation of Telomere Length. *The Journal of Biological Chem‐ istry*, Vol.279, No.27, (July 2004), pp. 28585-28591.

[75] Londoño-Vallejo, J. A. (2008). Telomere Instability and Cancer. *Biochimie*, Vol.90, No.

[76] Mackenney, V. J.; Barnes, D. E. & Lindahl, T. (1997). Specific Function of DNA Ligase I in Simian Virus 40 DNA Replication by Human Cell-Free Extracts is Mediated by the Amino-Terminal Non-Catalytic Domain. *The Journal of Biological Chemistry*, Vol.

[77] Makarov, V. L.; Hirose, Y. & Langmore, J. P. (1997). Long G Tails at Both Ends of Hu‐ man Chromosomes Suggest a C Strand Degradation Mechanism for Telomere Short‐

[78] Maringele, L. & Lydall, D. (2002). EXO1-Dependent Single-Stranded DNA at Telo‐ meres Activates Subsets of DNA Damage and Spindle Checkpoint Pathways in Bud‐ ding Yeast yku70Delta Mutants. *Genes and Development*, Vol.16, No.15, (August 2002),

[79] McElligott, R. & Wellinger, R. J. (1997). The Terminal DNA Structure of Mammalian Chromosomes. *The EMBO Journal*, Vol.16, No.12, (June 1997), pp. 3705-3714.

[80] Meeker, A. K. & Coffey, D. S. (1997). Telomerase: A Promising Marker of Biological Immortality of Germ, Stem, and Cancer Cells. A Review. *Biochemistry (Moscow)*, Vol.

[81] Mimitou, E. P. & Symington, L. S. (2009). DNA End Resection: Many Nucleases Make Light Work. *DNA Repair (Amsterdam)*, Vol.8, No.9, (September 2009), pp. 983-995.

[82] Muntoni, A. & Reddel R. R. (2005). The First Molecular Details of ALT in Human Tu‐ mor Cells. *Human Molecular Genetics*, Vol. 14, No.2, (October 2005), pp. 191-196.

[83] Murr, R.; Loizou, J. I., Yang, Y. G., Cuenin, C., Li, H., Wang, Z. Q. & Herceg, Z. (2006). Histone Acetylation by Trrap-Tip60 Modulates Loading of Repair Proteins and Repair of DNA Double-Strand Breaks. *Nature Cell Biology*, Vol.8, No.1, (January

[84] Nam, E. A. & Cortez, D. (2011). ATR Signalling: More than Meeting at the Fork. *Bio‐*

[85] Nosek, J.; Dinouël, N., Kovac, L. & Fukuhara, H. (1995). Linear Mitochondrial DNAs from Yeasts: Telomeres with Large Tandem Repetitions. *Molecular and General Genet‐*

[86] Nugent, C. I., Bosco, G., Ross, L. O., Evans, S. K., Salinger, A. P., Moore, J. K., Haber, J. E. & Lundblad, V. (1998). Telomere Maintenance is Dependent on Activities Re‐ quired for End Repair of Double-Strand Breaks. *Current Biology*, Vol.8, No.11, (May

*chemical Journal*, Vol.436, No.3, (June 2011), pp. 527-536.

*ics*, Vol.247, No.1, (April 1995), pp. 61-72.

1, (January 2008), pp. 73-82.

482 The Mechanisms of DNA Replication

pp. 1919-1933.

2006), pp. 91-99.

1998), p. 657-660.

272, No.17, (April 1997), pp. 11550-11556.

62, No.11, (November 1997), pp. 1323-1331.

ening. *Cell*, Vol.88, No.5, (March 1997), pp. 657-666.


[112] Sfeir, A. J.; Chai, W., Shay, J. W. & Wright, W. E. (2005). Telomere-End Processing: the Terminal Nucleotides of Human Chromosomes. *Molecular Cell*, Vol.18, No.1, (April 2005), pp. 131-138.

[99] Pedram, M.; Sprung, C. N., Gao, Q., Lo, A. W. I., Reynolds, G. E. & Murnane, J. P. (2006). Telomere Position Effect and Silencing of Transgenes near Telomeres in the Mouse. *Molecular and Cellular Biology*, Vol.26, No.5, (March 2006), pp. 1865-1878.

[100] Pennaneach, V.; Putnam, C. D. & Kolodner, R. D. (2006). Chromosome Healing by *de novo* Telomere Addition in *Saccharomyces cerevisiae*. *Molecular Microbiology*, Vol.59,

[101] Petersen, S.; Saretzki, G. & von Zglinicki, T. (1998). Preferential Accumulation of Sin‐ gle-Stranded Regions in Telomeres of Human Fibroblasts. *Experimental Cell Research*,

[102] Podgornaya, O. I.; Bugaeva, E. A., Voronin, A. P., Gilson, E. & Mitchell, A. R. (2000). Nuclear Envelope Associated Protein That Binds Telomeric DNAs. *Molecular Repro‐*

[103] Polotnianka, R. M.; Li, J. & Lustig, A. J. (1998). The Yeast Ku Heterodimer is Essential for Protection of the Telomere Against Nucleolytic and Recombinational Activities.

[104] Polyak, K.; Xia, Y., Zweier, J. L., Kinzler, K. W. & Vogelstein, B. (1997). A Model for p53-Induced Apoptosis. *Nature*, Vol.389, No.6648, (September 1997), pp. 300-305.

[105] Reardon, J. T. & Sancar, A. (2005). Nucleotide Excision Repair. *Progress in Nucleic*

[106] Rhyu, M. S. (1995). Telomeres, Telomerase, and Immortality. *Journal of the National*

[107] Richter, T.; Saretzki, G., Nelson, G., Melcher, M., Olijslagers, S. & von Zglinicki, T. (2007). TRF2 Overexpression Diminishes Repair of Telomeric Single-Strand Breaks and Accelerates Telomere Shortening in Human Fibroblasts. *Mechanisms of Ageing*

[108] Richter, T. & von Zglinicki, T. (2007). A Continuous Correlation between Oxidative Stress and Telomere Shortening in Fibroblasts. *Experimental Gerontology*, Vol.42, No.

[109] Sancar, A.; Lindsey-Boltz, L. A., Unsal-Kaçmaz, K. & Linn, S. (2004). Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. *Annual*

[110] Saretzki, G.; Sitte, N., Merkel, U., Wurm, R. E. & von Zglinicki, T. (1999). Telomere Shortening Triggers a p53-dependent Cell Cycle Arrest via Accumulation of G-rich Single Stranded DNA Fragments. *Oncogene*, Vol.18, No.37, (September 1999), pp.

[111] Seeberg, E.; Eide, L. & Bjørås, M. (1995). The Base Excision Repair Pathway. *Trends in*

*Biochemical Sciences*, Vol.20, No.10, (October 1995), pp. 391-397.

*duction and Development*, Vol.57, No.1, (September 2000), pp. 16-25.

*Current Biology*, Vol.8, No.14, (July 1998), pp. 831-834.

*Acid Research and Molecular Biology*, Vol.79, (n.d.), pp. 183-235.

*Cancer Institute*, Vol.87, No.12, (June 1995), pp. 884-894.

*and Development*, Vol.128, No.4, (April 2007), pp. 340-345.

*Review of Biochemistry*, Vol.73, (July 2004), pp. 39-85.

11, (November 2007), pp. 1039-1042.

5148-5158.

No.5, (March 2006), pp. 1357-1368.

484 The Mechanisms of DNA Replication

Vol.239, No.1, (February 1998), pp. 152-160.


[137] Zou, L. & Elledge, S. J. (2003). Sensing DNA Damage through ATRIP Recognition of RPA-ssDNA Complexes. *Science*, Vol.300, No.5625, (June 2003), pp. 1542-1548.

[125] von Zglinicki, T. (2000). Role of Oxidative Stress in Telomere Length Regulation and Replicative Senescence. *Annals of the New York Academy of Sciences*, Vol.908, (June

[126] von Zglinicki, T.; Pilger, R. & Sitte, N. (2000). Accumulation of Single-Strand Breaks is the Major Cause of Telomere Shortening in Human Fibroblasts. *Free Radical Biology*

[127] von Zglinicki, T. (2002). Oxidative Stress Shortens Telomeres. *Trends in Biochemical*

[128] Watson, J. D. (1972). Origin of Concatemeric T7 DNA. *Nature New Biology*, Vol.239,

[129] Wellinger, R. J.; Wolf, A. J. & Zakian, V. A. (1993). Saccharomyces Telomeres Acquire Single-Strand TG1-3 Tails Late in S Phase. *Cell*, Vol.72, No.1, (January 1993), pp.

[130] Williams, G. J.; Lees-Miller, S. P. & Tainer, J. A. (2010). Mre11-Rad50-Nbs1 Confor‐ mations and the Control of Sensing, Signaling, and Effector Responses at DNA Dou‐ ble-Strand Breaks. *DNA Repair (Amsterdam)*, Vol.9, No.12, (December 2010), pp.

[131] Wright, W. E.; Tesmer, V. M., Huffman, K. E., Levene, S. D. & Shay, J. W. (1997). Nor‐ mal Human Chromosomes Have Long G-rich Telomeric Overhangs at One End.

[132] Wu, P.; van Overbeek, M., Rooney, S. & de Lange, T. (2010). Apollo Contributes to G Overhang Maintenance and Protects Leading-End Telomeres. *Molecular Cell*, Vol.39,

[133] Ye, J. Z.; Donigian, J. R., van Overbeek, M., Loayza, D., Luo, Y., Krutchinsky, A. N., Chait, B. T. & de Lange T. (2004). TIN2 Binds TRF1 and TRF2 Simultaneously and Stabilizes the TRF2 Complex on Telomeres. *The Journal of Biological Chemistry*, Vol.

[134] Yuan, X.; Ishibashi, S., Hatakeyama, S., Saito, M., Nakayama, J., Nikaido, R., Haruya‐ ma, T., Watanabe, Y., Iwata, H., Iida, M., Sugimura, H., Yamada, N. & Ishikawa, F. (1999). Presence of Telomeric G-Strand Tails in the Telomerase Catalytic Subunit

TERT Knockout Mice. *Genes to Cells*, Vol.4, No.10, (October 1999), pp. 563-572.

[135] Zheng, L. & Shen, B. (2011). Okazaki Fragment Maturation: Nucleases Take Centre Stage. *Journal of Molecular Cell Biology*, Vol.3, No.1, (February 2011), pp. 23-30.

[136] Zhu, X. D.; Küster, B., Mann, M., Petrini, J. H. & de Lange, T. (2000). Cell-Cycle-Regu‐ lated Association of RAD50/MRE11/NBS1 with TRF2 and Human Telomeres. *Nature*

*Genes and Development*, Vol.11, No.21, (November 1997), pp. 2801-2809.

*and Medicine*, Vol.28, No.1, (January 2000), pp. 64-74.

*Sciences*, Vol.27, No.7, (July 2002), pp. 339-344.

No.94, (October 1972), pp. 197-201.

No.4, (August 2010), pp. 606-617.

279, No.45, (November 2004), pp. 47264-47271.

*Genetics*, Vol.25, No.3, (July 2000), pp. 347-352.

2000), pp. 99-110.

486 The Mechanisms of DNA Replication

51-60.

1299-1306.

[138] Zubko, M. K.; Guillard, S. & Lydall, D. (2004). Exo1 and Rad24 Differentially Regu‐ late Generation of ssDNA at Telomeres of *Saccharomyces cerevisiae* cdc13-1 Mutants. *Genetics*, Vol.168, No.1, (September 2004), pp. 103-115
