Werner syndrome protein: Functions in the response to DNA damage and replication stress in S-phase

Experimental Gerontology 42 (2007) 871–878 www.elsevier.com/locate/expgero

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Werner syndrome protein: Functions in the response to DNA damage and replication stress in S-phase Wen-Hsing Cheng, Meltem Muftuoglu, Vilhelm A. Bohr

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Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA Received 23 February 2007; received in revised form 23 April 2007; accepted 27 April 2007 Available online 10 May 2007

Abstract Werner syndrome (WS) is an excellent model system for the study of human aging. WRN, a nuclear protein mutated in WS, plays multiple roles in DNA metabolism. Our understanding about the metabolic regulation and function of this RecQ helicase has advanced greatly during the past decade, largely due to the availability of purified WRN protein, WRN knockdown cells, and WRN knockout mice. Recent biochemical and genetic studies indicate that WRN plays significant roles in DNA replication, DNA repair, and telomere maintenance. Interestingly, many WRN functions require handling of DNA ends during S-phase, and evidence suggests that WRN plays both upstream and downstream roles in the response to DNA damage. Future research should focus on the mechanism(s) of WRN in the regulation of the various DNA metabolism pathways and development of therapeutic approaches to treat premature aging syndromes such as WS. Published by Elsevier Inc. Keywords: Aging; Werner syndrome; DNA repair; Replication; Telomere; Base excision repair; Recombination

1. Werner syndrome (WS) as a model system to study normal aging Studies of progeroid syndromes have advanced our understanding of how defects in cellular responses to DNA damage contribute to the aging process. Werner syndrome (WS), the best studied progeroid syndrome, is an autosomal recessive disorder characterized by inactivating mutations in WRN, the gene encoding WS protein (WRN). WRN belongs to the conserved RecQ family of DNA helicases. Mutations in other RecQ family members in humans cause Bloom syndrome and Rothmund–Thomson syndrome, both of which also exhibit features of premature aging (for comprehensive reviews on RecQ helicases, see (Hickson, 2003; Opresko et al., 2004a)). WS is characterized by predisposition to aging-related pathologies, the most prevailing of which is bilateral ocular cataracts (Huang et al., 2006). In addition, type II diabetes, *

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0531-5565/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.exger.2007.04.011

arteriosclerosis, osteoporosis, and tumors of mesenchymal origin are commonly observed in WS patients (Martin and Oshima, 2000). Patients begin to show signs of accelerated aging after puberty. Based on the updated International Registry of Werner syndrome (www.wernersyndrome.org), the average age of death from WS is 54 years, usually as a result of cancer and arteriosclerosis (Huang et al., 2006). Noticeably, a splicing mutation that results in the deletion of exon 26 is found in 80% of WS patients of Japanese origin (Moser et al., 1999). Other WRN mutations include nonsense and frameshift mutations, some of which generate C-terminally truncated proteins that lack a nuclear localization signal and are degraded in the cytoplasm. Interestingly, 20% of the patients diagnosed with WS lack WRN mutations and are classified as atypical WS patients. Among atypical WS patients, 15% carry missense mutations in the Lamin A gene (Chen et al., 2003). Strikingly, a rare childhood syndrome of premature aging, Hutchinson–Gilford syndrome, is also associated with mutations in Lamin A and is characterized by genomic instability (Liu et al., 2005). Although patients with atypical WS

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and Lamin A mutations do not develop cataracts or diabetes, evidence from these two representative progeria syndromes lend support to the hypothesis that genomic instability is positively associated with the process of normal aging. Intriguingly, the pattern of altered gene expression in fibroblasts derived from WS patients is reminiscent of the array of changes in cells from naturally aged control individuals (Kyng et al., 2003). Moreover, the aging phenotypes observed in WS have been reproduced in a WRN knockout mouse model in a short telomere background (Chang et al., 2004; Du et al., 2004). WRN is also considered a caretaker-type tumor suppressor. As a gate-keeper, the p53 tumor suppressor promotes apoptosis that in principle would suppress carcinogenesis; however, such a pathway of cell death is attenuated in WS cells (Spillare et al., 1999). Epigenetic inactivation of WRN, due to CpG island promoter hypermethylation, has also been detected in some cancer cells (Agrelo et al., 2006). Although premature aging syndromes are phenocopies of normal human aging, clinical and molecular evidence strongly suggest that WS is a relevant model system for studying human aging and the relationship between aging and cancer. 2. Biochemical characteristics of WRN WRN contains a 3 0 –5 0 exonuclease domain, acidic regions, a 3 0 –5 0 helicase domain, a RecQ C-terminal (RQC) domain, a helicase and ribonuclease D C-terminal (HRDC) domain, and a nuclear localization signal (NLS) (Fig. 1). Electrophoretic mobility shift assays using WRN domain fragments suggest that the RQC and HRDC domains of WRN fold independently and bind DNA in a structure-specific manner (von Kobbe et al., 2003b). The WRN RQC domain prefers to bind DNA structures resembling replication intermediates (forked and Holliday junctions) (von Kobbe et al., 2003b). The RQC domain also plays a role in WRN protein–protein interactions with FEN-1, BLM, TRF2, and PARP-1 (Brosh et al., 2001; von Kobbe et al., 2002, 2003a; Opresko et al., 2002). These DNA- and protein-binding features are consistent with recent structural studies of a WRN aa 949–1079 fragment that contains the WRN RQC domain (Hu et al., 2005), and a WRN aa 1142–1242 fragment that contains the HRDC domain (Kitano et al., 2006). Specifically, the RQC domain includes a 20 aa winged helix subdomain that binds to DNA and protein (Hu et al., 2005). Amino acid substitution of Lys-1016 in the winged helix domain

decreases WRN binding to fork or bubble DNA substrates and markedly reduces WRN helicase activity on fork, D-loop, and Holliday junction substrates (Lee et al., 2005). Additional biochemical studies are needed to verify the structural data that the winged helix domain also binds to proteins. Structural and biochemical studies were also recently performed using a human WRN aa 38–236 fragment or a mouse WRN aa 31–238 fragment that contains the exonuclease domain. The results of these studies suggest that the exonuclease domain of WRN belongs to the DnaQ family, which shares a conserved replicative proofreading 3 0 –5 0 -exonuclease (Perry et al., 2006; Choi et al., 2007). The results also suggest that WRN exonuclease activity is activated upon substrate DNA binding in a Zn2+-dependent manner (Choi et al., 2007). This is consistent with results from biochemical assays demonstrating that Zn2+ and Mn2+ stimulate WRN exonuclease activity (Choudhary et al., 2004). To date, the structures of the helicase domain of WRN and full-length WRN have not yet been solved. Members of the RecQ family share a conserved 3 0 –5 0 helicase domain. Helicases separate complementary strands of nucleic acids in a reaction coupled to NTP hydrolysis. WRN is a DNA structure-specific helicase. The non-B form DNA G-quadruplexes and triple helix are highly preferred substrates for WRN helicase. The next most preferred substrates for WRN are DNA molecules that resemble recombination intermediates (D-loops, Holliday junctions, and three-way junctions), followed by structures associated with DNA replication (bubbles, forks, and flaps) and 3 0 -single-stranded DNA tailed dsDNA (Opresko et al., 2004a). One of the critical steps of the early response to DNA double strand breaks (DSBs) involves 5 0 -recession of blunt DNA ends to generate 3 0 -protruding ssDNA tails. Telomeres also have 3 0 -ssDNA tails that are thought to be folded and protected in the form of D-loops. It is notable that 3 0 -ssDNA tailed duplex is a good substrate for WRN helicase, but 5 0 -ssDNA tailed or blunt duplex molecules are not (Mohaghegh et al., 2001), suggesting that WRN may play an important role in telomere maintenance and DSB repair. This is consistent with the observation that the helicase activity of WRN is required for lagging strand DNA synthesis at telomeres (Crabbe et al., 2004). Biochemical analyses indicate that the WRN helicase activity on forked duplex DNA is activated by telomeric binding proteins TRF2 and POT1 and by the MRN complex (Opresko et al., 2002; Cheng et al., 2004). It has also

Fig. 1. Schematic representation of the domains of WRN. RQC, RecQ conserved C-terminal domain; HRDC, helicase and RNase D C-terminal domain; NLS, nuclear localization signal; aa, amino acids.

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been reported that the WRN helicase activity on a bubble substrate can be stimulated by Rad52 (Baynton et al., 2003). In contrast, WRN helicase activity on a Holliday junction DNA substrate is inhibited by interaction with Rad52 (Baynton et al., 2003), c-Abl tyrosine kinase or the DNA-PK complex (Cheng et al., 2003; Karmakar et al., 2002; Yannone et al., 2001). These results suggest that the WRN helicase activity may be modulated by post-translational modifications, specific DNA structures, or interactions with other proteins or protein–DNA complexes. WRN contains a 3 0 –5 0 -exonuclease domain. WRN exonuclease prefers to digest dsDNA with a 5 0 -overhang, but can also degrade blunt-ended dsDNA, if the substrate contains a fork, Holliday junction, or D-loop (Shen and Loeb, 2000; Orren et al., 2002). Notably, WRN exonuclease does not degrade blunt-ended dsDNA with a 3 0 -overhang or ssDNA. Thus, WRN exonuclease is expected to be inactive at unprotected, linear telomeres and at processed DNA DSBs (i.e. DSBs with 3 0 -protruding ssDNA tails). Interaction with Ku70/Ku80 or BRCA1 stimulate WRN exonuclease activity (Yannone et al., 2001; Li and Comai, 2000; Cooper et al., 2000; Cheng et al., 2006), whereas interaction with PARP-1 or BLM or phosphorylation by c-Abl inhibit WRN exonuclease activity (von Kobbe et al., 2004a, 2002; Li et al., 2004; Cheng et al., 2003). The exonuclease activity of WRN is not required for lagging strand DNA synthesis at telomeres (Crabbe et al., 2004). However, the WRN exonuclease activity is required to complement the DNA end-joining defect in WS cells (Perry et al., 2006), and can promote survival after cellular exposure to the DNA cross-linking agent cis-platinum (Swanson et al., 2004). Thus, in contrast to the WRN helicase activity, the WRN exonuclease activity may play important roles in specific DNA repair pathways and may play an accessory role in telomere maintenance. Biochemical evidence suggests that WRN exonuclease activity may be involved in shortening of the 3 0 -sstelomeric tails and in resolution of telomeric D-loops (Opresko et al., 2004b). Adding to the complexity of the intrinsic WRN catalytic activities, WRN helicase and exonuclease appear to act in a coordinated manner; furthermore, recent studies indicate that WRN also catalyzes ssDNA annealing (Opresko et al., 2001; Machwe et al., 2005b). Progression of the WRN exonuclease on one side of a replication fork is inhibited by the action of WRN helicase on the other side of the fork, as WRN helicase converts dsDNA to ssDNA that cannot be digested by the WRN exonuclease. On a model telomeric D-loop, the WRN helicase and exonuclease act simultaneously to release the 3 0 -invading tail (Opresko et al., 2004b). Although the telomere binding proteins TRF1 and TRF2 limit digestion by WRN (Opresko et al., 2004b), another report shows that TRF2 may recruit WRN to process telomeric D-loops (Machwe et al., 2004). Although the biological significance of WRN DNA strand annealing is unknown, it could in principle compete with

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WRN unwinding activity (Machwe et al., 2005b); a recent report suggests that WRN strand annealing activity may play a role in regression of stalled DNA replication forks (Machwe et al., 2006). It will be of interest to determine which domains of WRN are required for DNA strand annealing, and to determine the molecular mechanisms involved in regulating and coordinating distinct WRN catalytic functions. 3. Roles of WRN in S-phase 3.1. DNA replication In unstressed conditions, WS fibroblasts display fewer replication initiation sites than wild-type fibroblasts (Takeuchi et al., 1982; Crabbe et al., 2004; Laud et al., 2005; Poot et al., 1992), and the duration of S-phase is prolonged in primary WS cells (Takeuchi et al., 1982; Crabbe et al., 2004; Laud et al., 2005; Poot et al., 1992). DNA replication is not bidirectional in WS cells, resulting in marked asymmetry of fork progression (Rodriguez-Lopez et al., 2002). Biochemical and cellular studies suggest that WRN is involved in both unperturbed DNA replication and the response to replication stress (Fig. 2). The ringshaped PCNA protein slides on primed DNA and interacts with the replicative DNA polymerases d and e for processive DNA synthesis. Many proteins bind to PCNA to modulate DNA replication, DNA repair, and checkpoint control (Indiani and O’Donnell, 2006). In mice, WRN interacts with PCNA and co-purifies with the 17S multiprotein DNA replication complex (Lebel et al., 1999). Recent studies show that both the exonuclease and acidic regions of WRN interact physically with PCNA (Rodriguez-Lopez et al., 2003). WRN also interacts with DNA polymerase d and stabilizes stalled replication forks when DNA polymerase d encounters G-quadruplex structures (Kamath-Loeb et al., 2001). In addition, the Xenopus orthologue of WRN, FFA-1, is required for the formation of replication foci (Yan et al., 1998). There is evidence for a functional interaction between WRN and FEN-1, implicating WRN in the processing of Okazaki fragments during lagging strand DNA synthesis (Brosh et al., 2001). Consistent with this observation, biochemical analysis demonstrates that a truncated WRN containing the exonuclease domain binds preferentially to forked substrates (von Kobbe et al., 2003b). These results suggest that WRN plays a role in the normal DNA replication process. WRN plays an important role in the response to DNA damage during S-phase. WRN-deficient cells are hypersensitive to clastogens that inhibit DNA replication, including DNA interstrand cross-linking agents, topoisomerase inhibitors (CPT, for example), and hydroxyurea (HU) (Poot et al., 2001; Lebel and Leder, 1998; Pichierri et al., 2003; Bohr et al., 2001). In contrast, WS cells are only mildly sensitive to c-irradiation-induced DSBs (Poot et al., 2001; Yannone et al., 2001; Bohr et al., 2001). WRN helicase preferentially unwinds DNA substrates that

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Fig. 2. Roles of WRN in S-phase. WRN is involved in normal DNA replication, telomere maintenance, early responses to replication stress, and DNA DSB repair in S-phase. Proteins reported to interact with WRN in these specific pathways are shown.

resemble replicating DNA, including forks, bubbles, D-loops, Holliday junctions, and G-quartets (Opresko et al., 2004a). SGS1, the RecQ helicase in yeast, is required to stabilize stalled replication forks (Cobb et al., 2003). In this context, WRN strand annealing activity may promote fork stabilization and prevent inappropriate pairing or strand exchange between nascent strands (Machwe et al., 2005a). Replication forks that regress and form Holliday junction structures (also known as chicken foot structures) have been proposed as an initial step in the resolution of stalled replication forks. Because WRN can branch migrate RPA-coated Holliday junction substrates (Constantinou et al., 2000), WRN activities must be carefully regulated to assure a specific outcome with different DNA structures. It should also be kept in mind that DNA in the nucleus is usually associated with proteins. In this regard, it will be of interest to employ novel protein-containing DNA substrates to investigate the precise role(s) of WRN in DNA metabolism, as well as to determine its distinct catalytic activities during S-phase in cells with or without DNA damage. 3.2. Replicative senescence and telomere maintenance Cells derived from WS patients show many features of accelerated aging. In 1965, it was shown that primary WS cells senesce after fewer population doublings than normal primary cells (20 vs. 40–100) (Martin et al., 1965). The diminished replicative lifespan of WS cells is associated with a decline in the mitotic fraction after each duplication cycle (Faragher et al., 1993). Recent studies established that the premature replicative senescence of WS cells is caused by dysfunctional telomere maintenance. WRN associates with telomeric DNA and TRF1 during

S-phase in telomerase-deficient ALT cells but not in telomerase-positive HeLa cells (Opresko et al., 2004b). The budding yeast RecQ helicase Sgs1 also interacts with telomere components in ALT cells (Johnson et al., 2001). However, a higher rate of telomere loss may not completely explain the replicative senescence in primary WS cells. WRN helicase and exonuclease activities are required to resolve telomeric D-loops in vitro (Opresko et al., 2004b), suggesting a role for WRN in telomere recombination. Furthermore, WRN helicase activity is required to suppress sister chromatid exchange in telomeres (Laud et al., 2005), and for lagging strand DNA synthesis during telomere replication (Crabbe et al., 2004). Thus, both telomere shortening and dysfunction may lead to premature replicative senescence in primary WS cells. This is consistent with the observation that overexpression of telomerase prevents replicative senescence in WS cells (Wyllie et al., 2000). However, introduction of human telomerase reverse transcriptase (hTERT) in WS cells alters pattern of gene expression (Choi et al., 2001), suggesting that other factors may also regulate replicative senescence in WS cells. An understanding of the mechanism by which WRN participates in telomere metabolism in ALT cells is likely to provide insight into the cause of mesenchymal tumorigenesis in WS patients, because ALT cell lines are usually of mesenchymal origin. 3.3. An upstream role in response to replication stress In response to DNA damage, normal human diploid fibroblasts enter an irreversible state of proliferation arrest, known as stress-induced senescence. Recent studies showed that H2O2-induced oxidative DNA lesions accumulate in diploid WS fibroblasts, and that these damaged cells do

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not undergo cellular senescence (von Kobbe et al., 2004b). Recent reports also suggest that WRN could play an upstream role in the cellular response to DNA damage during S-phase. CAF1 is a histone chaperone that deposits histones H3 and H4 onto newly replicated DNA (Smith and Stillman, 1989). Jiao et al. showed that WRN is required for a replication-dependent chromatin assembly factor 1 (CAF1) complex to form nuclear foci after cellular exposure to the replication blocker HU (Jiao et al., 2006). Transfection of telomeric oligonucleotides with a 3 0 -overhang induces a DNA damage response, as evidenced by phosphorylation of p53 and H2AX in normal cells; interestingly, this response is compromised in WRN-deficient cells (Eller et al., 2006). Available evidence also suggests that WRN is involved in the response to replication stress in an ATM/ATR-dependent manner (Pichierri et al. 2001, 2003). Consistent with this notion, another RecQ helicase, BLM, is required for activation of ATM following prolonged exposure to HU (Davalos et al., 2004), and deletion of the yeast homolog of WRN, Sgs1, leads to defective S-phase checkpoint activation in stressed cells (Frei and Gasser, 2000). We thus propose an upstream role for WRN in the response to replication stress. Several lines of evidence support the view that WRN might play an upstream role in the response to DSBs at replication forks. WRN is required for activation of ATM and phosphorylation of downstream ATM substrates in cells with collapsed replication forks (Cheng and Bohr, unpublished). This resembles the role of the MRN complex, which is required for activation of ATM in response to DSBs induced by ionizing radiation or radiomimetic chemicals (Uziel et al., 2003; Carson et al., 2003; Costanzo et al., 2004; Lee and Paull, 2005; Dupre et al., 2006). Biochemical evidence also indicates that WRN binds preferentially to DNA substrates that resemble replication structures (von Kobbe et al., 2003b). These observations suggest that WRN may play a role in the rapid response to collapsed replication forks. It is also likely that WRN participates in the metabolism of collapsed replication forks after an initial stress response signal is generated and amplified. As such, WRN would at least partially meet the criteria for a DNA damage sensor in the context of a replication checkpoint (Petrini and Stracker, 2003). 3.4. A downstream role in response to replication stress Cellular and biochemical evidence indicate that WRN plays a role in the repair of DSBs and interstrand crosslinks (ICLs) during S-phase (Prince et al., 2001; Saintigny et al., 2002; Ahn et al., 2004; Cheng et al., 2006). An early report showed that WS lymphoblasts are hypersensitive to CPT-induced cell killing during S-phase (Poot et al., 1999). Using a neo gene selection assay, it was shown that WRN participates in the resolution of recombinational intermediates in a Rad51-dependent manner (Prince et al., 2001; Saintigny et al., 2002). Immunofluorescence studies also

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show that WRN co-localizes with Rad51 and RPA in cells exposed to CPT (Sakamoto et al., 2001). WRN cooperates with BRCA1 during processing of DNA ICLs, and the WRN-BRCA1 association increases 12 h after induction of DNA ICLs, when the majority of cells are in S-phase (Cheng et al., 2006). Therefore, WRN is likely involved in the repair of DNA DSBs and ICLs via homologous recombination. Indeed, WRN is shown to move along a Holliday junction substrate in the presence of RPA (Constantinou et al., 2000), suggesting a role for WRN during resolution of recombination intermediates. The DNA repair functions of WRN are likely to be different in different cellular contexts (i.e., at different stages of the cell cycle); this is consistent with the observation that WRN is required for the recovery from replication stress (such as exposure to HU or CPT) and it is phosphorylated in an ATR/ATM-dependent manner during S-phase in stressed cells (Pichierri et al., 2001, 2003). 4. Conclusions and perspectives A large body of evidence suggests that WRN is involved in the response to replication stress and in telomere maintenance during S-phase. In particular, WS cells are more sensitive to DNA ICLs than to any other type of DNA damage. DNA ICL repair in eukaryotic cells is less well understood than repair of other types of DNA damage. This is partially due to the complexity of ICL repair, which involves nucleotide excision repair, homologous recombination, post-replication repair, and cell cycle regulation (McHugh and Sarkar, 2006). During ICL processing, the ERCC1/XPF complex excises one strand of the crosslinked DNA independent DNA replication; however, the subsequent DNA DSB formation is largely confined to S-phase (Rothfuss and Grompe, 2004). Given the high susceptibility of WS cells to DNA ICLs and the possible role of WRN helicase in ICL unhooking (Cheng et al., 2006; Zhang et al., 2005), it will be important to elucidate the precise role of WRN in ICL repair. The role of WRN in signaling replication stress during S-phase, and its role in telomere recombination are also important areas for future research. To this end, DNA repair systems specific for telomeres would be very useful. Moreover, well-designed cellular and genetic studies are warranted to provide further understanding of the functional and physiological significance of the many observed WRN protein–protein interactions. Recently, Niedernhofer et al. created an Ercc1 knockout (Ercc1/) mouse that exhibits symptoms of progeria. ERCC1 forms a complex with XPF as the initial process for the repair of DNA ICLs. Remarkably, liver and kidney transcriptomes from young Ercc1/ mice, naturally aged wild-type mice, and young wild-type mice challenged with mitomycin C (an inducer of DNA ICLs) show similar changes in gene expression relative to appropriate controls (Niedernhofer et al., 2006). Similarly, transcriptomes from WS fibroblasts and human cells treated with c-irradiation

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show similar changes in the expression of stress–response genes (Kyng et al., 2005). These recent reports using models of human progeria shed light on a frequently asked question in the field of aging research: is aging the inevitable corollary of the biological clock or is it the consequence of inadequate DNA repair? Apparently, these two theories of aging can be at least partially reconciled. Nevertheless, pathways other than DNA repair may also contribute to the aging process, through yet to be clarified mechanisms. Although it remains a challenge to elucidate the mechanism of WRN action in the DNA DSB response, DNA replication, and telomere maintenance during S-phase, it will be helpful to develop novel tools to diagnose and possibly treat WS patients, potentially by manipulating WRN expression and function. Acknowledgements We thank Jason Aulds and Alexandra Gorgevska for comments. This work was supported by the Intramural Research Program for the NIH, National Institute on Aging. References Agrelo, R., Cheng, W.H., Setien, F., Ropero, S., Espada, J., Fraga, M.F., Herranz, M., Paz, M.F., Sanchez-Cespedes, M., Artiga, M.J., Guerrero, D., Castells, A., von Kobbe, C., Bohr, V.A., Esteller, M., 2006. Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer. Proc. Natl. Acad. Sci. USA 103, 8822–8827. Ahn, B., Harrigan, J.A., Indig, F.E., Wilson III, D.M., Bohr, V.A., 2004. Regulation of WRN helicase activity in human base excision repair. J. Biol. Chem. 279, 53465–53474. Baynton, K., Otterlei, M., Bjoras, M., von Kobbe, C., Bohr, V.A., Seeberg, E., 2003. WRN interacts physically and functionally with the recombination mediator protein RAD52. J. Biol. Chem. 278, 36476– 36486. Bohr, V.A., Souza, P.N., Nyaga, S.G., Dianov, G., Kraemer, K., Seidman, M.M., Brosh Jr., R.M., 2001. DNA repair and mutagenesis in Werner syndrome. Environ. Mol. Mutagen. 38, 227–234. Brosh, R.M., von Kobbe, C., Sommers, J.A., Karmakar, P., Opresko, P.L., Piotrowski, J., Dianova, I., Dianov, G.L., Bohr, V.A., 2001. Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. EMBO J. 20, 5791–5801. Carson, C.T., Schwartz, R.A., Stracker, T.H., Lilley, C.E., Lee, D.V., Weitzman, M.D., 2003. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J. 22, 6610–6620. Chang, S., Multani, A.S., Cabrera, N.G., Naylor, M.L., Laud, P., Lombard, D., Pathak, S., Guarente, L., DePinho, R.A., 2004. Essential role of limiting telomeres in the pathogenesis of Werner syndrome. Nat. Genet. 36, 877–882. Chen, L., Lee, L., Kudlow, B.A., Dos Santos, H.G., Sletvold, O., Shafeghati, Y., Botha, E.G., Garg, A., Hanson, N.B., Martin, G.M., Mian, I.S., Kennedy, B.K., Oshima, J., 2003. LMNA mutations in atypical Werner’s syndrome. Lancet 362, 440–445. Cheng, W.H., Kusumoto, R., Opresko, P.L., Sui, X., Huang, S., Nicolette, M.L., Paull, T.T., Campisi, J., Seidman, M., Bohr, V.A., 2006. Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links. Nucleic Acids Res. 34, 2751–2760. Cheng, W.H., von Kobbe, C., Opresko, P.L., Arthur, L.M., Komatsu, K., Seidman, M.M., Carney, J.P., Bohr, V.A., 2004. Linkage between Werner syndrome protein and the Mre11 complex via Nbs1. J. Biol. Chem. 279, 21169–21176.

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