**6. Gratuitous repair on undamaged DNA misfolds by multiple proteins**

DNA damage and repair are always active in living cells regardless of the proliferation status of the cells. And unpaired bases and the helix distortions/ junctions in most of the non-B DNA secondary structures can therefore be targets for the structure specific proteins working in DNA repair, e.g. mismatch repair, nucleotide excision repair etc., launching DNA repairs or activating checkpoints repair (Voineagu et al., 2009).

#### **6.1 Repair by singular pathway of DNA repair**

Small DNA loops/ bulges, triplex DNA may be readily corrected by an individual repair, such as a mismatch repair or a nucleotide excision repair. For example, helix distortion and/ or mismatched base pairs in a hairpin, which sometime also occurs with imperfect hairpin

(HEX1-N2) has high activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not have specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues (Lee & Wilson, 1999). FEN-1, a structure-specific endonuclease is essential for DNA replication and repair, removes RNA and DNA 5' flaps (Tsutakawa et al., 2011). FEN-1 was thought to be involved in hairpin structure processing, and was found to be involved in CNG triplet repeat stability in the lagging strand template (Spiro et al., 1999; Singh et al., 2007). Similarly, Deletions in PCNA, RPA, and the Bloom protein (BLM), a 3'-5' helicase can also increase CNG repeat expansion or deletion, which reportedly interacts with FEN-1 in cleaving flaps. Recently NucS from *Pyrococcus abyssi* was found to be the equivalent of FEN-1 that cleaves the flapped DNA in Okazaki frangment processing in the lagging strand DNA replication (Ren

SLX1 and SLX4 are other structure-specific endonucleases acting as heteromer that cleave branched DNA substrates, particularly simple-Y, 5'-flap, or replication fork structures. It also cleaves the strand bearing the 5' nonhomologous arm at the branch junction and generates ligatable nicked products from 5'-flap or replication fork substrates (Fricke & Brill, 2003). RAGs is a complex consisting of RAG1, RAG2, and HMGB1 that cleaves 3' overhangs in multiple locations at the duplex/ single-stranded transitions (Fugmann, 2001). RAGs complex is able to cleave different non-B DNA structures such as symmetric bubbles, heterologous loops and proposed triplex DNA. For example, RAGs complex cleaves the *bcl*-2 Mbr at 3' overhang and non-B DNA structures under physiological buffer conditions

In addition, many single-strand specific nucleases, like S1, P1, and mung bean nucleases, are also efficient at cleaving single stranded DNA in the non-B DNA structures, though at low pH. Since some non-B DNA structures, e.g. H-DNA and G4 DNA disclose an unstructured single-stranded DNA region, which therefore serve as substrates for those single-strand specific nucleases. Recently, a more specific nuclease that cuts single-stranded DNA 5' to a G4 domain was isolated from human cells. This enzyme, initially named G quartet nuclease 1 (GQN1) is thought to be involved in immunoglobulin heavy chain class switch recombination in B cells, does not digest single- or double-stranded DNA, Holliday junctions or tetraplex RNA. It specifically cuts single-stranded DNA located few nucleotides 5' to either G'2 or G4 domains (Sun et al., 2001). However, GQN1 cannot incise tetraplex RNA, showing a significant difference from a mouse cytoplasmic exoribonuclease

**6. Gratuitous repair on undamaged DNA misfolds by multiple proteins** 

DNA damage and repair are always active in living cells regardless of the proliferation status of the cells. And unpaired bases and the helix distortions/ junctions in most of the non-B DNA secondary structures can therefore be targets for the structure specific proteins working in DNA repair, e.g. mismatch repair, nucleotide excision repair etc., launching

Small DNA loops/ bulges, triplex DNA may be readily corrected by an individual repair, such as a mismatch repair or a nucleotide excision repair. For example, helix distortion and/ or mismatched base pairs in a hairpin, which sometime also occurs with imperfect hairpin

(Adachi & Tsujimoto, 1990; Fugmann, 2001; Raghavan et al., 2004, 2005).

(mXRN1p) which cleaves G4 RNA (Bashkirov et al., 1997).

**6.1 Repair by singular pathway of DNA repair** 

DNA repairs or activating checkpoints repair (Voineagu et al., 2009).

et al., 2009; Creze et al., 2011).

structures at CAG repeats, can be recognized by mismatch repair machinery (Yang, 2006). Msh2/ Msh3 complex in eukaryotic cells specifically binds CAG-hairpins, and the ATP-ase activity of the Msh2 / Msh3 complex can be altered by the binding. However, the repair is dependent on the number of loops/ bulges. A few of them may be repaired by MMR, but too many may not because of interfering MMR by multiple MutS binding, suggesting that repair on a particular non-B DNA conformation will be conditional, depending on locations and environments. Further, nucleotide excision repair (NER) proteins can bind intermolecular triplex, which are involved in the triplex mediated mutagenesis and recombination (Wang & Vasquez, 2006). In bacterial cells, NER proteins UvrB and UvrC were responsible for triplex-induced cell growth retardation. Given the likenesses of the intermolecular and intramolecular triplex, it is possible for NER contributing to the H-DNAinduced mutagenesis and recombination**.**

#### **6.2 Competitions among multiple repair proteins**

Apart from initiating an individual pathway of DNA repair, some non-B DNA structures can also be recognized by more than one repair proteins working in different repair pathways, resulting in competitions between proteins on same DNA structures.

Competition of repair proteins on a non-B DNA structure may be needed for a cooperative repair, setting up a cooperative new DNA repair to repair; in contrast, the competition may sometimes be internecine, failing in repair of either pathway. Under this circumstance, the repair on a non-B DNA structure by the compositing actions of the DNA structural recognition proteins would be compromised. For example, a stable hairpin may be needed for starting DNA replication, but such a stable hairpin would also be repaired by SbcCD or Mre11-Rad50, making a DNA break for homologous recombination to repair (Leach, 1994). Similarly, unwound DNA or small DNA loops may also be needed for DNA replication or for transcription. While they may also be recognized and bound by repair proteins, such as DNA mismatch and nucleotide-excision repair proteins, recombination proteins, instead of SSB/ RPA (Kirkpatrick & Petes, 1997).

A good demonstration for the internecine competition between multiple repair proteins was the foldings of TGG and AGG repeats in the lagging strand template in a replication fork (Pan & Leach, 2000; Pan et al., to be published results). TGG, AGG and CGG repeats are a group of NGG repeats which own significant potential of folding into non-B DNA secondary structures (Usdin, 1998; Pan & Leach, 2000). AGG repeats formed triplex (Suda et al., 1996; Mishima et al., 1996, 1997), homoduplex (Suda et al.,1995), tetra-duplex (Yang & Hurley, 2006), and a special G-quadruplex, known as tetrad:heptad:heptad:tetrad ((G:H:H:G) or (T:H:H:T)) (Matsugami et al., 2001a, 2001b, 2002, 2003), while CGG and TGG repeats formed pseudo-hairpin and tetra-duplex, respectively (Darlow & Leach, 1998; Usdin, 1998; Pan & Leach, 2000; Zemánek et al., 2005).

It was shown by Pan and Leach, that replication of TGG repeats in the lagging strand template experiences repeats misfolding, during which both MutS and SbcCD were found to affect the later processing by homologous recombination. Binding MutS to the non-B DNA structure formed by TGG repeats may stabilize the structure, while hindering SbcCD cleaving the structure. Interestingly, the roles of MutS and SbcCD in this case seemed complex, since TGG repeats can replicate either without MutS or SbcCD, suggesting that they also play same role in stabilizing the TGG repeat structure. In contrast, similar sized AGG repeats was found also to fold into non-B DNA structures in a similar lagging strand template of a replication fork.

The Gratuitous Repair on Undamaged DNA Misfold 419

The existence of cellular proteins that interact with non-B DNA structures provides both strong argument for the existence of non-B DNA structure formations in genomic DNA, and suggestion for cell having intrinsic response to the formation of non-B DNA structures. However, it seems that not all non-B DNA secondary structures, unless they make severe troublesome to DNA metabolism such as making DNA double strand breaks, or generating long single stranded region, were recognized as "DNA damage". Even if cruciforms / hairpins, triplexes, slipped conformations, quadruplexes, and left-handed Z-DNA have all been reported to be chromosomal targets for DNA repair, recombination, and aberrant DNA synthesis, leading to repeat expansion or genomic rearrangements associated with neurodegenerative and genomic disorders. Some of them may also raise more severe

The situations for a non-B DNA secondary structure intriguing a cellular response may be addressed at the competing recognition and processing by multiple repair proteins, resulting in incomplete / partial / opposing processing of the non-B DNA structure. Such intermediates may be recognized by proteins capable of activating a cellular response. Alternatively non-B DNA structure bears components that can be recognized by proteins capable of activating a cellular response (Voineagu et al., 2009). In support of this idea, DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 were found to play opposite roles in yeast gene targeting, a triple stranded DNA mediated process. During which Rad1, Msh2, and Msh3 facilitated forming triplex DNA, while Sgs1 prevented forming triplex DNA (Langston & Symington, 2005), therefore should a cellular response be intrigued in gene

The ssDNA region in a non-B DNA structure may likely be coated by single-stranded DNAbinding protein (RPA) directly, or RPA coats the ssDNA after the non-B DNA structure is processed. Either way makes a common intermediate of ssDNA-RPA that activates ATR signaling in response to all of the genotoxic lesions (Krejci et al., 2003; Hu et al., 2007). Indeed, the ssDNA-RPA complex has been found to be a common intermediate in the processing of many types of damaged DNA, including DSBs, UV-induced thymidine dimers, intrastrand cross-links, and mismatches in base-pairing (Ball et al., 2005; Choi et al., 2010 ). The RPA–ssDNA complex will promote the loading of the 9–1–1 and ATR–ATRIP complexes (Dore et al., 2009). The juxtaposition of these complexes allows ATR to phosphorylate Chk1, which then promotes cell cycle arrest, causing a cellular response to non-B DNA structure formation. Alternatively, ssDNA-RPA complex can recruit Cut5, by which ATR (ATR-ATRIP) (Mec1-Ddc2 in yeast), DNA polymerase α, Rad50-Mre11-Nbs1 (MRN) and clamp loader Rad24 (Rad17 in mammals) can all be recruited to the ssDNA-RPA

The purpose of activating DNA damage checkpoint in response to the formation of non-B DNA secondary structure is to regulate cell cycle events, for mediating appropriate repair and fork restart processes. While non-B DNA structure forming sequences per se are probably an infrequent trigger of DNA damage checkpoint responses, and, thus, should not be regarded as a real DNA damage by cells. There has extensive evidence suggesting that non-B DNA structure forming sequences can only induce checkpoint-triggering events when stable non-B DNA structures are adopted. The stable DNA structures may affect normal DNA metabolism, making DSBs or causing more severe effects on DNA metabolism, such as replication fork stalling, formation of nucleosome free sites (Chromosomal Fragile

**6.4 Cellular response to non-B DNA structures by activating checkpoints** 

targeting may have to wait for processing the structure-specific proteins.

(Cortez et al., 2001; Zou & Elledge, 2003; Robison et al., 2004).

Sites) etc.

response by cells (Voineagu et al., 2009).

However, the non-B DNA structure formed by AGG repeats was found to be incapable of binding with MutS protein, and being cleaved by SbcCD. This made consistence with the reports though AGG repeats belong to a same group of NGG trinucleotide repeats with TGG repeats, they form various G-rich DNA secondary structures, including quadruplex, triple helical, homoduplex and tetrad:heptad:heptad:tetrad ((G:H:H:G) or (T:H:H:T)). Obviously, some of these non-B DNA structures folded may not be recognized by MutS protein *in vivo*, making significant differences in DNA structure formation between AGG repeats and TGG repeats (Pan et al., unpublished results).

The examples of a coordinated repair by different repair proteins on the same non-B DNA structures are the repair of DNA loops by MMR and NER proteins (Kirkpatrick & Petes, 1997; Zhao et al., 2009, 2010). It has been found that both MSH2 and XPA proteins are involved in the instabilities of CAG repeats, possibly through some so far unidentified roles (Kirkpatrick & Petes, 1997; Lin & Wilson, 2009; Zhao etal., 2009, 2010). Knocking down both MSH2 and XPA proteins did not further reduce CAG repeat contraction, suggesting a new role for these proteins in the same pathway. Similarly, it has also been reported the MSH2 and XPA are also involved in H-DNA metabolism but once again the DNA structure may not be processed via canonical MMR or NER mechanisms (Zhao et al., 2009, 2010).

#### **6.3 Repair proteins can be defeated by DNA secondary structure**

It may be feasible by postulating that more non-B DNA structures might be formed by DNA sequences in the genomes. However the repair machinery in the cells may only be limited to a few types, such as those MMR, NER single / double strand breaks etc. It therefore raises a question as if all non-B DNA structures possibly form could be recognized and processed by those repair proteins? The answer to this question is presently unknown; however some of the known secondary structures cannot easily be repaired, including large DNA loops and the flapped DNA etc.

#### **6.3.1 Large loops**

Stable base pairing prevents recognition by repair enzymes of bases or junctions requiring repair. For example, in *E.coli*, small loops (or secondary structure) may allow mispairing of bases that are corrected by MMR enzymes, leading to loss of base interruption (Parker & Marinus, 1992; Carraway & Marinus, 1993). However, DNA loops made up of less than four unpaired bases are efficiently corrected by methyl-directed mismatch repair (MMR), but loops larger than that cannot be repaired effectively (Parker & Marinus, 1992; Carraway & Marinus, 1993; Fang et al., 2003). The reason for this inefficacy was found to be due to the failure in loop recognition using MutS proteins, leaving the large looped DNA unrepaired by MMR.

#### **6.3.2 Flapped DNA**

Flap endonuclease (RAD27 in *Saccharomyces cerevesiae*; FEN-1 in humans) can destabilize simple tandem repeat loci. The 5' to 3' flap endonuclease FEN-1/ RAD27 is a structurespecific nuclease required for Okazaki fragment processing in the lagging strand DNA replication. FEN-1, a structure-specific endonuclease is also thought to be involved in CNG triplet repeat stability. It has been reported that a stable hairpin formed by CTG or CAG repeats at the flap region can block the activity of FEN-1. Which then join the upstream Okazaki fragment, resulting in repeats expansion during the next cycle of replication, marking the activity of FEN-1 can be defeated by stable DNA structure (Spiro et al., 1999; Singh et al., 2007).

However, the non-B DNA structure formed by AGG repeats was found to be incapable of binding with MutS protein, and being cleaved by SbcCD. This made consistence with the reports though AGG repeats belong to a same group of NGG trinucleotide repeats with TGG repeats, they form various G-rich DNA secondary structures, including quadruplex, triple helical, homoduplex and tetrad:heptad:heptad:tetrad ((G:H:H:G) or (T:H:H:T)). Obviously, some of these non-B DNA structures folded may not be recognized by MutS protein *in vivo*, making significant differences in DNA structure formation between AGG repeats and TGG

The examples of a coordinated repair by different repair proteins on the same non-B DNA structures are the repair of DNA loops by MMR and NER proteins (Kirkpatrick & Petes, 1997; Zhao et al., 2009, 2010). It has been found that both MSH2 and XPA proteins are involved in the instabilities of CAG repeats, possibly through some so far unidentified roles (Kirkpatrick & Petes, 1997; Lin & Wilson, 2009; Zhao etal., 2009, 2010). Knocking down both MSH2 and XPA proteins did not further reduce CAG repeat contraction, suggesting a new role for these proteins in the same pathway. Similarly, it has also been reported the MSH2 and XPA are also involved in H-DNA metabolism but once again the DNA structure may

It may be feasible by postulating that more non-B DNA structures might be formed by DNA sequences in the genomes. However the repair machinery in the cells may only be limited to a few types, such as those MMR, NER single / double strand breaks etc. It therefore raises a question as if all non-B DNA structures possibly form could be recognized and processed by those repair proteins? The answer to this question is presently unknown; however some of the known secondary structures cannot easily be repaired, including large DNA loops and

Stable base pairing prevents recognition by repair enzymes of bases or junctions requiring repair. For example, in *E.coli*, small loops (or secondary structure) may allow mispairing of bases that are corrected by MMR enzymes, leading to loss of base interruption (Parker & Marinus, 1992; Carraway & Marinus, 1993). However, DNA loops made up of less than four unpaired bases are efficiently corrected by methyl-directed mismatch repair (MMR), but loops larger than that cannot be repaired effectively (Parker & Marinus, 1992; Carraway & Marinus, 1993; Fang et al., 2003). The reason for this inefficacy was found to be due to the failure in loop

Flap endonuclease (RAD27 in *Saccharomyces cerevesiae*; FEN-1 in humans) can destabilize simple tandem repeat loci. The 5' to 3' flap endonuclease FEN-1/ RAD27 is a structurespecific nuclease required for Okazaki fragment processing in the lagging strand DNA replication. FEN-1, a structure-specific endonuclease is also thought to be involved in CNG triplet repeat stability. It has been reported that a stable hairpin formed by CTG or CAG repeats at the flap region can block the activity of FEN-1. Which then join the upstream Okazaki fragment, resulting in repeats expansion during the next cycle of replication, marking the activity of FEN-1 can be defeated by stable DNA structure (Spiro et al., 1999;

recognition using MutS proteins, leaving the large looped DNA unrepaired by MMR.

not be processed via canonical MMR or NER mechanisms (Zhao et al., 2009, 2010).

**6.3 Repair proteins can be defeated by DNA secondary structure** 

repeats (Pan et al., unpublished results).

the flapped DNA etc.

**6.3.1 Large loops** 

**6.3.2 Flapped DNA** 

Singh et al., 2007).

#### **6.4 Cellular response to non-B DNA structures by activating checkpoints**

The existence of cellular proteins that interact with non-B DNA structures provides both strong argument for the existence of non-B DNA structure formations in genomic DNA, and suggestion for cell having intrinsic response to the formation of non-B DNA structures. However, it seems that not all non-B DNA secondary structures, unless they make severe troublesome to DNA metabolism such as making DNA double strand breaks, or generating long single stranded region, were recognized as "DNA damage". Even if cruciforms / hairpins, triplexes, slipped conformations, quadruplexes, and left-handed Z-DNA have all been reported to be chromosomal targets for DNA repair, recombination, and aberrant DNA synthesis, leading to repeat expansion or genomic rearrangements associated with neurodegenerative and genomic disorders. Some of them may also raise more severe response by cells (Voineagu et al., 2009).

The situations for a non-B DNA secondary structure intriguing a cellular response may be addressed at the competing recognition and processing by multiple repair proteins, resulting in incomplete / partial / opposing processing of the non-B DNA structure. Such intermediates may be recognized by proteins capable of activating a cellular response. Alternatively non-B DNA structure bears components that can be recognized by proteins capable of activating a cellular response (Voineagu et al., 2009). In support of this idea, DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 were found to play opposite roles in yeast gene targeting, a triple stranded DNA mediated process. During which Rad1, Msh2, and Msh3 facilitated forming triplex DNA, while Sgs1 prevented forming triplex DNA (Langston & Symington, 2005), therefore should a cellular response be intrigued in gene targeting may have to wait for processing the structure-specific proteins.

The ssDNA region in a non-B DNA structure may likely be coated by single-stranded DNAbinding protein (RPA) directly, or RPA coats the ssDNA after the non-B DNA structure is processed. Either way makes a common intermediate of ssDNA-RPA that activates ATR signaling in response to all of the genotoxic lesions (Krejci et al., 2003; Hu et al., 2007). Indeed, the ssDNA-RPA complex has been found to be a common intermediate in the processing of many types of damaged DNA, including DSBs, UV-induced thymidine dimers, intrastrand cross-links, and mismatches in base-pairing (Ball et al., 2005; Choi et al., 2010 ). The RPA–ssDNA complex will promote the loading of the 9–1–1 and ATR–ATRIP complexes (Dore et al., 2009). The juxtaposition of these complexes allows ATR to phosphorylate Chk1, which then promotes cell cycle arrest, causing a cellular response to non-B DNA structure formation. Alternatively, ssDNA-RPA complex can recruit Cut5, by which ATR (ATR-ATRIP) (Mec1-Ddc2 in yeast), DNA polymerase α, Rad50-Mre11-Nbs1 (MRN) and clamp loader Rad24 (Rad17 in mammals) can all be recruited to the ssDNA-RPA (Cortez et al., 2001; Zou & Elledge, 2003; Robison et al., 2004).

The purpose of activating DNA damage checkpoint in response to the formation of non-B DNA secondary structure is to regulate cell cycle events, for mediating appropriate repair and fork restart processes. While non-B DNA structure forming sequences per se are probably an infrequent trigger of DNA damage checkpoint responses, and, thus, should not be regarded as a real DNA damage by cells. There has extensive evidence suggesting that non-B DNA structure forming sequences can only induce checkpoint-triggering events when stable non-B DNA structures are adopted. The stable DNA structures may affect normal DNA metabolism, making DSBs or causing more severe effects on DNA metabolism, such as replication fork stalling, formation of nucleosome free sites (Chromosomal Fragile Sites) etc.

The Gratuitous Repair on Undamaged DNA Misfold 421

generation of genomic instability. Strikingly, unusual DNA structures were often found to trigger some kinds of repair actions or avoidance responses that promote their removal of the structures once formed. Under this later circumstance, it becomes obvious that formation of non-B DNA structures *in vivo* was somehow similar to the appearances of some real DNA damages as induced by environmental DNA damaging agents. Certain unusual DNA structures have unpaired bases and regions with helix distortions/junctions etc., which may experience unprovoked repair in cells. Therefore triggering cellular responses of a non-B DNA structure is subject to its morphological/ topological properties, which could attract recognizing repair proteins. In fact, a non-B DNA structure is often recognized by more than one repair proteins, such as the proteins working in MMR, NER and recombination. Questions rose therefore as if individual pathways of DNA repair accounts enough for the repair of the non-B DNA structures? Or does it need multiple proteins working in different repair pathways reconstitute synthesized pathway(s) to repair? Nevertheless, progress in this field seems support an idea that enzymes/ proteins that recognize and/ or process the possible non-B DNA structures may be different because of the non-B DNA structures formed. Proteins that have been found to associate with non-B DNA instability might take part in an unexpected way in processing the non-B DNA structures. Therefore studies in the coming future may have to focus on the identifications of the types of non-B DNA structures that elicit certain kinds of mutations and the enzyme systems involved. It could be expected that more diseases will be recognized as because of mutations at non-B DNA structures. Also, strategies will have to make toward developing

Adachi, M. & Tsujimoto. Y. (1990). Potential Z-DNA elements surround the breakpoints of

Alvarez, D., Novac, O., Callejo, M., Ruiz, M. T., Price, G. B. & Zannis-Hadjopoulos, M.

Antony, S., Arimondo, P. B., Sun, J-S. & Pommier, Y. (2004). Position- and orientation-

Arimondo, P. B., Riou, J-F., Mergny, J-L., Tazi, J., Sun, J-S., Garestier, T. & Hélène, C. (2000).

Ashton, T. M., Mankouri, H. W., Heidenblut, A., McHugh, P. J. & Hickson, I. D. (2011).

Bachrati, C. Z. & Hickson, I. D. (2003). RecQ helicases: suppressors of tumorigenesis and

Bacolla, A., Jaworski, A., Connors, T. D. & Wells, R. D. (2001). Pkd1 unusual DNA

Bacolla, A. & Wells, R. D. (2009). Non-B DNA conformations as determinants of

chromosome translocation within the 5' flanking region of *bcl*-2 gene. *Oncogene,*

(2002).14-3-3sigma is a cruciform DNA binding protein and associates in vivo with

specific enhancement of topoisomerase I cleavage complexes by triplex DNA

Interaction of human DNA topoisomerase I with G‐quartet structures. *Nucleic Acids* 

Pathways for Holliday Junction Processing during Homologous Recombination in

conformations are recognized by nucleotide excision repair. *J Biol Chem.,* 276:18597–

therapeutics to appease the devastating effects of the syndromes.

origins of DNA replication. *J Cell Biochem.* 87(2):194-207.

*Saccharomyces cerevisiae*. *Mol Cell Biol.,* 31(9):1921-1933.

mutagenesis and human disease. *Mol Carcinog.,* 48:273-285.

premature aging. *Biochem J.,* 374(Pt 3):577-606.

structures. *Nucleic Acids Res.*, 32 (17): 5163-5173.

**8. References** 

5:1653–1657.

18604.

*Res.*, 28 (24): 4832-4838.

Consisting with that, mutations in checkpoint genes, such as Mec1, Ddc2, Rad9, Rad17, Rad24, or Rad53, produce repeat instabilities by a CAG~70, including both expansion and contraction instabilities. These suggested that DNA structure formed by long CAG repeats activated checkpoints in eukaryotes (Lahiri et al., 2004; Sundararajan & Freudenreich, 2011). Similarly, a CAG175 repeat on plasmids can also be recognized as ''DNA damage'' in *E. coli*, as witnessed by inducing SOS response (Majchrzak et al., 2006).

Surprisingly, it was found that even those shorter CAG repeats (containing 13–20 triplets) can also intrigue DNA damage checkpoint. By which repeats expansion can be prevented when the repeats formed non-B structures, suggesting that cells have endowed the checkpoint mechanism of responding to non-B DNA structure formation (Razidlo & Lahue, 2008).

Another example as intriguing cellular response for non-B DNA structure formation by derived structure processing is also found with human PKD1 gene. The 2.5-kb polypurine– polypyrimidine tract in intron 21 in human PKD1 gene potentially forms H-DNA structure, contributing to the high mutation rate of the PKD1 gene (Bacolla et al., 2001;Patel et al., 2004). A plasmid carrying this polypurine–polypyrimidine tract induced a stronge SOS response and severely delayed the host cell growth, resulting in a dramatic decrease in colony formation (Patel et al., 2004). However, the effect was largely reduced without UvrA (100-fold decrease in colony formation), and nearly vanished without UvrB or UvrC. These suggested the polypurine–polypyrimidine repeat sequence or the structure formed by the repeats per se was not involved in the effects, while the NER processing was essential (Bacolla et al., 2001).

#### **6.5 Mre11-Rad50-Nbs1 (MRN)/ Mre11-Rad50-Xrs2 (MRX)**

Apart from the nucleolytic activity, MRN / MRX can also play roles in activating the checkpoints as mentioned above (van den Bosch, et al., 2003; Sundararajan & Freudenreich, 2011). It was believed that a single stranded region in a non-B DNA structure forms ssDNA-RPA to the amount of triggering a checkpoint response (normally exceeds 300 bp). One way of Rad50-Mre11-Nbs1 (MRN) contributing to checkpoint response might be through Cut5 recruitment. Rad50-Mre11-Nbs1 (MRN) can be recruited to the single stranded region in the non-B DNA structure, and then participates in ATR checkpoint. Alternatively Rad50-Mre11- Nbs1 (MRN) can also secure DNA replication as implicated by its ortholog SbcCD in *E.coli*  (Darmon et al., 2007; Zahra et al., 2007). Indeed, the MRN / MRX complex has been co localized in the replication machinery. In this context, the resection role of MRN / MRX on DSB initiated recombination repair may be no more necessary as long as the checkpoints mechanism prevented the DSB formation by checkpoint proteins (Mimitou & Symington, 2008; Zhu et al., 2008).

Non-B DNA structure forming sequences are potential triggers of DNA damage checkpoint responses mainly by inducing replication fork stalling and chromosomal breaks. Since the non-B DNA structures have specific DNA conformations at the damaged site, which may influence the checkpoint signaling, and the dynamics of checkpoint activation are likely to differ at different types of non-B DNA structure forming sequences.

#### **7. Future perspectives**

Many lines of evidence suggest that unusual DNA structures can form *in vivo* and play significant roles in DNA metabolism, while they may also serve as a source for the generation of genomic instability. Strikingly, unusual DNA structures were often found to trigger some kinds of repair actions or avoidance responses that promote their removal of the structures once formed. Under this later circumstance, it becomes obvious that formation of non-B DNA structures *in vivo* was somehow similar to the appearances of some real DNA damages as induced by environmental DNA damaging agents. Certain unusual DNA structures have unpaired bases and regions with helix distortions/junctions etc., which may experience unprovoked repair in cells. Therefore triggering cellular responses of a non-B DNA structure is subject to its morphological/ topological properties, which could attract recognizing repair proteins. In fact, a non-B DNA structure is often recognized by more than one repair proteins, such as the proteins working in MMR, NER and recombination. Questions rose therefore as if individual pathways of DNA repair accounts enough for the repair of the non-B DNA structures? Or does it need multiple proteins working in different repair pathways reconstitute synthesized pathway(s) to repair? Nevertheless, progress in this field seems support an idea that enzymes/ proteins that recognize and/ or process the possible non-B DNA structures may be different because of the non-B DNA structures formed. Proteins that have been found to associate with non-B DNA instability might take part in an unexpected way in processing the non-B DNA structures. Therefore studies in the coming future may have to focus on the identifications of the types of non-B DNA structures that elicit certain kinds of mutations and the enzyme systems involved. It could be expected that more diseases will be recognized as because of mutations at non-B DNA structures. Also, strategies will have to make toward developing therapeutics to appease the devastating effects of the syndromes.
