**The MCM and RecQ Helicase Families: Ancient Roles in DNA Replication and Genomic Stability Lead to Distinct Roles in Human Disease**

Dianne C. Daniel\*, Ayuna V. Dagdanova and Edward M. Johnson

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52961

### **1. Introduction**

#### **1.1. Rationale for comparison of MCM and RecQ helicase families**

DNA helicases are currently organized into superfamilies based on their sequence structures and 3-D conformations. Within each superfamily, there are members that have further evolved for specialized functions [1]. There is conservation of RecQ proteins from bacteria to humans. Whereas bacteria have one RecQ helicase, humans have evolved at least five differ‐ ent proteins [2]. The RecQ members belong to the helicase Superfamily II, and as such have the characteristic Rec fold [1]. In this chapter, we will focus on RecQ family members WRN, BLM and RECQL4 (RecQ protein-like 4), which is also referred to in the literature as RECQ4. Eukaryotic and archaeal MCMs belong to the helicase Superfamily VI, and have the AAA+ (ATPases associated with diverse cellular activities) fold [1, 3, 4, 5]. Both Rec and AAA+ folds are based on the ancestral ASCE (additional strand conserved E) fold or an alpha-betaalpha domain necessary for nucleoside triphosphate binding and catalysis [1, 6, 7]. A ration‐ ale for comparison of the RecQ and MCM family members relates to the importance of their activities for genomic integrity. The WRN and BLM proteins as well as other members of the RecQ family are characterized by this feature [8]. Both WRN and BLM are involved in DNA repair and a role for WRN in telomere homeostasis in humans is well established [2, 9]. MCM2-7 proteins, along with cofactors, are thought to function as the eukaryotic replicative helicase [10]. MCM8 [11, 12] and MCM9 [13, 14] are more recently discovered and their roles are less well defined. Although data point to a role for MCM8 in DNA replication, that role may be specialized in higher organisms. In human cells, MCM10 is recruited to chromoso‐

© 2013 Daniel\* et al.; 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 Daniel\* et al.; 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.

mal domains before they replicate and studies in yeast suggest a role in DNA replication, but not as a helicase [15-18]. Members of each family are essential for chromosome homeo‐ stasis. When replication forks stall, there may be involvement of members of each family. These proteins have interlocking functions since, for example, a stalled replication fork with attendant MCM proteins can lead to a DNA double-strand break (DSB), which requires RecQ proteins for repair [19].

### **2. RecQ and MCM family structures**

Breaks in double-strand DNA can occur during DNA replication, at specific loci (e.g., at te‐ lomeres) and during meiosis. RecQ helicases are implicated in DNA repair based on their involvement in such processes as DNA end resection, branch migration, D-loop processing, Holliday Junction (HJ) and double Holliday junction (dHJ) resolution [2]. RecQ proteins function at multiple steps, both early and late, during repair of DSB [19]. RecQ helicases travel in a 3' to 5' direction on ssDNA [20]. The RecQ proteins have an ancient lineage based on an ancestral ASCE fold (αβα domain) of distant relation to P-loop NTPase folds. RecQ structural domains include a conserved core helicase domain for binding and hydrolysis of nucleoside triphosphate that is equivalent to the Walker A and Walker B boxes seen in MCM proteins [21]. They have a helicase and RNAase D C-terminal (HRDC) domain thought to mediate structure-specific nucleic acid binding, double HJ dissolution and pro‐ tein-protein interactions. They also have a RecQ C-terminal (RQC) domain thought to medi‐ ate interactions with other proteins, structure-specific nucleic acid binding and metal cofactor binding [22]. Acidic regions present in many RecQ proteins aid in protein-protein interactions. There are also nuclear localization signals in some RecQ proteins (e.g., *H. sapi‐ ens* WRN and BLM). There are two RecQ members with an exonuclease domain, one of which is *H. sapiens* WRN. Two members have been functionally characterized as having an N-terminal strand exchange domain, one of which is *H. sapiens* BLM [2].

RECQL4 has been reported to have ssDNA binding and DNA strand-annealing activities. In this single study, RECQL4 did not display substrate unwinding or resolution of substrates resembling replication or recombination intermediates [23]. Recognizable HRDC and RQC domains that are important in BLM activity are missing in RECQL4 [22, 24]. As observed, the ssDNA annealing activity would allow RECQL4 to function during synthesis-dependent strand annealing (SDSA) along with another helicase. RECQL4 could help direct pathway choice during HR in DSB repair through aiding ssDNA annealing activity in non-homolo‐ gous end joining (NHEJ) [19]. Thus, RECQL4 is similar to other RecQ helicases in its core helicase domain, but its function as a helicase is unclear [23, 25, 26].

AAA+ proteins, including MCMs, have a core molecular motor. Like the RecQ proteins, the AAA+ fold is also based on the ancestral ASCE fold. Acquisition of a catalytic glutamate (Fig. 1) to initiate efficient hydrolysis of ATP marked the emergence of the ASCE division from the ancestral P-loop fold [7]. For further discussion of the glutamate "switch" in AAA+ proteins, see reference [27]. Mechanisms of action are diverse, although members are typi‐ cally oligomeric ring assemblies with inter-subunit communication. The central AAA+ mo‐ tor has been adapted in evolution through structural changes to the core module and through domains added either N- or C-terminal to the AAA+ core. Activities are facilitated by recognition of protein partners functioning in these diverse events. Thus, AAA+ proteins display a variety of macromolecular remodelling events that are energy-driven by nucleo‐ tide hydrolysis thought to be occurring throughout what is typically a hexameric complex assembly [28, 29]. The conserved Walker A and Walker B motifs within the central module mediate ATP-binding and hydrolysis [7, 30-32]. MCM proteins have two active site motifs, the P-loop domain and the lid. Motifs in the P-loop include Walker A, Walker B and Sensor 1. The lid domain contains the arginine finger and Sensor 2. A catalytic site is created by a dimer interface that employs a *cis* P-loop from one subunit and a *trans* lid from the adjacent subunit [4, 33, 34]. A similar catalytic site created at the interfaces between adjacent mono‐ mers is also characteristic of the RecQ ATPase core [21].

mal domains before they replicate and studies in yeast suggest a role in DNA replication, but not as a helicase [15-18]. Members of each family are essential for chromosome homeo‐ stasis. When replication forks stall, there may be involvement of members of each family. These proteins have interlocking functions since, for example, a stalled replication fork with attendant MCM proteins can lead to a DNA double-strand break (DSB), which requires

Breaks in double-strand DNA can occur during DNA replication, at specific loci (e.g., at te‐ lomeres) and during meiosis. RecQ helicases are implicated in DNA repair based on their involvement in such processes as DNA end resection, branch migration, D-loop processing, Holliday Junction (HJ) and double Holliday junction (dHJ) resolution [2]. RecQ proteins function at multiple steps, both early and late, during repair of DSB [19]. RecQ helicases travel in a 3' to 5' direction on ssDNA [20]. The RecQ proteins have an ancient lineage based on an ancestral ASCE fold (αβα domain) of distant relation to P-loop NTPase folds. RecQ structural domains include a conserved core helicase domain for binding and hydrolysis of nucleoside triphosphate that is equivalent to the Walker A and Walker B boxes seen in MCM proteins [21]. They have a helicase and RNAase D C-terminal (HRDC) domain thought to mediate structure-specific nucleic acid binding, double HJ dissolution and pro‐ tein-protein interactions. They also have a RecQ C-terminal (RQC) domain thought to medi‐ ate interactions with other proteins, structure-specific nucleic acid binding and metal cofactor binding [22]. Acidic regions present in many RecQ proteins aid in protein-protein interactions. There are also nuclear localization signals in some RecQ proteins (e.g., *H. sapi‐ ens* WRN and BLM). There are two RecQ members with an exonuclease domain, one of which is *H. sapiens* WRN. Two members have been functionally characterized as having an

N-terminal strand exchange domain, one of which is *H. sapiens* BLM [2].

helicase domain, but its function as a helicase is unclear [23, 25, 26].

RECQL4 has been reported to have ssDNA binding and DNA strand-annealing activities. In this single study, RECQL4 did not display substrate unwinding or resolution of substrates resembling replication or recombination intermediates [23]. Recognizable HRDC and RQC domains that are important in BLM activity are missing in RECQL4 [22, 24]. As observed, the ssDNA annealing activity would allow RECQL4 to function during synthesis-dependent strand annealing (SDSA) along with another helicase. RECQL4 could help direct pathway choice during HR in DSB repair through aiding ssDNA annealing activity in non-homolo‐ gous end joining (NHEJ) [19]. Thus, RECQL4 is similar to other RecQ helicases in its core

AAA+ proteins, including MCMs, have a core molecular motor. Like the RecQ proteins, the AAA+ fold is also based on the ancestral ASCE fold. Acquisition of a catalytic glutamate (Fig. 1) to initiate efficient hydrolysis of ATP marked the emergence of the ASCE division from the ancestral P-loop fold [7]. For further discussion of the glutamate "switch" in AAA+ proteins, see reference [27]. Mechanisms of action are diverse, although members are typi‐

RecQ proteins for repair [19].

60 The Mechanisms of DNA Replication

**2. RecQ and MCM family structures**

**Figure 1.** Comparison of conserved ATPase motifs in RecQ proteins WRN, BLM and RECQL4 to the Walker A and Walk‐ er B boxes of MCM8. MCM8 was chosen as the MCM for comparison because it has a canonical GKS Walker A [12] and the signature MCM IDEFDKM Walker B ATP-binding domains. In the Walker B motif, note the conserved structural fea‐ tures and the conserved DE motif (containing aspartate, D, and the catalytic glutamate, E).

### **3. RecQ and MCM helicases: association with disease and aging**

#### **3.1. BLM and Bloom syndrome, WRN and Werner syndrome, RECQL4 and Rothmund-Thomson syndrome**

As a group, mutations in the RecQ helicases lead to adult segmental progeria with ab‐ normalities in development, predisposition to cancer and acceleration of aging processes. Three of the RecQ family members are associated with rare autosomal recessive diseases [19]. These disorders Werner syndrome (WS) [35], Bloom syndrome (BS) [36] and Roth‐ mund-Thomson syndrome (RTS) [37, 38] are caused by mutations in the genes coding for WRN, BLM and RECQL4, respectively. RTS is a heterogeneous disorder with about 60% of cases resulting from mutations in the *RECQL4* gene [37]. Mutations in *RECQL4* can also lead to two other disease phenotypes [39], but only RTS will be discussed here. The RecQ deficiency diseases are associated with cancer predisposition and several char‐ acteristics of aging [8, 20, 26, 40]. In BS cells, there is a 10-fold elevation in frequency of homologous recombination (HR), and reciprocal exchanges occur between homologous chromosomes and sister chromatids [41, 42]. WS cells, on the other hand, display large chromosome deletions and an increase in illegitimate recombination [43]. A higher fre‐ quency of chromosomal aberrations is reported for cells from RTS [44]. These deficien‐ cies thus provide hints as to the cellular activities of these three helicases. Clinical features of these diseases, as referenced above, are as follows.

BS manifests in pleiotropic phenotypes such as growth retardation leading to proportional dwarfism, erythema with light sensitivity, skin lesions with hypo- and hyperpigmentation, immunodeficiency, susceptibility to type II diabetes, male infertility, female sub-fertility, re‐ ports of mental retardation, cancer predisposition (all types but at an earlier age of diagnosis than in the normal population).

WS leads to short stature and early onset age-related diseases, including greying hair, alope‐ cia, bilateral cataracts, osteoporosis, arteriosclerosis, atherosclerosis, skin atrophy, hypogo‐ nadism, type II diabetes mellitus and susceptibility to tumors, especially those of mesenchymal origin (sarcomas).

RTS manifests as early growth deficiency, congenital bone defects, poikiloderma, cataracts, greying hair, alopecia, hypogonadism, and some increased susceptibility to cancer, especial‐ ly osteogenic sarcomas.

#### **3.2. MCMs and genomic stability**

The MCM proteins 2-7 are necessary for DNA replication in yeast [45, 46], and this basic life function extends in evolution to a single MCM protein in archaea [47-49]. MCM2-7 are also essential for replication in *Xenopus* [50, 51] and have been proposed as a licensing factor for initiation of eukaryotic replication [52, 53]. The MCM proteins 2-8 have an identical Walker B-box motif of IDEFDKM. The MCM2-7 complex is enigmatic in that MCMs 4, 6 and 7 func‐ tion alone as a heterohexameric helicase [54, 55]. For a discussion of individual MCM subu‐ nit arrangements and activities, see the references [33, 34]. MCM2-7 have now been shown to have helicase activity *in vitro* [56], and to be components of a holo-helicase Cdc45/ MCM2-7/GINS (CMG) complex [57, 58]. The MCMs require a clamp-loading factor to as‐ semble as a multimeric ring on DNA, and this function is fulfilled in known cases by the protein Cdc6 [59-64] although the regulation of this step in the formation of the CMG com‐ plex proceeds through multiple pathways [57, 58]. Various papers have dealt with the func‐ tion of MCM proteins in DNA replication [10, 46, 65-68], and regarding their processive mechanism of DNA unwinding [27, 56, 58, 69, 70] and only certain lingering, disease-related questions will be considered here. A summary statement can be made regarding known re‐ lationships between MCM and RecQ helicases. Members of the MCM protein family are es‐ sential for the life-creating process of DNA replication, whereas members of the RecQ family are essential for the life-prolonging maintenance of the genome.

Due to their essential roles, it is not surprising that there are few diseases directly ascribed to defects in MCMs 2-7. This does, however, bring up an unresolved MCM enigma: there are more MCM proteins than are required to form initiation complexes at cellular origins active within a given round of DNA replication [71]. In addition, MCM proteins in human cells re‐ main at peak levels in G2 phase of the cell cycle, after DNA replication is complete [59, 72, 73] This high copy number could be an aspect of securing sufficient protein quantities for basic function. A role for MCM proteins in transcription has also been proposed, and this remains to be defined [74]; for further discussion see reference [46]. Redundancy of functions among MCM members is also a factor to be considered. Intriguingly, knockdown of MCM8 [75], not even present in yeast [12], retards S-phase approximately 25% in human cells in culture [75], suggesting a specialized function in higher eukaryotes critical for basic replication. It has been suggested that the excess MCMs license dormant origins, which are used under conditions of stress upon normally functional origins [71]. An overview provides a discussion of recent work connecting dormant origins of replication and tumor suppression based on the role of dormant origins during fork restart after repair of DNA damage [76].

chromosome deletions and an increase in illegitimate recombination [43]. A higher fre‐ quency of chromosomal aberrations is reported for cells from RTS [44]. These deficien‐ cies thus provide hints as to the cellular activities of these three helicases. Clinical

BS manifests in pleiotropic phenotypes such as growth retardation leading to proportional dwarfism, erythema with light sensitivity, skin lesions with hypo- and hyperpigmentation, immunodeficiency, susceptibility to type II diabetes, male infertility, female sub-fertility, re‐ ports of mental retardation, cancer predisposition (all types but at an earlier age of diagnosis

WS leads to short stature and early onset age-related diseases, including greying hair, alope‐ cia, bilateral cataracts, osteoporosis, arteriosclerosis, atherosclerosis, skin atrophy, hypogo‐ nadism, type II diabetes mellitus and susceptibility to tumors, especially those of

RTS manifests as early growth deficiency, congenital bone defects, poikiloderma, cataracts, greying hair, alopecia, hypogonadism, and some increased susceptibility to cancer, especial‐

The MCM proteins 2-7 are necessary for DNA replication in yeast [45, 46], and this basic life function extends in evolution to a single MCM protein in archaea [47-49]. MCM2-7 are also essential for replication in *Xenopus* [50, 51] and have been proposed as a licensing factor for initiation of eukaryotic replication [52, 53]. The MCM proteins 2-8 have an identical Walker B-box motif of IDEFDKM. The MCM2-7 complex is enigmatic in that MCMs 4, 6 and 7 func‐ tion alone as a heterohexameric helicase [54, 55]. For a discussion of individual MCM subu‐ nit arrangements and activities, see the references [33, 34]. MCM2-7 have now been shown to have helicase activity *in vitro* [56], and to be components of a holo-helicase Cdc45/ MCM2-7/GINS (CMG) complex [57, 58]. The MCMs require a clamp-loading factor to as‐ semble as a multimeric ring on DNA, and this function is fulfilled in known cases by the protein Cdc6 [59-64] although the regulation of this step in the formation of the CMG com‐ plex proceeds through multiple pathways [57, 58]. Various papers have dealt with the func‐ tion of MCM proteins in DNA replication [10, 46, 65-68], and regarding their processive mechanism of DNA unwinding [27, 56, 58, 69, 70] and only certain lingering, disease-related questions will be considered here. A summary statement can be made regarding known re‐ lationships between MCM and RecQ helicases. Members of the MCM protein family are es‐ sential for the life-creating process of DNA replication, whereas members of the RecQ

Due to their essential roles, it is not surprising that there are few diseases directly ascribed to defects in MCMs 2-7. This does, however, bring up an unresolved MCM enigma: there are more MCM proteins than are required to form initiation complexes at cellular origins active within a given round of DNA replication [71]. In addition, MCM proteins in human cells re‐ main at peak levels in G2 phase of the cell cycle, after DNA replication is complete [59, 72, 73]

family are essential for the life-prolonging maintenance of the genome.

features of these diseases, as referenced above, are as follows.

than in the normal population).

62 The Mechanisms of DNA Replication

mesenchymal origin (sarcomas).

**3.2. MCMs and genomic stability**

ly osteogenic sarcomas.

Mutations in MCM family members have been studied in yeast and mouse models, and they confirm an essential role for MCM2-7 in DNA replication [34, 45, 77, 78]. A human MCM4 mutation (destabilizing the MCM2-7 complex) was recently reported concurrently by two groups who studied consanguineous families, and the resulting phenotype was found to be associated with immune deficiency (NK cells), adrenal insufficiency and short stature [79, 80]. Patient fibroblasts showed chromosome fragility [79]. The susceptibility of these pa‐ tients to cancer is not currently known [80]. An *MCM8* disruption and alternative splice form have been noted in hepatic carcinoma [11] and choriocarcinoma [12], respectively. Al‐ though not necessarily a cause of disease, the MCM proteins may be useful tools in diagno‐ ses [81-85]. Elevated levels of MCM proteins 2-7 have been observed in several cancers [81, 84, 85]. In contrast, reduced levels of MCM8 mRNA have been reported in colon carcinoma [12]. Nuclear MCM7 is a good marker for proliferating cells [86], and MCMs 2, 5 or 7 may be an alternative to the Ki-67 marker to distinguish certain hyperproliferative disorders [83].

#### *3.2.1. A structural domain deleted from MCM8 and present in WRN and BLM*

A brief discussion of MCM8 is included in this section because it contains a motif that may be structurally similar to one found in BLM and WRN and is linked to neoplasia. Human MCM8 has a splice variant that results in a 16 amino acid (aa) deletion in a location between the Zn finger of MCM8 and its Walker A box [12], Fig. 2. Thus far this deletion has been detected by various different groups only in cases of choriocarcinoma. MCM8 with this same sequence deleted (Fig. 2) has, however, been detected in several higher eukaryotes other than humans, suggesting that the variant does have, or perhaps lacks, a function. That function is as yet un‐ known, but clues to it may be gleaned from a comparison of the MCM8 deletion with sequen‐ ces from WRN and BLM. These RecQ proteins have a counterpart 16 aa domain with partial sequence homology and notable structural homology, as denoted in Fig. 2. This sequence in the RecQ proteins is located in a different orientation to that of MCM8 with regard to the heli‐ case Walker A and B boxes (Fig. 2). In each case the first 8 aa of this sequence are highly charg‐ ed and, in WRN, BLM and MCM8, contain a polar S. The 9th aa in this deletion is a conserved C. The remaining 7 aa contain a preponderance of aromatic and hydrophobic residues. This configuration of charged and aromatic residues is characteristic of known single-stranded nu‐ cleic acid binding proteins [87]. Among MCMs, this 16 aa domain is identifiable in the single MCM of *Sulfolobus solfataricus*, in which it has been implicated as a single-stranded DNA- binding "finger" [88]. Mutations of the positively charged amino acids strongly reduce singlestranded DNA binding of this MCM. In contrast, in BLM the polar S residue is thought to be involved in ATP binding [89]. Because of the aromatic nature of a portion of this domain, binding to one or more DNA nucleotide bases may be involved as a common link between functions of these 16 aa in RECQ and MCM helicases.

**Figure 2.** Comparison of placement of selected structural motifs in human RecQ and MCM8 helicases. MCM8 has a splice variant that results in a 16 aa deletion in choriocarcinoma [12]. The Walker A and B boxes and the MCM Zn finger (ZnF) domains are indicated by filled rectangles. The MCM8 variant 2 deletion (Var2 del) is indicated by an open rectangle, as is its partially-homologous counterpart (del hom) in WRN and BLM. This conservatively structured do‐ main consists of an N-terminal highly charged sequence followed by a conserved C and an aromatic-hydrophobic se‐ quence. The open box is located in a different orientation relative to Walker A and B boxes in WRN vs. MCM8. The positions of black and white boxes are approximately to scale.

### **4. Supportive roles for WRN, BLM helicases and RECQL4 during replication elongation**

During normal metabolism, such as in mitochondrial respiration, endogenous reactive oxy‐ gen species (ROS) are produced that lead to oxidative DNA modifications. In addition to en‐ dogenous mutagens, there are also environmental mutagens that damage the DNA [90]. Furthermore, during replication and transcription, duplex DNA is transiently opened, and there is an opportunity for non-B DNA structures to form in the genomic DNA [40]. RecQ helicases act on recombination intermediates, on preferred substrates including those resem‐ bling G-quadruplex DNA [91, 92], D-loops [93], HJ [94] and double HJ [95]. RecQ helicases may play a regulatory role in both pro- and anti-HR events. They function at the interface of HR with the stressed replication fork and may affect repair pathway choice. There is little evidence for a specific clear cut role, but for a discussion of proposed mechanistic models of RecQ protein function, see the references [19, 20].

#### **4.1. BLM and RECQL4**

binding "finger" [88]. Mutations of the positively charged amino acids strongly reduce singlestranded DNA binding of this MCM. In contrast, in BLM the polar S residue is thought to be involved in ATP binding [89]. Because of the aromatic nature of a portion of this domain, binding to one or more DNA nucleotide bases may be involved as a common link between

**Figure 2.** Comparison of placement of selected structural motifs in human RecQ and MCM8 helicases. MCM8 has a splice variant that results in a 16 aa deletion in choriocarcinoma [12]. The Walker A and B boxes and the MCM Zn finger (ZnF) domains are indicated by filled rectangles. The MCM8 variant 2 deletion (Var2 del) is indicated by an open rectangle, as is its partially-homologous counterpart (del hom) in WRN and BLM. This conservatively structured do‐ main consists of an N-terminal highly charged sequence followed by a conserved C and an aromatic-hydrophobic se‐ quence. The open box is located in a different orientation relative to Walker A and B boxes in WRN vs. MCM8. The

During normal metabolism, such as in mitochondrial respiration, endogenous reactive oxy‐ gen species (ROS) are produced that lead to oxidative DNA modifications. In addition to en‐ dogenous mutagens, there are also environmental mutagens that damage the DNA [90]. Furthermore, during replication and transcription, duplex DNA is transiently opened, and there is an opportunity for non-B DNA structures to form in the genomic DNA [40]. RecQ helicases act on recombination intermediates, on preferred substrates including those resem‐ bling G-quadruplex DNA [91, 92], D-loops [93], HJ [94] and double HJ [95]. RecQ helicases

**4. Supportive roles for WRN, BLM helicases and RECQL4 during**

functions of these 16 aa in RECQ and MCM helicases.

64 The Mechanisms of DNA Replication

positions of black and white boxes are approximately to scale.

**replication elongation**

In BS cells, there are S-phase defects in DNA replication involving abnormal replication inter‐ mediates, and replication elongation is slower [96-98]. These cells don't recover well from in‐ duced fork stalling and accumulate DSB [99, 100]. In human cells, RECQL4 interacts with the MCM2-7 complex, Cdc45 and GINS. The interaction is facilitated by MCM10 [25]. BLM and RECQL4 are at their highest levels during S phase. At stalled replication forks, BLM physical‐ ly associates with Rad51 and p53, and BLM and p53 function synergistically in HR [101]. BLM colocalizes in foci with PCNA and with the BASC (BRCA1-associated genome surveillance complex) [102]. BLM is phosphorylated by ATR [103]. When replication forks are stalled by use of hydroxyurea (HU), BLM colocalizes with Chk1 and p53BP1 foci. Chk1 is required for BLM and 53BP1 foci formation. Thus Chk1 may recruit BLM to stalled forks [104]. This impli‐ cates RecQ proteins as DNA damage checkpoint mediators in response to stalled forks. *In vi‐ tro* studies with use of substrates similar to Okazaki fragments showed BLM stimulation of flap endonuclease [105] (a protein that functions in lagging strand synthesis [106]). BLM func‐ tions to prevent the association of homologous sequences in the displaced flap DNA of the Okazaki fragment and the sister chromatid [107-110]. D-loops are formed when a ssDNA tail invades a homologous duplex, and BLM has a preference for dissociating D-loops with a 5' in‐ vaded end suggesting a selection of recombination intermediates that are not extended by polymerase [93, 111, 112]. BLM is able to disrupt the initial Rad51 filament formation step to destabilize recombinase-nucleoprotein filaments. D-loops are susceptible to BLM activity when Rad51 is in an inactive form (ADP-bound) [112, 113]. BLM physically associates with CAF-1 (chromatin assembly factor I) largest subunit, and the colocalization of these two pro‐ teins occurs at sites of DNA synthesis. BLM inhibits CAF-1 function in chromatin assembly during DNA repair *in vitro*, and inhibits its mobilization after damage induction *in vivo* [114]. Mammalian WRN and BLM interact [115], and they both interact with RPA [116-118] and p53 [119-121]. Based on coimmunoprecipitation there is limited BLM and WRN interaction, but they may function in the same pathway during HR [20]. BLM helicase activity is stimulated by its binding to the RPA70 kDa subunit [116]. In mouse spermatocytes during meiotic pro‐ phase, BLM and RPA are nuclear colocalized [122]. This suggests a potential role for these proteins in resolution of recombination intermediates during meiosis [8]. BLM has a prefer‐ ence for unwinding G-quadruplex structures versus HJ [92]. Both mammalian WRN and BLM bind to G-quadruplex structures, which are roadblocks to polymerases [123, 124].

Aberrant replication intermediates arise in cells lacking WRN and BLM [97, 125]. Such unre‐ solved replication or recombination structures lead to incomplete chromosome segregation. BLM, topoisomerase 3 alpha (Topo3α) and BLAP75/RMI1 (for BLM-associated polypeptide/ RecQ-mediated genome instability) or a BLM-Topo3α-BLAP75/RMI1 complex localizes to resulting anaphase bridges [126]. A helicase known as PICH arrives first, followed by the resolution activity of BLM [127]. As helicases unwind duplex DNA, torsional stress pro‐ duced in the DNA may require relief through topoisomerase activity, and such activity fi‐ nally decatenates interlocked DNA molecules [128, 129]. *In vitro* studies show that BLM can partner with Topo3α to resolve dHJ and prevent sequence exchange through resolution of this recombination intermediate [95]. The double-junction dissolution reaction requires the HRDC domain of BLM [24]. An additional protein, BLAP75/DMI1 mediates formation of the "dissolvasome" (BLM, Topo3α and BLAP75/RMI1) [130-132]. Mammalian BLAP18/RMI2 has also been found to be part of this complex [133].

#### **4.2. WRN**

Over 50 distinct *WRN* mutations have been reported, most of which lead to premature ter‐ mination of translation [19]. Recent missense mutations in the exonuclease domain in one patient compromised protein stability [134]. Most mutations in WS patients occur in the WRN C-terminal domain, which could disrupt the WRN/p53 interaction [20, 134]. Such pre‐ mature termination could also disrupt the Del hom sequence shown in Fig. 2. No WS muta‐ tions have been reported that eliminate only helicase or only exonuclease activity. Both activities are compromised in the development of WS [134]. WS fibroblasts undergo replica‐ tive senescence prematurely [135-139]. Telomere defects in WS cells suggest WRN activity in human telomere homeostasis [19]. Telomeres are needed to avoid loss of genetic material. They are important for chromosome end replication and for protection of the ends from en‐ zymatic attack [140]. Human telomeres contain 5 to 20 kb of the repetitive sequence TTAGGG [141]. At the terminal there are 100-200 bp of 3' ssDNA overhang. This overhang can anneal with telomere DNA to form a stable D-loop leading to a structure referred to as a 't-loop' [142, 143]. Alternatively, this free unannealed end may form G-quadruplex DNA [144]. Human WRN functions in lagging-strand synthesis, and in the replication of telomeric G-rich DNA ends [145]. *C. elegans* WRN-1 can disrupt D-loops [146] and human WRN can prevent aberrant recombination [147]. WRN 3' to 5' exonuclease is stimulated by the inter‐ acting Ku70/Ku86 complex supporting a role for WRN in DNA repair [148]. Evidence sug‐ gests that in the absence of telomerase, WRN and BLM have a role in the ALT (Alternate Lengthening of Telomeres) pathway for telomere maintenance [149, 150]. In biochemical ex‐ periments, WRN releases a 3' invading tail from a telomeric type D-loop by coordinated WRN helicase and exonuclease activities [149].

WRN and BLM catalytic activities are comparable except for 3' to 5' exonuclease activity of WRN [151-153], which BLM lacks. On dsDNA and on RNA-DNA hybrids, the WRN exonu‐ clease activity degrades a 3' recessed end. This activity can remove only one mismatched NT at the end of the recessed 3' DNA and can initiate exonuclease activity from a gap or nick [154, 155]. The exonuclease activity of WRN can degrade abnormal DNA structures suggest‐ ing that WRN helicase and exonuclease activities are involved in resolution of aberrant DNA structures at stalled forks [156]. Human WRN interacts with proteins involved in DNA replication. WRN coimmunoprecipitates with PCNA and topoisomerase 1 [157]. WRN functionally and physically interacts with RPA [117], and it functionally interacts with DNA polymerase delta [152]. WS cells accumulate recombination intermediates that impede cell growth [158]. In cells treated with HU, WRN colocalizes with RPA foci and is thought to dis‐ sociate recombination intermediates at the stalled forks [94, 147]. WRN stimulates polymer‐ ase delta activity in the absence of its processivity factor PCNA. This suggests a role for WRN in recruiting polymerase delta for replication restart at blocked or collapsed forks [152]. RPA can stimulate the processivity of WRN. The stimulation of WRN by RPA is due to protein-protein interactions as opposed to enhanced ATPase activity [117, 118].

resolution activity of BLM [127]. As helicases unwind duplex DNA, torsional stress pro‐ duced in the DNA may require relief through topoisomerase activity, and such activity fi‐ nally decatenates interlocked DNA molecules [128, 129]. *In vitro* studies show that BLM can partner with Topo3α to resolve dHJ and prevent sequence exchange through resolution of this recombination intermediate [95]. The double-junction dissolution reaction requires the HRDC domain of BLM [24]. An additional protein, BLAP75/DMI1 mediates formation of the "dissolvasome" (BLM, Topo3α and BLAP75/RMI1) [130-132]. Mammalian BLAP18/RMI2

Over 50 distinct *WRN* mutations have been reported, most of which lead to premature ter‐ mination of translation [19]. Recent missense mutations in the exonuclease domain in one patient compromised protein stability [134]. Most mutations in WS patients occur in the WRN C-terminal domain, which could disrupt the WRN/p53 interaction [20, 134]. Such pre‐ mature termination could also disrupt the Del hom sequence shown in Fig. 2. No WS muta‐ tions have been reported that eliminate only helicase or only exonuclease activity. Both activities are compromised in the development of WS [134]. WS fibroblasts undergo replica‐ tive senescence prematurely [135-139]. Telomere defects in WS cells suggest WRN activity in human telomere homeostasis [19]. Telomeres are needed to avoid loss of genetic material. They are important for chromosome end replication and for protection of the ends from en‐ zymatic attack [140]. Human telomeres contain 5 to 20 kb of the repetitive sequence TTAGGG [141]. At the terminal there are 100-200 bp of 3' ssDNA overhang. This overhang can anneal with telomere DNA to form a stable D-loop leading to a structure referred to as a 't-loop' [142, 143]. Alternatively, this free unannealed end may form G-quadruplex DNA [144]. Human WRN functions in lagging-strand synthesis, and in the replication of telomeric G-rich DNA ends [145]. *C. elegans* WRN-1 can disrupt D-loops [146] and human WRN can prevent aberrant recombination [147]. WRN 3' to 5' exonuclease is stimulated by the inter‐ acting Ku70/Ku86 complex supporting a role for WRN in DNA repair [148]. Evidence sug‐ gests that in the absence of telomerase, WRN and BLM have a role in the ALT (Alternate Lengthening of Telomeres) pathway for telomere maintenance [149, 150]. In biochemical ex‐ periments, WRN releases a 3' invading tail from a telomeric type D-loop by coordinated

WRN and BLM catalytic activities are comparable except for 3' to 5' exonuclease activity of WRN [151-153], which BLM lacks. On dsDNA and on RNA-DNA hybrids, the WRN exonu‐ clease activity degrades a 3' recessed end. This activity can remove only one mismatched NT at the end of the recessed 3' DNA and can initiate exonuclease activity from a gap or nick [154, 155]. The exonuclease activity of WRN can degrade abnormal DNA structures suggest‐ ing that WRN helicase and exonuclease activities are involved in resolution of aberrant DNA structures at stalled forks [156]. Human WRN interacts with proteins involved in DNA replication. WRN coimmunoprecipitates with PCNA and topoisomerase 1 [157]. WRN functionally and physically interacts with RPA [117], and it functionally interacts with DNA polymerase delta [152]. WS cells accumulate recombination intermediates that impede cell

has also been found to be part of this complex [133].

WRN helicase and exonuclease activities [149].

**4.2. WRN**

66 The Mechanisms of DNA Replication

### **5. Unification of BLM, WRN, RECQL4 and MCM2-7 activities in DNA replication and recombination/repair**

#### **5.1. BLM: Role in DNA damage response with a complex role in inhibiting or promoting HR**

BLM is found mostly in fine granules throughout the nucleoplasm at highest levels during S and G2 phases of the cell cycle. Its focal localization is in PML nuclear bodies (PML-NB). The name PML derives from the promyelocytic leukemia protein, PML [159-162]. This protein forms the structural groundwork of the PML bodies, which store various nuclear proteins [163]. These PML-NB store repair proteins (e.g., Topo3α, MRN and p53) and may be involved in sensing DNA damage [163]. By regulating the availability of repair proteins, response can be directed to DNA damage sites. Trafficking of proteins to the PML-NB is regulated by su‐ moylation [164]. The sumoylation pathway involves E1, E2 and E3 enzymes, which regulate respectively, SUMO activation, SUMO conjugation and targeting of specific substrates for su‐ moylation through ligation [165]. BLM contains a motif for SUMO binding that would facili‐ tate its integration into this repair protein storage network [166]. In addition, BLM is SUMO-1 and SUMO-2 modified [167]. When mutants are prepared in which the SUMO-binding se‐ quences are deleted, BLM cannot localize to PML-NB [168]. When mutants are prepared that do not allow BLM localization to PML-NB, there is about a two-fold increase in sister chroma‐ tid exchange. These findings indicate that there is a need for BLM-SUMO interaction in order for BLM to localize to PML-NB, and that BLM activity, such as its accumulation at stalled rep‐ lication forks, may be regulated by this specific localization [168].

At sites of DSB, repair foci form. A central player, H2AX, is phosphorylated when DSBs are induced, and this phosphorylation involves ATM, ATR and DNA-PK. Over one million bp are then marked by phosphorylated H2AX (γH2AX) on each side of the break [112, 168-171]. γH2AX recruits additional repair proteins to the damage site [172]. In studies where normal S-phase cells are treated with DNA damaging agents (HU, UV and cross-linking agents), BLM responds by leaving the PML-NBs to relocate to repair foci and colocalize with the marker γH2AX [101, 173]. BLM interacts physically and functionally with γH2AX as well as with ATM and ATR [103, 173-175]. In damage that is S-phase specific, BLM associates with the complex ATR/CHK1/53BP1, which gathers at repair foci as an early response to the damage. Based on kinetic studies, BLM may facilitate BRCA1 and MRN complex localization at repair foci in S phase [173], which may involve BLM regulation of p53 in these foci [101]. This early function of BLM at repair foci may allow for BML to influence the choice of repair pathways and facilitate a BLM function in anti-recombination at stalled forks that perhaps involves SU‐ MO regulation [19].

As discussed in the previous section, BLM interacts with the recombinase, Rad51. Rad51 cat‐ alyzes the pairing of a ssDNA tail and a homologous stretch of dsDNA to promote strand exchange early in the HR pathway [111]. Following ionizing radiation, Rad51 foci contained BLM [159]. Rad51 functions in HR, and localizes to ssDNA when DSB are induced [176]. BML can displace the recombinase from the ssDNA filament [112], which can be viewed as an anti-recombinagenic function [19]. In Rad51-associated D-loops, BLM can interact with Rad51 and unwind DNA in front of the polymerase [177]. This could favor SDSA leading to pro-recombinagenic function [19, 177]. BLM, however, also functions in resolution of Gquadruplex DNA structure and has higher binding affinity for it compared to HJ. BLM heli‐ case activity is required for resolution of this structure [92]. Whereas the BLM/Rad51 interaction would represent an early event in a DNA damage/repair process, the formation of HJ, on the other hand, is a late event in HR. As discussed in section 5.1, the BLM-Topo3α-BLAP75/RMI1 complex functions to "dissolve" dHJ by convergent fork migration to gener‐ ate non-cross-over products [178]. This is facilitated by Topo3α relief of superhelicity and by its ability to cleave and rejoin one strand of a DNA duplex. In the absence of BLM, Topo3α activity would involve break and rejoining activities instead of dissolution, which could lead to crossover events and an increase in sister chromatid exchange in BS cells [19]. BLM that is mutated to be unable to interact with Topo3α can only partially rescue the frequency of sister chromatid exchange [168]. Thus, together in a complex, BLM and Topo3α achieve dissolution of a recombinogenic intermediate. BLM has been proposed to regulate ploidy based on a role along with other members of this complex in resolution of anaphase bridges [126]. BLM has been found in complexes with mismatch repair protein MLH1 [179, 180]. It has been found to be present in large complexes containing not only BLM-Topo3a-BLAF75, but also additional factors. These additional factors could include several BLAF factors, pro‐ teins from the FA (Fanconi anemia) pathway, RPA, and mismatch repair protein MLH1. [181, 182]. The interactive role of these pathway components remains to be determined.

#### **5.2. WRN: Helicase and exonuclease activities in concert**

When WRN biochemical activities were compared using nonhydrolyzable ATPγS to inhibit only WRN helicase activity or aa substitutions to eliminate only the WRN exonuclease activi‐ ty, the exonuclease activity was shown to function in degradation of the leading strand on replication fork-type substrates and in degradation of the annealed telomere overhangs on substrates resembling D-loop structures. WRN binding proteins were inhibitory [149, 183]. In addition, WRN was found to degrade ssDNA substrates longer than 40 nt with dependence upon the helicase activity [183]. WRN activities were also explored using WS fibroblasts. In WS cells, WRN and the enzyme telomerase are able to reverse the phenotype of excess chro‐ mosome fusion. In these cells, the anaphase bridges were missing telomere DNA. Dominant negative telomerase was not able to rescue the phenotype indicating that a stable telomere length was needed for rescue [184]. Telomeres are stabilized by a complex of proteins that bind DNA, known as the shelterin complex [185]. This complex consists of TRF1, TRF2 (dou‐ ble-strand DNA binding proteins), and POT1 (single-strand DNA binding protein) as well as adaptor proteins. The coordinate action of these proteins in the presence of telomerase is needed to regulate telomere length. Without telomerase, telomeres shorten in length each cell division [185]. In S phase, WRN colocalizes with TRF2 [145, 186]. TRF1 and TRF2 limit WRN exonuclease activity on synthetic telomere D-loops [149]. WRN helicase is stimulated by sin‐ gle-stranded DNA binding proteins, RPA or POT1. These proteins modulate WRN exonu‐ clease degradation of the 3' overhang [149, 187]. The hypothesis is that WRN could unwind D-loops to facilitate leading strand synthesis through telomeric DNA, and there is also the possibility that WRN prevents interchromosomal interactions between telomeres [19].

#### *5.2.1. WRN activities in response to G-quadruplex structures in the lagging strand*

and facilitate a BLM function in anti-recombination at stalled forks that perhaps involves SU‐

As discussed in the previous section, BLM interacts with the recombinase, Rad51. Rad51 cat‐ alyzes the pairing of a ssDNA tail and a homologous stretch of dsDNA to promote strand exchange early in the HR pathway [111]. Following ionizing radiation, Rad51 foci contained BLM [159]. Rad51 functions in HR, and localizes to ssDNA when DSB are induced [176]. BML can displace the recombinase from the ssDNA filament [112], which can be viewed as an anti-recombinagenic function [19]. In Rad51-associated D-loops, BLM can interact with Rad51 and unwind DNA in front of the polymerase [177]. This could favor SDSA leading to pro-recombinagenic function [19, 177]. BLM, however, also functions in resolution of Gquadruplex DNA structure and has higher binding affinity for it compared to HJ. BLM heli‐ case activity is required for resolution of this structure [92]. Whereas the BLM/Rad51 interaction would represent an early event in a DNA damage/repair process, the formation of HJ, on the other hand, is a late event in HR. As discussed in section 5.1, the BLM-Topo3α-BLAP75/RMI1 complex functions to "dissolve" dHJ by convergent fork migration to gener‐ ate non-cross-over products [178]. This is facilitated by Topo3α relief of superhelicity and by its ability to cleave and rejoin one strand of a DNA duplex. In the absence of BLM, Topo3α activity would involve break and rejoining activities instead of dissolution, which could lead to crossover events and an increase in sister chromatid exchange in BS cells [19]. BLM that is mutated to be unable to interact with Topo3α can only partially rescue the frequency of sister chromatid exchange [168]. Thus, together in a complex, BLM and Topo3α achieve dissolution of a recombinogenic intermediate. BLM has been proposed to regulate ploidy based on a role along with other members of this complex in resolution of anaphase bridges [126]. BLM has been found in complexes with mismatch repair protein MLH1 [179, 180]. It has been found to be present in large complexes containing not only BLM-Topo3a-BLAF75, but also additional factors. These additional factors could include several BLAF factors, pro‐ teins from the FA (Fanconi anemia) pathway, RPA, and mismatch repair protein MLH1. [181, 182]. The interactive role of these pathway components remains to be determined.

When WRN biochemical activities were compared using nonhydrolyzable ATPγS to inhibit only WRN helicase activity or aa substitutions to eliminate only the WRN exonuclease activi‐ ty, the exonuclease activity was shown to function in degradation of the leading strand on replication fork-type substrates and in degradation of the annealed telomere overhangs on substrates resembling D-loop structures. WRN binding proteins were inhibitory [149, 183]. In addition, WRN was found to degrade ssDNA substrates longer than 40 nt with dependence upon the helicase activity [183]. WRN activities were also explored using WS fibroblasts. In WS cells, WRN and the enzyme telomerase are able to reverse the phenotype of excess chro‐ mosome fusion. In these cells, the anaphase bridges were missing telomere DNA. Dominant negative telomerase was not able to rescue the phenotype indicating that a stable telomere length was needed for rescue [184]. Telomeres are stabilized by a complex of proteins that bind DNA, known as the shelterin complex [185]. This complex consists of TRF1, TRF2 (dou‐ ble-strand DNA binding proteins), and POT1 (single-strand DNA binding protein) as well as

**5.2. WRN: Helicase and exonuclease activities in concert**

MO regulation [19].

68 The Mechanisms of DNA Replication

At the telomere, the G-rich strand is duplicated by lagging-strand synthesis. It may assume a G-quadruplex structure, which would interrupt the replication fork. Experimentally, when inhibiting WRN by use of overexpression of a dominant-negative WRN, the helicase defi‐ ciency leads to loss almost entirely affecting the sister telomere on the lagging strand [145]. WRN interaction with FEN-1 (the flap endonuclease involved in the processing of Okazaki fragments, [188]) could assist in the maturation of these fragments [105, 189]. Data suggest that WRN may function in concert with lagging strand synthesis perhaps in unwinding complex structure at the telomere. WRN interacts physically with DNA polymerase delta to stimulate its activity [190]. WRN can prevent stalling of polymerases delta at telomere se‐ quence *in vitro* [191]. WRN stimulation of polymerase delta happens only in the absence of PCNA, which suggests that WRN has a role at stalled forks rather than in the regulation of processive DNA synthesis [19]. WRN, as opposed to BLM, is specific for G-quadruplex structure in the trinucleotide repeat of Fragile X syndrome [192].

#### *5.2.2. WRN and Ku in suppression of aberrant recombination at telomeres*

Proteins involved in the NHEJ path for repair of DSBs that are found at telomeres include Ku heterodimer, DNA-PK, MRN complex and ATM [193]. Through interaction with NBS1, WRN colocalizes with two of these components, Ku and the MRN complex [194]. Under un‐ stable conditions, Ku can suppress sister chromatid exchange at telomeres [195]. Mouse knockout studies show that WRN normally suppresses aberrant recombination at telomeres [196].

#### *5.2.3. WRN activities in base excision repair and interstrand cross link repair by HR*

WS cells show increased sensitivity to alkylating agents and to agents that increase ROS. Human fibroblasts in which WRN has been knocked down respond to oxidative stress with an increase in DNA damage [197]. These observations support a role for WRN in repair of such damage [198]. DNA damage resulting from oxidation, alkylation, methylation and de‐ amination is repaired by the base excision repair (BER) pathway. WRN interacts with pro‐ teins in this pathway. In addition, WRN helicase activity stimulates and is stimulated by polymerase beta activity [199]. There is evidence that WRN activity in BER is regulated by PARP-1, but the reader is referred to references [200, 201]. BRCA1 and BRCA2 play an im‐ portant role in DSB repair in the HR subpathway [202, 203]. HR is a pathway for repairing interstrand crosslinks (ICL). Cells deficient in either WRN or BRCA1 are hypersensitive to induction of ICLs [204, 205]. A physical interaction of WRN with BRCA1 enhances the activ‐ ities of WRN [206]. In addition, data from WRN and/or BRCA1 knockdown studies indicate that BRCA1 may act cooperatively with WRN in HR during ICL repair [206].

#### **5.3. RECQL4**

Data from *Recql4-/-* mice indicate a role for mouse RECQL4 in genomic stability and in the promoting cohesion between sister chromatids [207]. Depleting the *X. laevis* homologue of RTS, xRTS, in *Xenopus* egg extracts led to reduced DNA synthesis and an inhibition of RPA stabilization of ssDNA prior to polymerase loading at unwound origins. The addition of pu‐ rified human RECQL4 could reverse this effect [208]. A nonhelical region in the N terminus of xRTS could be important in initiation of DNA replication based on its interaction with the Cut5 protein and the importance of xRTS in loading DNA polymerase alpha onto chromatin [209]. The N terminus of xRTS is not homologous to that of RECQL4 in mammals, however, and this role may not be conserved [19]. RECQL4, along with Ctf4 and MCM10, has been shown to be required for stable association of the CMG complex in human cells. In this study, Cut5/TopBP1 was not required for CMG stabilization [210]. In the *Xenopus* replication model, RECQL4 binds to chromatin that has been processed to resemble DSBs with a de‐ pendence on RPA and ATM activity [211]. Chromatin immunoprecipitation experiments show that RECQL4 functions on DNA in close association with Ku [212]. In HeLa cells hu‐ man RECQL4 forms a complex with Rad51 and colocalizes with Rad51 foci formed after treatment with etoposide [212]. PML-NB contain a portion of RECQL4 [212]. It is also found in the nucleolus [213]. When using a T7 phage display screen, RECQL4 was found to inter‐ act with PARP-1, and the association influenced the nuclear localization of RECQL4. PARP-1 has a role in RECQL4 movement to the nucleolus from the nucleoplasm. When oxi‐ dative stress is induced, as opposed to other types of damage induction, RECQL4 increas‐ ingly localizes in the nucleolus. This trafficking is inhibited by inhibition of PARP [213]. RTS cells decrease proliferation and synthesis of DNA when exposed to hydrogen peroxide [214]. Lack of proper response to ROS could lead to premature aging as see in RTS. PARP is also involved in an end-joining pathway of DNA repair [215, 216]. The role of RECQL4 in its interaction with PARP-1 is not known (for further discussion see reference [19]).

#### **5.4. MCM2-7**

Replication fork stalling, can lead to DSB and chromosomal rearrangements. An S phase checkpoint is triggered by these events and there is a block to elongation. When this occurs, the proteins Mrc1, Tof1, and Csm3 (M/T/C complex) interact with the MCM2-7 complex to stabilize the replication fork. When the M/T/C complex is missing, the replisome continues, but synthesis stops. This may be partially due to loss of DNA polymerase epsilon from the fork [217-219]. Studies in yeast provide insight into these activities. The M/T/C complex as‐ sociates with MCM2-7 [218, 220-222] and also with polymerase epsilon [221, 223]. These physical interactions permit communication between polymerase epsilon and the MCM complex [223]. The M/T/C complex may be part of the normal replication fork protein en‐ tourage [218, 220, 221, 224]. Mrc1 and Cdc45 coimmunoprecipitate, indicating an interaction of Mrc1 with the The Replication Progression Complex core, which includes Cdc45, the MCMs and GINS [223]. In each cell cycle Mrc loads onto replication origins along with the polymerases. This occurs after the Replication Progression Complex forms. Mrc migrates with the replication forks. [218, 219, 224-226]. Tof1, like Mrc, also coimmunoprecipitates with Cdc45 [227]. The exact mechanism of action of the M/T/C complex is not known. In a yeast study, the Tof1 homologue could switch regulation between pro- and anti-recombina‐ tion activities in a site-specific manner [228]. Data indicate that a Mrc1 and MCM6-C termi‐ nal interaction senses alkylated DNA damage [221]. The other two subunits Tof1 and Csm3, may function to sense other types of damage. Although the helicase domains of MCM2-7 are conserved, the N and C terminals are divergent. Other negative regulators could differen‐ tially bind to these regions to regulate powering of elongation by the MCM2-7 helicase dur‐ ing times of stress [10]. A future question relates to the extent to which leading or lagging strand polymerase arrest is associated with formation of ssDNA, fork regression and forma‐ tion of abnormal DNA structures [66]. These data indicate a functional connection between the MCM proteins, which act at stalled forks, and the RecQ proteins, which facilitate repair of the resulting damage.

### **6. Summary**

induction of ICLs [204, 205]. A physical interaction of WRN with BRCA1 enhances the activ‐ ities of WRN [206]. In addition, data from WRN and/or BRCA1 knockdown studies indicate

Data from *Recql4-/-* mice indicate a role for mouse RECQL4 in genomic stability and in the promoting cohesion between sister chromatids [207]. Depleting the *X. laevis* homologue of RTS, xRTS, in *Xenopus* egg extracts led to reduced DNA synthesis and an inhibition of RPA stabilization of ssDNA prior to polymerase loading at unwound origins. The addition of pu‐ rified human RECQL4 could reverse this effect [208]. A nonhelical region in the N terminus of xRTS could be important in initiation of DNA replication based on its interaction with the Cut5 protein and the importance of xRTS in loading DNA polymerase alpha onto chromatin [209]. The N terminus of xRTS is not homologous to that of RECQL4 in mammals, however, and this role may not be conserved [19]. RECQL4, along with Ctf4 and MCM10, has been shown to be required for stable association of the CMG complex in human cells. In this study, Cut5/TopBP1 was not required for CMG stabilization [210]. In the *Xenopus* replication model, RECQL4 binds to chromatin that has been processed to resemble DSBs with a de‐ pendence on RPA and ATM activity [211]. Chromatin immunoprecipitation experiments show that RECQL4 functions on DNA in close association with Ku [212]. In HeLa cells hu‐ man RECQL4 forms a complex with Rad51 and colocalizes with Rad51 foci formed after treatment with etoposide [212]. PML-NB contain a portion of RECQL4 [212]. It is also found in the nucleolus [213]. When using a T7 phage display screen, RECQL4 was found to inter‐ act with PARP-1, and the association influenced the nuclear localization of RECQL4. PARP-1 has a role in RECQL4 movement to the nucleolus from the nucleoplasm. When oxi‐ dative stress is induced, as opposed to other types of damage induction, RECQL4 increas‐ ingly localizes in the nucleolus. This trafficking is inhibited by inhibition of PARP [213]. RTS cells decrease proliferation and synthesis of DNA when exposed to hydrogen peroxide [214]. Lack of proper response to ROS could lead to premature aging as see in RTS. PARP is also involved in an end-joining pathway of DNA repair [215, 216]. The role of RECQL4 in its

that BRCA1 may act cooperatively with WRN in HR during ICL repair [206].

interaction with PARP-1 is not known (for further discussion see reference [19]).

Replication fork stalling, can lead to DSB and chromosomal rearrangements. An S phase checkpoint is triggered by these events and there is a block to elongation. When this occurs, the proteins Mrc1, Tof1, and Csm3 (M/T/C complex) interact with the MCM2-7 complex to stabilize the replication fork. When the M/T/C complex is missing, the replisome continues, but synthesis stops. This may be partially due to loss of DNA polymerase epsilon from the fork [217-219]. Studies in yeast provide insight into these activities. The M/T/C complex as‐ sociates with MCM2-7 [218, 220-222] and also with polymerase epsilon [221, 223]. These physical interactions permit communication between polymerase epsilon and the MCM complex [223]. The M/T/C complex may be part of the normal replication fork protein en‐ tourage [218, 220, 221, 224]. Mrc1 and Cdc45 coimmunoprecipitate, indicating an interaction of Mrc1 with the The Replication Progression Complex core, which includes Cdc45, the MCMs and GINS [223]. In each cell cycle Mrc loads onto replication origins along with the

**5.3. RECQL4**

70 The Mechanisms of DNA Replication

**5.4. MCM2-7**

BLM, WRN and RECQL4 act during events that stress the advancing replication fork pro‐ viding relief through DNA damage repair and through resolution of aberrant replication/ recombination intermediates, including those present at the telomere. At checkpoint, the replication proteins at a stalled fork are held stable through communication that occurs due to proteins that bind and signal to both the MCM complex and polymerase(s). This would allow repair proteins such as WRN and BLM helicases and RECQL4 to resolve the stress and thus aid in fork restart. Advancing our knowledge of the RecQ and MCM family activi‐ ties and the mechanisms and signalling behind these activities will increase our understand‐ ing of cancer and aging and perhaps enlighten us regarding how to accommodate these challenges to human health.

### **Acknowledgments**

This. work was supported by NIH funding to EMJ and resources derived from grant fund‐ ing from Virginia's Commonwealth Health Research Board to DCD.

### **Author details**

Dianne C. Daniel\*, Ayuna V. Dagdanova and Edward M. Johnson

\*Address all correspondence to: danieldc@evms.edu

Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia, USA

### **References**


[15] Fien K, Cho YS, Lee JK, Raychaudhuri S, Tappin I, Hurwitz J. Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p. J Biol Chem. 2004 Apr 16;279(16):16144-53. 14766746

**References**

72 The Mechanisms of DNA Replication

888- e1. 18775318

11257107

nu Rev Genet. 2010;44:393-417. 21047263

life. Nat Struct Biol. 2003 Mar;10(3):148-50. 12605215

[1] Berger JM. SnapShot: nucleic acid helicases and translocases. Cell. 2008 Sep 5;134(5):

[2] Bernstein KA, Gangloff S, Rothstein R. The RecQ DNA helicases in DNA repair. An‐

[3] Kelman Z, Hurwitz J. Structural lessons in DNA replication from the third domain of

[4] Erzberger JP, Berger JM. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu Rev Biophys Biomol Struct. 2006;35:93-114. 16689629

[5] Koonin EV. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eu‐ karyotic DNA replication. Nucleic Acids Res. 1993 Jun 11;21(11):2541-7. 8332451

[6] Iyer LM, Leipe DD, Koonin EV, Aravind L. Evolutionary history and higher order classification of AAA+ ATPases. J Struct Biol. 2004 Apr-May;146(1-2):11-31. 15037234

[7] Leipe DD, Koonin EV, Aravind L. Evolution and classification of P-loop kinases and

[8] Mohaghegh P, Hickson ID. DNA helicase deficiencies associated with cancer predis‐ position and premature ageing disorders. Hum Mol Genet. 2001 Apr;10(7):741-6.

[9] Brosh RM, Jr., Bohr VA. Human premature aging, DNA repair and RecQ helicases.

[10] Bochman ML, Schwacha A. The Mcm complex: unwinding the mechanism of a repli‐

[11] Gozuacik D, Chami M, Lagorce D, Faivre J, Murakami Y, Poch O, Biermann E, Knip‐ pers R, Brechot C, Paterlini-Brechot P. Identification and functional characterization of a new member of the human Mcm protein family: hMcm8. Nucleic Acids Res.

[12] Johnson EM, Kinoshita Y, Daniel DC. A new member of the MCM protein family en‐ coded by the human MCM8 gene, located contrapodal to GCD10 at chromosome

[13] Lutzmann M, Maiorano D, Mechali M. Identification of full genes and proteins of MCM9, a novel, vertebrate-specific member of the MCM2-8 protein family. Gene.

[14] Yoshida K. Identification of a novel cell-cycle-induced MCM family protein MCM9.

cative helicase. Microbiol Mol Biol Rev. 2009 Dec;73(4):652-83. 19946136

band 20p12.3-13. Nucleic Acids Res. 2003 Jun 1;31(11):2915-25. 12771218

Biochem Biophys Res Commun. 2005 Jun 3;331(2):669-74. 15850810

related proteins. J Mol Biol. 2003 Oct 31;333(4):781-815. 14568537

Nucleic Acids Res. 2007;35(22):7527-44. 18006573

2003 Jan 15;31(2):570-9. 12527764

2005 Dec 5;362:51-6. 16226853


[44] Vasseur F, Delaporte E, Zabot MT, Sturque MN, Barrut D, Savary JB, Thomas L, Tho‐ mas P. Excision repair defect in Rothmund Thomson syndrome. Acta Derm Venere‐ ol. 1999 Mar;79(2):150-2. 10228638

[30] Ogura T, Wilkinson AJ. AAA+ superfamily ATPases: common structure--diverse

[31] Saraste M, Sibbald PR, Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430-4. 2126155

[32] Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the al‐ pha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Embo J. 1982;1(8):945-51. 6329717

[33] Davey MJ, Indiani C, O'Donnell M. Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture. J Biol Chem. 2003 Feb 14;278(7):

[34] Schwacha A, Bell SP. Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. Mol Cell. 2001

[35] Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, Martin GM, Mulligan J, Schellenberg GD. Positional cloning of the

[36] Bloom D. Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs; probably a syndrome entity. AMA Am J Dis Child. 1954 Dec;88(6):754-8.

[37] Kitao S, Lindor NM, Shiratori M, Furuichi Y, Shimamoto A. Rothmund-thomson syn‐ drome responsible gene, RECQL4: genomic structure and products. Genomics. 1999

[38] Siitonen HA, Sotkasiira J, Biervliet M, Benmansour A, Capri Y, Cormier-Daire V, Crandall B, Hannula-Jouppi K, Hennekam R, Herzog D, Keymolen K, Lipsanen-Ny‐ man M, Miny P, Plon SE, Riedl S, Sarkar A, Vargas FR, Verloes A, Wang LL, Kaariai‐ nen H, Kestila M. The mutation spectrum in RECQL4 diseases. Eur J Hum Genet.

[39] Hanada K, Hickson ID. Molecular genetics of RecQ helicase disorders. Cell Mol Life

[40] Karow JK, Wu L, Hickson ID. RecQ family helicases: roles in cancer and aging. Curr

[41] German J. Bloom syndrome: a mendelian prototype of somatic mutational disease.

[43] Shen JC, Loeb LA. The Werner syndrome gene: the molecular basis of RecQ helicase-

[42] German J. Bloom's syndrome. Dermatol Clin. 1995 Jan;13(1):7-18. 7712653

deficiency diseases. Trends Genet. 2000 May;16(5):213-20. 10782115

Werner's syndrome gene. Science. 1996 Apr 12;272(5259):258-62. 8602509

function. Genes Cells. 2001 Jul;6(7):575-97. 11473577

4491-9. 12480933

74 The Mechanisms of DNA Replication

13206391

Nov;8(5):1093-104. 11741544

Nov 1;61(3):268-76. 10552928

2009 Feb;17(2):151-8. 18716613

Sci. 2007 Sep;64(17):2306-22. 17571213

Opin Genet Dev. 2000 Feb;10(1):32-8. 10679384

Medicine (Baltimore). 1993 Nov;72(6):393-406. 8231788


ing circle DNA synthesis. Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):6042-7. 22474384


[71] Woodward AM, Gohler T, Luciani MG, Oehlmann M, Ge X, Gartner A, Jackson DA, Blow JJ. Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. J Cell Biol. 2006 Jun 5;173(5):673-83. 16754955

ing circle DNA synthesis. Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):6042-7.

[59] Kinoshita Y, Johnson EM. Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle. J

[60] Schepers A, Diffley JF. Mutational analysis of conserved sequence motifs in the bud‐ ding yeast Cdc6 protein. J Mol Biol. 2001 May 11;308(4):597-608. 11350163

[61] Nevis KR, Cordeiro-Stone M, Cook JG. Origin licensing and p53 status regulate Cdk2

[62] Coverley D, Pelizon C, Trewick S, Laskey RA. Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 de‐

[63] Alexandrow MG, Hamlin JL. Cdc6 chromatin affinity is unaffected by serine-54 phosphorylation, S-phase progression, and overexpression of cyclin A. Mol Cell Biol.

[64] Shin JH, Grabowski B, Kasiviswanathan R, Bell SD, Kelman Z. Regulation of mini‐ chromosome maintenance helicase activity by Cdc6. J Biol Chem. 2003 Sep

[65] Daniel DC, Johnson EM. Addressing the enigma of MCM8 in DNA replication. In: Kušić-Tišma J, ed. Fundamental Aspects of DNA Replication: INTECH 2011:37-52. DOI: 10.5772/21177. http://www.intechopen.com/books/fundamental-aspects-of-dna-

[66] Forsburg SL. The MCM helicase: linking checkpoints to the replication fork. Biochem

[67] Masai H, You Z, Arai K. Control of DNA replication: regulation and activation of eu‐ karyotic replicative helicase, MCM. IUBMB Life. 2005 Apr-May;57(4-5):323-35.

[68] Costa A, Onesti S. The MCM complex: (just) a replicative helicase? Biochem Soc

[69] Brewster AS, Wang G, Yu X, Greenleaf WB, Carazo JM, Tjajadi M, Klein MG, Chen XS. Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase. Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20191-6.

[70] Makarova KS, Koonin EV, Kelman Z. The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukar‐

activity during G(1). Cell Cycle. 2009 Jun 15;8(12):1952-63. 19440053

pendent process. J Cell Sci. 2000 Jun;113 ( Pt 11):1929-38. 10806104

replication/addressing-the-enigma-of-mcm8-in-dna-replication

Soc Trans. 2008 Feb;36(Pt 1):114-9. 18208397

Trans. 2008 Feb;36(Pt 1):136-40. 18208401

yotes. Biol Direct. 2012;7:7. 22329974

Biol Chem. 2004 Aug 20;279(34):35879-89. 15190069

2004 Feb;24(4):1614-27. 14749377

26;278(39):38059-67. 12837750

16036617

19073923

22474384

76 The Mechanisms of DNA Replication


[96] Hand R, German J. A retarded rate of DNA chain growth in Bloom's syndrome. Proc Natl Acad Sci U S A. 1975 Feb;72(2):758-62. 1054854

[83] Freeman A, Morris LS, Mills AD, Stoeber K, Laskey RA, Williams GH, Coleman N. Minichromosome maintenance proteins as biological markers of dysplasia and ma‐

[84] Gonzalez MA, Pinder SE, Callagy G, Vowler SL, Morris LS, Bird K, Bell JA, Laskey RA, Coleman N. Minichromosome maintenance protein 2 is a strong independent prognostic marker in breast cancer. J Clin Oncol. 2003 Dec 1;21(23):4306-13. 14645419

[85] Hunt DP, Freeman A, Morris LS, Burnet NG, Bird K, Davies TW, Laskey RA, Cole‐ man N. Early recurrence of benign meningioma correlates with expression of minichromosome maintenance-2 protein. Br J Neurosurg. 2002 Feb;16(1):10-5. 11928726

[86] Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, John‐ son EM, Daniel DC, Kinoshita Y, Amini S, Gordon J. Puralpha Is Essential for Postna‐ tal Brain Development and Developmentally Coupled Cellular Proliferation As Revealed by Genetic Inactivation in the Mouse. Mol Cell Biol. 2003 Oct 1;23(19):

[87] Bergemann AD, Johnson EM. The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

[88] Liu W, Pucci B, Rossi M, Pisani FM, Ladenstein R. Structural analysis of the Sulfolo‐ bus solfataricus MCM protein N-terminal domain. Nucleic Acids Res. 2008 Jun;

[89] Rong SB, Valiaho J, Vihinen M. Structural basis of Bloom syndrome (BS) causing mu‐ tations in the BLM helicase domain. Mol Med. 2000 Mar;6(3):155-64. 10965492

[90] Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr

[91] Sun H, Karow JK, Hickson ID, Maizels N. The Bloom's syndrome helicase unwinds

[92] Huber MD, Lee DC, Maizels N. G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition. Nucleic Acids Res. 2002 Sep 15;30(18):

[93] van Brabant AJ, Ye T, Sanz M, German IJ, Ellis NA, Holloman WK. Binding and melting of D-loops by the Bloom syndrome helicase. Biochemistry. 2000 Nov

[94] Karow JK, Constantinou A, Li JL, West SC, Hickson ID. The Bloom's syndrome gene product promotes branch migration of holliday junctions. Proc Natl Acad Sci U S A.

[95] Wu L, Hickson ID. The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature. 2003 Dec 18;426(6968):870-4. 14685245

G4 DNA. J Biol Chem. 1998 Oct 16;273(42):27587-92. 9765292

lignancy. Clin Cancer Res. 1999;5(8):2121-32.

6857-75. 12972605

78 The Mechanisms of DNA Replication

Mol Cell Biol. 1992;12:1257-65.

36(10):3235-43. 18417534

22;362(6422):709-15. 8469282

28;39(47):14617-25. 11087418

2000 Jun 6;97(12):6504-8. 10823897

3954-61. 12235379


together process branchmigrating DNA structures associated with the replication fork. Mol Biol Cell. 2004 Feb;15(2):734-50. 14657243


[121] Wang XW, Tseng A, Ellis NA, Spillare EA, Linke SP, Robles AI, Seker H, Yang Q, Hu P, Beresten S, Bemmels NA, Garfield S, Harris CC. Functional interaction of p53 and BLM DNA helicase in apoptosis. J Biol Chem. 2001 Aug 31;276(35):32948-55. 11399766

together process branchmigrating DNA structures associated with the replication

[109] Sharma S, Sommers JA, Gary RK, Friedrich-Heineken E, Hubscher U, Brosh RM, Jr. The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordina‐

[110] Sharma S, Sommers JA, Wu L, Bohr VA, Hickson ID, Brosh RM, Jr. Stimulation of flap endonuclease-1 by the Bloom's syndrome protein. J Biol Chem. 2004 Mar

[111] Bachrati CZ, Borts RH, Hickson ID. Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. Nucleic Acids Res. 2006;34(8):2269-79. 16670433

[112] Bugreev DV, Yu X, Egelman EH, Mazin AV. Novel pro- and anti-recombination ac‐ tivities of the Bloom's syndrome helicase. Genes Dev. 2007 Dec 1;21(23):3085-94.

[113] Bugreev DV, Brosh RM, Jr., Mazin AV. RECQ1 possesses DNA branch migration ac‐

[114] Jiao R, Bachrati CZ, Pedrazzi G, Kuster P, Petkovic M, Li JL, Egli D, Hickson ID, Stagljar I. Physical and functional interaction between the Bloom's syndrome gene product and the largest subunit of chromatin assembly factor 1. Mol Cell Biol. 2004

[115] von Kobbe C, Karmakar P, Dawut L, Opresko P, Zeng X, Brosh RM, Jr., Hickson ID, Bohr VA. Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins. J Biol Chem. 2002 Jun 14;277(24):22035-44. 11919194 [116] Brosh RM, Jr., Li JL, Kenny MK, Karow JK, Cooper MP, Kureekattil RP, Hickson ID, Bohr VA. Replication protein A physically interacts with the Bloom's syndrome pro‐ tein and stimulates its helicase activity. J Biol Chem. 2000 Aug 4;275(31):23500-8.

[117] Brosh RM, Jr., Orren DK, Nehlin JO, Ravn PH, Kenny MK, Machwe A, Bohr VA. Functional and physical interaction between WRN helicase and human replication

[118] Shen JC, Gray MD, Oshima J, Loeb LA. Characterization of Werner syndrome pro‐ tein DNA helicase activity: directionality, substrate dependence and stimulation by

[119] Blander G, Kipnis J, Leal JF, Yu CE, Schellenberg GD, Oren M. Physical and function‐ al interaction between p53 and the Werner's syndrome protein. J Biol Chem. 1999 Oct

[120] Spillare EA, Robles AI, Wang XW, Shen JC, Yu CE, Schellenberg GD, Harris CC. p53 mediated apoptosis is attenuated in Werner syndrome cells. Genes Dev. 1999 Jun

replication protein A. Nucleic Acids Res. 1998 Jun 15;26(12):2879-85. 9611231

protein A. J Biol Chem. 1999 Jun 25;274(26):18341-50. 10373438

tivity. J Biol Chem. 2008 Jul 18;283(29):20231-42. 18495662

tion of WRN and PCNA. Nucleic Acids Res. 2005;33(21):6769-81. 16326861

fork. Mol Biol Cell. 2004 Feb;15(2):734-50. 14657243

12;279(11):9847-56. 14688284

Jun;24(11):4710-9. 15143166

8;274(41):29463-9. 10506209

1;13(11):1355-60. 10364153

18003860

80 The Mechanisms of DNA Replication

10825162


[145] Crabbe L, Verdun RE, Haggblom CI, Karlseder J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science. 2004 Dec 10;306(5703): 1951-3. 15591207

[133] Singh TR, Ali AM, Busygina V, Raynard S, Fan Q, Du CH, Andreassen PR, Sung P, Meetei AR. BLAP18/RMI2, a novel OB-fold-containing protein, is an essential compo‐ nent of the Bloom helicase-double Holliday junction dissolvasome. Genes Dev. 2008

[134] Huang S, Lee L, Hanson NB, Lenaerts C, Hoehn H, Poot M, Rubin CD, Chen DF, Yang CC, Juch H, Dorn T, Spiegel R, Oral EA, Abid M, Battisti C, Lucci-Cordisco E, Neri G, Steed EH, Kidd A, Isley W, Showalter D, Vittone JL, Konstantinow A, Ring J, Meyer P, Wenger SL, von Herbay A, Wollina U, Schuelke M, Huizenga CR, Leistritz DF, Martin GM, Mian IS, Oshima J. The spectrum of WRN mutations in Werner syn‐

[135] Davis T, Singhrao SK, Wyllie FS, Haughton MF, Smith PJ, Wiltshire M, Wynford-Thomas D, Jones CJ, Faragher RG, Kipling D. Telomere-based proliferative lifespan barriers in Werner-syndrome fibroblasts involve both p53-dependent and p53-inde‐

[136] Epstein CJ, Martin GM, Schultz AL, Motulsky AG. Werner's syndrome a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine (Baltimore). 1966 May;45(3):177-221. 5327241 [137] Faragher RG, Kill IR, Hunter JA, Pope FM, Tannock C, Shall S. The gene responsible for Werner syndrome may be a cell division "counting" gene. Proc Natl Acad Sci U S

[138] Schulz VP, Zakian VA, Ogburn CE, McKay J, Jarzebowicz AA, Edland SD, Martin GM. Accelerated loss of telomeric repeats may not explain accelerated replicative de‐

[139] Tahara H, Tokutake Y, Maeda S, Kataoka H, Watanabe T, Satoh M, Matsumoto T, Su‐ gawara M, Ide T, Goto M, Furuichi Y, Sugimoto M. Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Ep‐

[140] Sandell LL, Zakian VA. Loss of a yeast telomere: arrest, recovery, and chromosome

[141] Nurnberg P, Thiel G, Weber F, Epplen JT. Changes of telomere lengths in human in‐

[142] Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T. Mammalian telomeres end in a large duplex loop. Cell. 1999 May 14;97(4):503-14.

[143] Murti KG, Prescott DM. Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops. Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14436-9.

[144] Williamson JR, Raghuraman MK, Cech TR. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell. 1989 Dec 1;59(5):871-80. 2590943

cline of Werner syndrome cells. Hum Genet. 1996 Jun;97(6):750-4. 8641691

stein-Barr virus. Oncogene. 1997 Oct 16;15(16):1911-20. 9365237

tracranial tumours. Hum Genet. 1993 Mar;91(2):190-2. 8462979

pendent mechanisms. J Cell Sci. 2003 Apr 1;116(Pt 7):1349-57. 12615976

drome patients. Hum Mutat. 2006 Jun;27(6):558-67. 16673358

Oct 15;22(20):2856-68. 18923083

82 The Mechanisms of DNA Replication

A. 1993 Dec 15;90(24):12030-4. 8265666

loss. Cell. 1993 Nov 19;75(4):729-39. 8242745

10338214

10588723


[172] Bassing CH, Alt FW. H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. Cell Cycle. 2004 Feb;3(2):149-53. 14712078

[158] Prince PR, Emond MJ, Monnat RJ, Jr. Loss of Werner syndrome protein function pro‐ motes aberrant mitotic recombination. Genes Dev. 2001 Apr 15;15(8):933-8. 11316787

[159] Bischof O, Kim SH, Irving J, Beresten S, Ellis NA, Campisi J. Regulation and localiza‐ tion of the Bloom syndrome protein in response to DNA damage. J Cell Biol. 2001

[160] Dutertre S, Ababou M, Onclercq R, Delic J, Chatton B, Jaulin C, Amor-Gueret M. Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase. On‐

[161] Sanz MM, Proytcheva M, Ellis NA, Holloman WK, German J. BLM, the Bloom's syn‐ drome protein, varies during the cell cycle in its amount, distribution, and co-locali‐ zation with other nuclear proteins. Cytogenet Cell Genet. 2000;91(1-4):217-23.

[162] Zhong S, Hu P, Ye TZ, Stan R, Ellis NA, Pandolfi PP. A role for PML and the nuclear body in genomic stability. Oncogene. 1999 Dec 23;18(56):7941-7. 10637504

[163] Dellaire G, Ching RW, Ahmed K, Jalali F, Tse KC, Bristow RG, Bazett-Jones DP. Pro‐ myelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and

[164] Matunis MJ, Zhang XD, Ellis NA. SUMO: the glue that binds. Dev Cell. 2006 Nov;

[165] Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355-82.

[166] Shen TH, Lin HK, Scaglioni PP, Yung TM, Pandolfi PP. The mechanisms of PML-nu‐

[167] Eladad S, Ye TZ, Hu P, Leversha M, Beresten S, Matunis MJ, Ellis NA. Intra-nuclear trafficking of the BLM helicase to DNA damage-induced foci is regulated by SUMO

[168] Hu P, Beresten SF, van Brabant AJ, Ye TZ, Pandolfi PP, Johnson FB, Guarente L, Ellis NA. Evidence for BLM and Topoisomerase IIIalpha interaction in genomic stability.

[169] Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A crit‐ ical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA

[170] Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM. Char‐ acteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Bi‐

[171] Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol. 1999 Sep 6;146(5):905-16. 10477747

clear body formation. Mol Cell. 2006 Nov 3;24(3):331-9. 17081985

modification. Hum Mol Genet. 2005 May 15;14(10):1351-65. 15829507

Hum Mol Genet. 2001 Jun 1;10(12):1287-98. 11406610

ol. 2003 Jun;81(3):123-9. 12897845

damage. Curr Biol. 2000 Jul 27-Aug 10;10(15):886-95. 10959836

Apr 16;153(2):367-80. 11309417

11173860

84 The Mechanisms of DNA Replication

15189146

11(5):596-7. 17084352

cogene. 2000 May 25;19(23):2731-8. 10851073

ATR. J Cell Biol. 2006 Oct 9;175(1):55-66. 17030982


mere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway. Genes Dev. 2005 Nov 1;19(21):2560-70. 16264192

[197] Harrigan JA, Wilson DM, 3rd, Prasad R, Opresko PL, Beck G, May A, Wilson SH, Bohr VA. The Werner syndrome protein operates in base excision repair and cooper‐ ates with DNA polymerase beta. Nucleic Acids Res. 2006;34(2):745-54. 16449207

[184] Crabbe L, Jauch A, Naeger CM, Holtgreve-Grez H, Karlseder J. Telomere dysfunc‐ tion as a cause of genomic instability in Werner syndrome. Proc Natl Acad Sci U S A.

[185] de Lange T. Shelterin: the protein complex that shapes and safeguards human telo‐

[186] Opresko PL, von Kobbe C, Laine JP, Harrigan J, Hickson ID, Bohr VA. Telomerebinding protein TRF2 binds to and stimulates the Werner and Bloom syndrome heli‐

[187] Opresko PL, Mason PA, Podell ER, Lei M, Hickson ID, Cech TR, Bohr VA. POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates. J Bi‐

[188] Kao HI, Campbell JL, Bambara RA. Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation. J Biol Chem. 2004

[189] Sharma S, Sommers JA, Brosh RM, Jr. In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication. Hum Mol Genet. 2004 Oct

[190] Szekely AM, Chen YH, Zhang C, Oshima J, Weissman SM. Werner protein recruits DNA polymerase delta to the nucleolus. Proc Natl Acad Sci U S A. 2000 Oct

[191] Kamath-Loeb AS, Loeb LA, Johansson E, Burgers PM, Fry M. Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat se‐

[192] Fry M, Loeb LA. Human werner syndrome DNA helicase unwinds tetrahelical struc‐ tures of the fragile X syndrome repeat sequence d(CGG)n. J Biol Chem. 1999 Apr

[193] Riha K, Heacock ML, Shippen DE. The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology. Annu Rev Genet.

[194] Cheng WH, von Kobbe C, Opresko PL, Arthur LM, Komatsu K, Seidman MM, Car‐ ney JP, Bohr VA. Linkage between Werner syndrome protein and the Mre11 complex

[195] Celli GB, Denchi EL, de Lange T. Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination. Nat Cell Biol. 2006

[196] Laud PR, Multani AS, Bailey SM, Wu L, Ma J, Kingsley C, Lebel M, Pathak S, DePin‐ ho RA, Chang S. Elevated telomere-telomere recombination in WRN-deficient, telo‐

quence. J Biol Chem. 2001 May 11;276(19):16439-46. 11279038

via Nbs1. J Biol Chem. 2004 May 14;279(20):21169-76. 15026416

2007 Feb 13;104(7):2205-10. 17284601

86 The Mechanisms of DNA Replication

meres. Genes Dev. 2005 Sep 15;19(18):2100-10. 16166375

cases. J Biol Chem. 2002 Oct 25;277(43):41110-9. 12181313

ol Chem. 2005 Sep 16;280(37):32069-80. 16030011

Dec 3;279(49):50840-9. 15448135

1;13(19):2247-61. 15282207

10;97(21):11365-70. 11027336

30;274(18):12797-802. 10212265

2006;40:237-77. 16822175

Aug;8(8):885-90. 16845382


[219] Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shira‐ hige K. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature. 2003 Aug 28;424(6952):1078-83. 12944972

[208] Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG, Venkitaraman AR. Initiation of DNA replication requires the RECQL4 protein mutated in Roth‐

[209] Matsuno K, Kumano M, Kubota Y, Hashimoto Y, Takisawa H. The N-terminal non‐ catalytic region of Xenopus RecQ4 is required for chromatin binding of DNA poly‐ merase alpha in the initiation of DNA replication. Mol Cell Biol. 2006 Jul;26(13):

[210] Im JS, Ki SH, Farina A, Jung DS, Hurwitz J, Lee JK. Assembly of the Cdc45-Mcm2-7- GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 pro‐

[211] Kumata Y, Tada S, Yamanada Y, Tsuyama T, Kobayashi T, Dong YP, Ikegami K, Murofushi H, Seki M, Enomoto T. Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts. Biochim Biophys Acta. 2007

[212] Petkovic M, Dietschy T, Freire R, Jiao R, Stagljar I. The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability. J Cell Sci. 2005 Sep

[213] Woo LL, Futami K, Shimamoto A, Furuichi Y, Frank KM. The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress. Exp

[214] Werner SR, Prahalad AK, Yang J, Hock JM. RECQL4-deficient cells are hypersensi‐ tive to oxidative stress/damage: Insights for osteosarcoma prevalence and heteroge‐ neity in Rothmund-Thomson syndrome. Biochem Biophys Res Commun. 2006 Jun

[215] Wang M, Wu W, Wu W, Rosidi B, Zhang L, Wang H, Iliakis G. PARP-1 and Ku com‐ pete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic

[216] Audebert M, Salles B, Calsou P. Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoin‐

[217] Bailis JM, Luche DD, Hunter T, Forsburg SL. Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome sta‐

[218] Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson RD, Labib K. GINS maintains association of Cdc45 with MCM in replisome progres‐ sion complexes at eukaryotic DNA replication forks. Nat Cell Biol. 2006 Apr;8(4):

teins. Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15628-32. 19805216

mund-Thomson syndrome. Cell. 2005 Jun 17;121(6):887-98. 15960976

4843-52. 16782873

88 The Mechanisms of DNA Replication

Apr;1773(4):556-64. 17320201

15;118(Pt 18):4261-9. 16141230

23;345(1):403-9. 16678792

358-66. 16531994

Cell Res. 2006 Oct 15;312(17):3443-57. 16949575

Acids Res. 2006;34(21):6170-82. 17088286

ing. J Biol Chem. 2004 Dec 31;279(53):55117-26. 15498778

bility. Mol Cell Biol. 2008 Mar;28(5):1724-38. 18180284

