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Making ends meet in old age: DSB repair and aging
Mechanisms of Ageing and Development 126 (2005) 621–628 www.elsevier.com/locate/mechagedev
Mini-review
Making ends meet in old age: DSB repair and aging Vera Gorbunova *, Andrei Seluanov Department of Biology, University of Rochester, River Campus Box 270211, Rochester, NY 14627, USA Received 22 December 2004; received in revised form 14 February 2005; accepted 14 February 2005 Available online 24 March 2005
Abstract Accumulation of somatic mutations has long been considered as a major cause of aging and age-related diseases such as cancer. Genomic rearrangements, which arise from aberrant repair of DNA breaks, are the most characteristic component of the mutation spectra in aging cells and tissues. The studies conducted in the past few years provide further support for the role of DNA double-strand break (DSB) repair in aging and cellular senescence. Evidence was obtained that in addition to accumulation of mutations the efficiency and fidelity of repair declines with age. We propose that DNA damage and age-related decline of DNA repair form a vicious cycle leading to amplification of damage and progression of aging, and discuss a hypothesis on how the interplay between the two pathways of DSB repair, homologous recombination and nonhomologous end joining, may contribute to the aging process. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: DNA repair; Aging; Double-strand DNA breaks; Homologous recombination; Nonhomologous DNA end joining
1. Accumulation of mutations The vital information about cellular structure and function is encoded in the DNA. Cellular DNA is constantly subjected to damage, which comes from the environment and as a result of cellular metabolism. A disruption of one essential DNA sequence may kill the cell. To deal with the damage cells have evolved elaborate DNA repair machinery. DNA repair is not perfect and frequently results in a mutation. In contrast to a DNA lesion, mutation becomes a permanent part of a DNA sequence and is transmitted to the following generations of cells. Having some level of mutagenesis is important as mutations are the driving force of evolution, however, except for those rare mutations that confer selective advantage, mutations are almost always deleterious. So, what happens to the DNA during aging? Accumulation of mutations has been studied extensively in human and mouse using various genetic systems. HPRT gene has been the most popular, since it provided a convenient selectable system to study mutations in cultured cells isolated * Corresponding author. Tel.: +1 585 275 7740; fax: +1 585 275 2070. E-mail address: [email protected] (V. Gorbunova).
from individuals of different age (Dempsey et al., 1993; Finette et al., 1994; Jones et al., 1995; Morley, 1998; Morley et al., 1982). These studies indicated accumulation of mutations with age in both human and mouse. A different set of assay systems based on the use of mice with transgenic reporter genes allowed the study of mutation frequency without additional in vitro cell culture. The assay systems are based on the chromosomally integrated lacZ (Vijg et al., 1997) or lacI (Kohler et al., 1991; Stuart and Glickman, 2000; Stuart et al., 2000) reporter gene cloned into a shuttle vector. Mutations arising within the reporter gene can be rescued from different mouse tissues, transferred to E. coli and sequenced. Using these mice, it was demonstrated that point mutations accumulate with age (Dolle et al., 1997; Dolle et al., 2000; Stuart and Glickman, 2000; Stuart et al., 2000; Vijg, 2000) and furthermore, the mutation rate is higher in old animals (Stuart and Glickman, 2000). An important conclusion from these studies is that the accumulation of mutations during lifespan will eventually reduce fitness and may be responsible for the aging phenotype. Furthermore, the increase of mutation rate suggests that the DNA repair systems become less efficient with age, which further contributes to the loss of fitness.
0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.02.008
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2. Genome rearrangements – a signature of old cells Increased incidence of cancer with age (DePinho, 2000) is the best-known evidence of age-related genomic instability. Chromosomal abnormalities are a hallmark of most tumors, and genomic instability is believed to be a prerequisite for tumorogenesis (Nowell, 1976). Genome stability during normal aging has been studied by cytogenetic methods (Curtis and Crowley, 1963; Martin et al., 1985; Ramsey et al., 1995; Tucker et al., 1999). An increase in the frequency of chromosomal aberrations such as translocations, insertions, dicentrics and acentric fragments was observed in human lymphocytes (Ramsey et al., 1995), and an increase in the frequency of translocations was observed in mouse lympocytes (Tucker et al., 1999) and in mouse kidney cells (Martin et al., 1985). Interestingly, nearly all the increase in chromosomal aberrations was observed above the age of 50 in human and above the age of 60 weeks in mice. The presence of a threshold at a similar relative point in the lifespan of two different species suggests that biological processes associated with aging, rather than accumulated damage is responsible for agerelated genomic instability. Southern blot analysis of HLA-A locus in human lymphocytes revealed similar accumulation of deletions and mitotic recombination events in older individuals (Grist et al., 1992). Interestingly, genomic instability has also been reported in old yeast cells (McMurray and Gottschling, 2003), suggesting that age-related genomic instability is a general phenomenon from unicellular to higher organisms. Transgenic mice carrying lacZ gene developed by Vijg and colleagues (Dolle et al., 1997) provide unique opportunity in addition to point mutations to monitor genomic rearrangements, and also to analyze the events on the sequence level. This has been instrumental to uncover the role of genome rearrangements in the aging process (Vijg and Dolle, 2002). Analysis of mutation spectra in these mice has shown that genome rearrangements were a characteristic component of the mutation spectra in old animals. Some of the rearrangements were very large, up to 66 Mb, with one breakpoint in the reporter gene and the other in the mouse flanking sequence (Dolle et al., 1997). Approximately, half of the breakpoints mapped to the chromosome carrying the reporter gene and another half of the events involved other chromosomes. Therefore, a complete repertoire of genome rearrangements was observed including deletions, inversions, and translocations. No regions of extended homology were found at the breakpoints, suggesting that the rearrangements may have resulted from erroneous non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). From these studies, it is possible to speculate that the fidelity of DSB repair deteriorates with aging, and also that perturbations of the end-joining pathway make the most contributions to this process.
3. Accumulation of DSB foci in senescent cells and in old mice Several studies using variations of the comet assay have shown that the number of DSBs increases in senescent cells (Chevanne et al., 2003) and in tissues of old mice (Singh et al., 2001). Recently, these results received a graphic confirmation, as it became possible to visualize the sites of DSBs in situ by monitoring nuclear foci of phosphorylated histone H2AX (g-H2AX). Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gamma H2AX foci (Rogakou et al., 1999). The foci then recruit DSB repair and signaling factors to the broken site (Paull et al., 2000). It was shown that g-H2AX foci form at telomeres uncapped through inhibition of telomere binding protein TRF2 (Takai et al., 2003), since inhibition of TRF2 induces senescence in primary fibroblasts (Smogorzewska and de Lange, 2002), these results suggest that DSB signaling is involved in induction of senescence. It was further demonstrated that replicatively senescent normal cells (Bakkenist et al., 2004; d’Adda di Fagagna et al., 2003; Sedelnikova et al., 2004) and aging mice (Sedelnikova et al., 2004) accumulate g-H2AX foci, which co-localize with DSB repair factors. Inactivation of DNA damage checkpoint kinases in senescent cells restored cell cycle progression into S phase (d’Adda di Fagagna et al., 2003), which is strong evidence that senescence is induced by DSB DNA damage checkpoint. There has been some controversy as to the location of the DSB foci, since d’Adda di Fagagna et al. (2003) demonstrated association of the foci with shortened telomeres, while the later (Sedelnikova et al., 2004) study did not find significant association with telomeres. It is likely, that accumulation of DSB at both telomeric and nontelomeric sites contributes to induction of senescence, and regardless of the location of the DSBs, the recent studies have proven that cellular senescence can be considered as cellular response to DNA damage (von Zglinicki et al., 2005).
4. Pathways of DSB repair DSBs in DNA are repaired by two major mechanisms: homologous recombination (HR) and non-homologous end joining. During HR-mediated repair of DSB, the sister chromatid is used as a template to copy the missing information into the broken locus. Repair by HR is mediated by hRAD51 protein with the help of other members of RAD52 epistasis group, single-strand binding protein RPA, BRCA1, BRCA2, and MRE11–RAD50–NBS1 nuclease complex (for detailed review see Helleday, 2003; Jackson, 2002). Since sister chromatids are identical to each other, DNA damage can be repaired faithfully with no genetic
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consequence. In contrast, the NHEJ pathway simply fuses two broken ends with little or no regard for sequence homology. NHEJ starts with binding of Ku heterodimer (Ku70–Ku80) to the broken DNA ends (Lieber, 1999). Ku facilitates recruitment of Artemis–DNA–PKcs complex, which is thought to trim single stranded overhangs (Lieber et al., 2003). Next, the ends are covalently joined by XRCC4–DNA ligase IV complex (Lieber et al., 2003). A few nucleotides at each end of the DNA break are lost in most instances during NHEJ event (Critchlow and Jackson, 1998; Lieber et al., 2003). NHEJ may also be associated with larger deletions or insertions of filler DNA (Gorbunova and Levy, 1997; Liang et al., 1998; Lin and Waldman, 2001a; Lin and Waldman, 2001b; Sargent et al., 1997). A crucial component of the DNA DSB signaling cascade in mammalian cells is the protein kinase, ATM (for reviews see Abraham, 2001; Shiloh, 2001). ATM is recruited to and activated at sites of DNA DSBs (Andegeko et al., 2001). Once activated, ATM then phosphorylates various downstream substrates, including p53, the checkpoint kinase CHK2, BRCA1 and NBS1 (for detailed reviews see refs. Abraham, 2001; Andegeko et al., 2001; Bartek et al., 2001; Khanna and Jackson, 2001; Shiloh, 2001; Zhou and Elledge, 2000). Mutations in DSB repair genes are often lethal. Remarkably, many of the viable mutations in DSB repair factors, which are discussed in the next section, lead to accelerated aging and premature senescence in culture, which further supports the role of DSB repair in aging.
5. Mutations in DSB repair factors cause premature senescence The premature aging syndrome, Werner syndrome, is caused by a mutation in a RecQ DNA helicase/nuclease (WRN) named after the disease. Werner patients display an increased risk for atherosclerosis, cancer, osteoporosis and type two diabetes, as well as early graying and loss of hair, atrophy of the skin and cataracts (Epstein et al., 1966; Goto, 1997). Cultured somatic cells from Werner patients have shortened replicative lifespan, display increased rates of genome rearrangements (Fukuchi et al., 1989) and extensive deletions during NHEJ (Oshima et al., 2002). Recent studies of WRN-interacting proteins further implicated WRN in DSB repair (reviewed in Bohr, 2002; Chen and Oshima, 2002; Comai and Li, 2004; Fry, 2002; Hickson, 2003; Opresko et al., 2003). WRN forms a tight complex with Ku heterodimer (Karmakar et al., 2002b; Li and Comai, 2000; Orren et al., 2001), and serves as a substrate for DNA–PKcs (Karmakar et al., 2002a; Yannone et al., 2001), which are the central components of NHEJ. WRN also interacts with the components of HR pathway. It binds RAD52 (Baynton et al., 2003) and colocalizes with RAD51 and RPA following DNA damage (Sakamoto et al., 2001). It was also shown that WRN associates with MRE11 complex via binding to NBS1 (Cheng et al., 2004).
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Another example of a mutation in DSB repair gene causing premature aging is Ku86. Ku86 knock out mice (Difilippantonio et al., 2000; Vogel et al., 1999) are completely defective in NHEJ and display various symptoms of accelerated aging including osteopososis, atropic skin, liver pathology, sepsis, cancer and shortened life-span (Vogel et al., 1999). DNA–PKcs is another key component of NHEJ pathway (Lieber et al., 2003). DNA–PKcsdeficient mice have shortened life-span, and show early onset of lymphomas, which is a common malignancy in old mice, loss of bone density, and kyphosis (abnormally increased curvature of the spine) (Espejel et al., 2004). ERCC1 is a structure-specific endonuclease that functions in HR repair of DSBs caused by DNA crosslinks (Niedernhofer et al., 2004). Disruption of mouse ERCC1 results in DNA repair deficiency, greatly reduced life span, abnormalities in liver, kidney and spleen, and lack of subcutaneous fat (Weeda et al., 1997). Similarly to other mutations in DNA repair genes, ERCC1 deficiency caused early onset of cellular senescence in primary fibroblasts (Weeda et al., 1997). Mutations of Atm gene in human result in the genetic disorder ataxia-telangiectasia (A-T). The hallmarks of A-T are neurodegeneration, immunodeficiency, genomic instability, cancer predisposition, and premature aging (Shiloh and Kastan, 2001). Fibroblasts from A-T patients are hypersensitive to DSBs, and have shortened replicative life-span (Shiloh et al., 1982). p53 protein provides further link between DSB repair and senescence. p53 is a major regulator of DNA damage response (Ko and Prives, 1996; Levine, 1997), it also plays a central role in induction of senescence (Itahana et al., 2001), and has been implicated in regulating HR (Bertrand et al., 1997; Dudenhoffer et al., 1999; Mekeel et al., 1997; Xia et al., 1997), and NHEJ (Akyuz et al., 2002; Bill et al., 1997; Lin et al., 2003; Okorokov et al., 2002; Tang et al., 1999). Heterozygous mice that express N-terminally truncated form of p53 have shortened life-span and display a number of premature aging phenotypes, possibly due to abnormal activation of p53 (Dumble et al., 2004; Maier et al., 2004; Tyner et al., 2002). Although telomerase is not a DSB repair factor per se, telomerase inactivation in mice leads to genomic instability (Rudolph et al., 1999) mediated by aberrant DSB repair of uncapped telomeres. What is the mechanism by which mutations in DSB repair genes or DSB DNA damage in general cause premature senescence? Defective genome maintenance may have multiple consequences. Deficient DNA repair is likely to result in genomic rearrangements, which may cause cancer if an oncogene or a tumor suppressor is involved, or cumulatively may impair transcription of essential genes and result in senescence (Vijg, 2004). DSBs may also induce the DNA damage response, which in turn may lead to senescence (von Zglinicki et al., 2005). Alternatively, DSBs may induce apoptosis. Elevated apoptosis will result in exhaustion of the stem cell pool, and lead to loss of organ
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cellularity and senescence. It is likely that all three pathways, impair transcription, cellular senescence, and apoptosis contribute to premature aging induced by DNA damage.
6. Why do old cells display genomic instability? We discussed the mechanisms that lead to premature senescence in DSB repair mutants, but what leads to increased mutation rate, chromosomal aberrations and genomic instability in normal aging? Impaired apoptosis is likely to contribute to age-related genomic instability. We have shown that presenescent human fibroblasts are unable to stabilize p53 in response to DNA damage, and consequently, do not undergo apoptosis when subjected to genotoxic stress (Seluanov et al., 2001). Down-regulation of DNA-damage induced apoptosis was also observed in vivo in old rats, where it was linked to alterations in MAP kinase signaling (Suh, 2002; Suh et al., 2002). p53 acquires a specific phosphorylation pattern in senescent cells, which may affect both its trans-activation and DNA repair activities (Webley et al., 2000). Altered DNA damage response is likely to lead to the changes in DSB repair in senescent cells. Another possibility, which has not received enough attention, is that DSB repair itself may decline with age. We have recently analyzed senescence-related changes in NHEJ in normal human fibroblasts (Seluanov et al., 2004). The efficiency of NHEJ was quantified in young, pre-senescent, and senescent fibroblasts using fluorescent reporter substrate. The efficiency of NHEJ was reduced up to 4.5 fold in presenescent and senescent relative to young cells. Decline of NHEJ efficiency has also been reported in rat brain during in vivo aging (Ren and de Ortiz, 2002). Strikingly, we observed that end joining in old cells was associated with extended deletions. Formation of extended deletions can be explained by competition between exonucleases and NHEJ activities at the sites of DSBs. The drop in the efficiency of end joining may allow more time for exonuclease degradation of the ends, leading to larger deletions. These results indicate that end joining becomes less efficient and more error-prone in senescent cells (Seluanov et al., 2004). Diminished efficiency and fidelity of NHEJ in old cells may explain the previously observed accumulation of DSBs (Bakkenist et al., 2004; Chevanne et al., 2003; d’Adda di Fagagna et al., 2003; Sedelnikova et al., 2004; Singh et al., 2001) and genomic rearrangements (Curtis and Crowley, 1963; Dolle et al., 1997; Grist et al., 1992; Ramsey et al., 1995; Tucker et al., 1999) in senescent cells and mice. It is tempting to propose that error-prone DSB repair and errorprone NHEJ, in particular, are responsible for genomic rearrangements and increased incidence of cancer with age. Formation of extended deletions at the sites of DSBs may lead to loss of genetic material and cell transformation. In addition to larger deletions at the site of a break, the persistence of DSBs due to lower efficiency of NHEJ could
Fig. 1. Relation between DNA damage, repair, genomic instability and aging. DNA damage causes mutations and genomic rearrangements and triggers cellular responses such as DNA damage response and apoptosis. Mutations and genomic rearrangements impair transcription of essential genes, induction of DNA damage response leads to cellular senescence, and increased apoptosis may result in exhaustion of the stem cell reserves. All these events result in functional decline, ultimately leading to aging. Agerelated changes, in turn, make DNA repair less efficient and more errorprone. The vicious cycle continues leading to amplification of damage and progression of aging.
lead to joining of inappropriate ends, giving rise to genomic rearrangements. In the model shown in Fig. 1, DNA damage elicits multiple cellular responses that ultimately lead to aging. However, aging itself may result in compromised DNA repair, which will cause even more damage, as depicted by the long reversed arrow (Fig. 1). This may be a selfamplifying process, and it is not clear yet what triggers the vicious cycle. There could be a putative regulatory process that shuts down genome maintenance systems past a certain age, or alternatively accumulation of a certain level of spontaneous damage may affect the function of DNA repair.
7. Interplay between HR and NHEJ in aging As we discussed above, the two pathways of DSB repair, HR and NHEJ differ in their fidelity. HR is usually precise, since the missing information is copied from genetically identical sister chromatid, while NHEJ may be associated
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with deletions or insertions of filler DNA. We (Gorbunova and Levy, 1997) and others (Liang et al., 1998; Lin and Waldman, 2001a; Lin and Waldman, 2001b; Sargent et al., 1997) have shown that filler DNA can be large and complex, with DNA sequences from different genomic locations being copied into the break site, leading to genomic rearrangements. Genomic rearrangements observed in aging cells are consistent with the products of NHEJ (Vijg and Dolle, 2002), suggesting that this repair pathway is responsible for age-related genomic instability. Both HR and NHEJ pathways are utilized to repair DSBs in mammalian cells. The exact mechanism that determines which pathway will be used at any given time is still unclear. It has recently been shown in yeast and in mammalian cells that the choice of DSB repair pathway depends on cell cycle stage (Ferreira and Cooper, 2004; Kruger et al., 2004; Rothkamm et al., 2003; Saleh-Gohari and Helleday, 2004). HR requires the repair template, and although the homologous chromosome is a potential repair template for HR, it is used three orders of magnitude less frequently than the sister chromatid (Johnson and Jasin, 2001). Therefore, it is safe for the cell to use HR when the sister chromatid is present. Inappropriate HR between repeated sequences may lead to gross genomic rearrangements. In a recent study, HR occurred at only low frequency in G1/G0 cells despite the presence of a homologous template on the same chromosome, which lead to the proposal that either recombination proteins are absent outside the 4N phase, or there may be factors that suppress HR in G1/G0 cells (Saleh-Gohari and Helleday, 2004). WRN protein interacts with components of both HR (Baynton et al., 2003; Cheng et al., 2004; Sakamoto et al., 2001) and NHEJ pathways (Karmakar et al., 2002a; Karmakar et al., 2002b; Li and Comai, 2000; Orren et al., 2001; Yannone et al., 2001), and the products of DSB repair are altered in Werner syndrome cells (Monnat and Saintigny, 2004; Prince et al., 2001). This led to an elegant hypothesis that WRN may play a role in the control DSB repair by suppressing NHEJ, and facilitating recruitment of error-free HR machinery during the S and G2 phases of cell cycle (Comai and Li, 2004). Thus, it is possible to speculate that activation of NHEJ contributes to premature senescence in WS cells. We propose, that a similar process may take place during normal aging. As postmitotic and senescent cells accumulate in the aging body, the mode of repair will shift from HR toward NHEJ. Since, the absolute efficiency of NHEJ declines with age (Seluanov et al., 2004), and NHEJ becomes the only available DSB repair pathway in senescent cells, this will result in greater accumulation of damage. Errors in HR and utilization of NHEJ would result in accumulation of genomic rearrangements, which lead to functional decline and age-associated diseases (Fig. 2). In agreement with this hypothesis, is the observation that HR is more efficient in embryonic than in adult cells (Arbones et al., 1994). Further studies of the DSB repair pathways with respect to aging are needed to test this model. It will be
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Fig. 2. Hypothetical model explaining how alterations of the ratio between HR and NHEJ lead to genomic instability during aging. Aging organisms accumulate nondividing cells. This may result in a higher proportion of DSBs repaired via error-prone NHEJ pathway, since the precise HR repair pathway only operates in dividing cells where the sister chromatid is available as repair template. Utilization of NHEJ pathway increases the frequency of deletions and genomic rearrangements leading to functional decline. WRN protein may play a role in suppressing the error-prone NHEJ and activating HR.
interesting to see how the ratio between HR and NHEJ changes during aging and cellular senescence, and whether stimulation of an error-free HR pathway may prevent agerelated genomic instability.
Acknowledgements We thank Dr. Thomas Eickbush for critically reading the manuscript. Work in the author’s laboratory is supported by the Ellison Medical Foundation and a start-up fund from the University of Rochester.
References Abraham, R.T., 2001. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177–2196. Akyuz, N., Boehden, G.S., Susse, S., Rimek, A., Preuss, U., Scheidtmann, K.H., Wiesmuller, L., 2002. DNA substrate dependence of p53mediated regulation of double-strand break repair. Mol. Cell. Biol. 22, 6303–6317. Andegeko, Y., Moyal, L., Mittelman, L., Tsarfaty, I., Shiloh, Y., Rotman, G., 2001. Nuclear retention of ATM at sites of DNA double strand breaks. J. Biol. Chem. 276, 38224–38230. Arbones, M.L., Austin, H.A., Capon, D.J., Greenburg, G., 1994. Gene targeting in normal somatic cells: inactivation of the interferon-gamma receptor in myoblasts. Nat. Genet. 6, 90–97. Bakkenist, C.J., Drissi, R., Wu, J., Kastan, M.B., Dome, J.S., 2004. Disappearance of the telomere dysfunction-induced stress response in fully senescent cells. Cancer Res. 64, 3748–3752. Bartek, J., Falck, J., Lukas, J., 2001. CHK2 kinase—a busy messenger. Nat. Rev., Mol. Cell. Biol. 2, 877–886. 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. Bertrand, P., Rouillard, D., Boulet, A., Levalois, C., Soussi, T., Lopez, B.S., 1997. Increase of spontaneous intrachromosomal homologous recom-
626
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628
bination in mammalian cells expressing mutant p53 protein. Oncogene 14, 1117–1122. Bill, C.A., Yu, Y., Miselis, N.R., Little, J.B., Nickoloff, J.A., 1997. A role for p53 in DNA end rejoining by human cell extracts. Mutat. Res. 385, 21– 29. Bohr, V.A., 2002. Human premature aging syndromes and genomic instability. Mech. Ageing Dev. 123, 987–993. Chen, L., Oshima, J., 2002. Werner syndrome. J. Biomed. Biotechnol. 2, 46–54. 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. Chevanne, M., Caldini, R., Tombaccini, D., Mocali, A., Gori, G., Paoletti, F., 2003. Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians. Biogerontology 4, 97–104. Comai, L., Li, B., 2004. The Werner syndrome protein at the crossroads of DNA repair and apoptosis. Mech. Ageing Dev. 125, 521–528. Critchlow, S.E., Jackson, S.P., 1998. DNA end-joining: from yeast to man. Trends Biochem. Sci. 23, 394–398. Curtis, H., Crowley, C., 1963. Chromosome aberrations in the liver cells in relation to the somatic mutation theory of aging. Radiat. Res. 19, 337– 344. d’Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N.P., Jackson, S.P., 2003. A DNA damage checkpoint response in telomere-initiated senescence. Nature 426, 194–198. Dempsey, J.L., Pfeiffer, M., Morley, A.A., 1993. Effect of dietary restriction on in vivo somatic mutation in mice. Mutat. Res. 291, 141–145. DePinho, R.A., 2000. The age of cancer. Nature 408, 248–254. Difilippantonio, M.J., Zhu, J., Chen, H.T., Meffre, E., Nussenzweig, M.C., Max, E.E., Ried, T., Nussenzweig, A., 2000. DNA repair protein Ku80 suppresses chromosomal aberratons and malignant transformation. Nature 404, 510–514. Dolle, M.E.T., Giese, H., Hopkins, C.L., Martus, H.-J., Hausdorf, J.M., Vijg, J., 1997. Rapid accumulation of genome rearrangements in liver but not in brain of old mice. Nat. Genet. 17, 431–434. Dolle, M.E.T., Snyder, W.K., Gossen, J.A., Lohman, P.H.M., Vijg, J., 2000. Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine. Proc. Natl. Acad. Sci. U.S.A. 97, 8403–8408. Dudenhoffer, C., Kurth, M., Janus, F., Depport, W., Wiesmuller, L., 1999. p53 domains involved in the control of recombination. Oncogene 18, 5773–5784. Dumble, M., Gatza, C., Tyner, S., Venkatachalam, S., Donehower, L.A., 2004. Insights into aging obtained from p53 mutant mouse models. Ann. N. Y. Acad. Sci. 1019, 171–177. Epstein, C.J., Martin, G.M., Schultz, A.L., Motulsky, A.G., 1966. Werner’s syndrome a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine (Baltimore) 45, 177–221. Espejel, S., Martin, M., Klatt, P., Martin-Caballero, J., Flores, J.M., Blasco, M.A., 2004. Shorter telomeres, accelerated ageing and increased lymphoma in DNA–PKcs-deficient mice. EMBO Rep. 5, 503–509. Ferreira, M.G., Cooper, J.P., 2004. Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle. Genes Dev. 18, 2249–2254. Finette, B.A., Sullivan, L.M., O’Neill, J.P., Nicklas, J.A., Vacek, P.M., Albertini, R.J., 1994. Determination of hprt mutant frequencies in Tlymphocytes from a healthy pediatric population: statistical comparison between newborn, children and adult mutant frequencies, cloning efficiency and age. Mutat. Res. 308, 223–231. Fry, M., 2002. The Werner syndrome helicase-nuclease—one protein, many mysteries. Sci. Aging Knowl. Environ. 2002, re2. Fukuchi, K., Martin, G.M., Monnat, R.J., 1989. Mutator phenotype of Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci. U.S.A. 86, 5893–5897.
Gorbunova, V., Levy, A.A., 1997. Nonhomologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res. 25, 4650–4657. Goto, M., 1997. Hierarchical deterioration of body systems in Werner’s syndrome: implications for normal ageing. Mech. Ageing Dev. 98, 239– 254. Grist, S.A., McCarron, M., Kutlaca, A., Turner, D.R., Morley, A.A., 1992. In vivo human somatic mutation: frequency and spectrum with age. Mutat. Res. 266, 189–196. Helleday, T., 2003. Pathways for mitotic homologous recombination in mammalian cells. Mutat. Res. 532, 103–115. Hickson, I.D., 2003. RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3, 169–178. Itahana, K., Dimri, G., Campisi, J., 2001. Regulation of cellular senescence by p53. Eur. J. Biochem. 268, 2784–2791. Jackson, S.P., 2002. Sensing and repairing DNA double-strand breaks. Carcinogenesis 23, 687–696. Johnson, R.D., Jasin, M., 2001. Double-strand-break-induced homologous recombination in mammalian cells. Biochem. Soc. Trans. 29, 196–201. Jones, I.M., Thomas, C.B., Tucker, B., Thompson, C.L., Pleshanov, P., Vorobtsova, I., Moore II, D.H., 1995. Impact of age and environment on somatic mutation at the hprt gene of T lymphocytes in humans. Mutat. Res. 338, 129–139. Karmakar, P., Piotrowski, J., Brosh Jr., R.M., Sommers, J.A., Miller, S.P., Cheng, W.H., Snowden, C.M., Ramsden, D.A., Bohr, V.A., 2002a. Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation. J. Biol. Chem. 277, 18291–18302. Karmakar, P., Snowden, C.M., Ramsden, D.A., Bohr, V.A., 2002b. Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus. Nucleic Acids Res. 30, 3583–3591. Khanna, K.K., Jackson, S.P., 2001. DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254. Ko, L.J., Prives, C., 1996. p53: puzzle and paradigm. Genes Dev. 10, 1054– 1072. Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Sorge, J.A., Putman, D.L., Short, J.M., 1991. Spectra of spontaneous and mutagen-induced mutations in the LacI gene in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 88, 7958–7962. Kruger, I., Rothkamm, K., Lobrich, M., 2004. Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells. Nucleic Acids Res. 32, 2677–2684. Levine, A.J., 1997. p53, the cellular gatekeeper for growth and division. Cell 88, 323–331. Li, B., Comai, L., 2000. Functional interaction between Ku and the werner syndrome protein in DNA end processing. J. Biol. Chem. 275, 28349– 28352. Liang, F., Han, M., Romanienko, P.J., Jasin, M., 1998. Homology-directed repair is a major double strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 95, 5172–5177. Lieber, M.R., 1999. The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes. Genes Cells 4, 77–85. Lieber, M.R., Ma, Y., Pannicke, U., Schwarz, K., 2003. Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev., Mol. Cell. Biol. 4, 712–720. Lin, Y., Waldman, A.S., 2001a. Capture of DNA sequences at double strand breaks in mammalian chromosomes. Genetics 195, 1665–1674. Lin, Y., Waldman, A.S., 2001b. Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA. Nucleics Acids Res. 29, 3975–3981. Lin, Y., Waldman, B.C., Waldman, A.S., 2003. Suppression of highfidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha, a chemical inhibitor of p53. DNA Repair (Amst.) 2, 1–11.
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628 Maier, B., Gluba, W., Bernier, B., Turner, T., Mohammad, K., Guise, T., Sutherland, A., Thorner, M., Scrable, H., 2004. Modulation of mammalian life span by the short isoform of p53. Genes Dev. 18, 306–319. Martin, G.M., Smith, A.C., Ketterer, D.J., Ogburn, C.E., Disteche, C.M., 1985. Increased chromosomal aberrations in first metaphases of cells isolated from the kidneys of aged mice. Isr. J. Med. Sci. 21, 296–301. McMurray, M.A., Gottschling, D.E., 2003. An age-induced switch to a hyper-recombinational state. Science 301, 1908–1911. Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.-M., DeFrank, J.S., Powell, S.N., 1997. Inactivation of p53 results in high rates of homologous recombination. Oncogene 14, 1847–1857. Monnat, R.J., Jr., Saintigny, Y., 2004. Werner syndrome protein—unwinding function to explain disease. Sci. Aging Knowl. Environ. 2004, re3. Morley, A., 1998. Somatic mutation and aging. Ann. N. Y. Acad. Sci. 854, 20–22. Morley, A.A., Cox, S., Holliday, R., 1982. Human lymphocytes resistant to 6-thioguanine increase with age. Mech. Ageing Dev. 19, 21–26. Niedernhofer, L.J., Odijk, H., Budzowska, M., van Drunen, E., Maas, A., Theil, A.F., de Wit, J., Jaspers, N.G., Beverloo, H.B., Hoeijmakers, J.H., Kanaar, R., 2004. The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks. Mol. Cell. Biol. 24, 5776–5787. Nowell, P.C., 1976. The clonal evolution of tumor cell populations. Science 194, 23–28. Okorokov, A.L., Warnock, L., Milner, J., 2002. Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential mechanism of DNA double-strand break repair modulation. Carcinogenesis 23, 549–557. Opresko, P.L., Cheng, W.H., von Kobbe, C., Harrigan, J.A., Bohr, V.A., 2003. Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process. Carcinogenesis 24, 791–802. Orren, D.K., Machwe, A., Karmakar, P., Piotrowski, J., Cooper, M.P., Bohr, V.A., 2001. A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA. Nucleic Acids Res. 29, 1926– 1934. Oshima, J., Huang, S., Pae, C., Campisi, J., Schiestl, R.H., 2002. Lack of WRN results in extensive deletion at nonhomologous joining ends. Cancer Res. 62, 547–551. Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., Bonner, W.M., 2000. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886– 895. Prince, P.R., Emond, M.J., Monnat Jr., R.J., 2001. Loss of Werner syndrome protein function promotes aberrant mitotic recombination. Genes Dev. 15, 933–938. Ramsey, M.J., Moore, D.H., Briner, J.F., Lee, D.A., Olsen, L.A., Senft, J.R., Tucker, J.D., 1995. The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. Mutat. Res. 338, 95–106. Ren, K., de Ortiz, S.P., 2002. Non-homologous DNA end joining in the mature rat brain. J. Neurochem. 80, 949–959. Rogakou, E.P., Boon, C., Redon, C., Bonner, W.M., 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916. Rothkamm, K., Kruger, I., Thompson, L.H., Lobrich, M., 2003. Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol. Cell. Biol. 23, 5706–5715. Rudolph, K.L., Chang, S., Lee, H.W., Blasco, M., Gottlieb, G.J., Greider, C., DePinho, R.A., 1999. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701–712. Sakamoto, S., Nishikawa, K., Heo, S.J., Goto, M., Furuichi, Y., Shimamoto, A., 2001. Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. Genes Cells 6, 421–430.
627
Saleh-Gohari, N., Helleday, T., 2004. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 32, 3683–3688. Sargent, R.G., Branneman, M.A., Wilson, J.H., 1997. Repair of site-specific double-strand breaks in mammalian chromosome by homologous and illegitimate recombination. Mol. Cell. Biol. 17, 267–277. Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner, W.M., Barrett, J.C., 2004. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170. Seluanov, A., Gorbunova, V., Falcovitz, A., Sigal, A., Milyavsky, M., Zurer, I., Shohat, G., Goldfinger, N., Rotter, V., 2001. Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53. Mol. Cell. Biol. 21, 1552–1564. Seluanov, A., Mittelman, D., Pereira-Smith, O.M., Wilson, J.H., Gorbunova, V., 2004. DNA end joining becomes less efficient and more errorprone during cellular senescence. Proc. Natl. Acad. Sci. U.S.A. 101, 7624–7629. Shiloh, Y., 2001. ATM and ATR: networking callular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77. Shiloh, Y., Kastan, M.B., 2001. ATM: genome stability, neuronal development, and cancer cross paths. Adv. Cancer Res. 83, 209–254. Shiloh, Y., Tabor, E., Becker, Y., 1982. Colony-forming ability of ataxiatelangiectasia skin fibroblasts is an indicator of their early senescence and increased demand for growth factors. Exp. Cell Res. 140, 191–199. Singh, N.P., Ogburn, C.E., Wolf, N.S., van Belle, G., Martin, G.M., 2001. DNA double-strand breaks in mouse kidney cells with age. Biogerontology 2, 261–270. Smogorzewska, A., de Lange, T., 2002. Different telomere damage signaling pathways in human and mouse cells. EMBO J. 21, 4338–4348. Stuart, G.R., Glickman, B.W., 2000. Through a glass, darkly: reflections of mutation from lacI transgenic mice. Genetics 155, 1359–1367. Stuart, G.R., Oda, Y., de Boer, J.G., Glickman, B.W., 2000. Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice. Genetics 154, 1291–1300. Suh, Y., 2002. Cell signaling in aging and apoptosis. Mech. Ageing Dev. 123, 881–890. Suh, Y., Lee, K.A., Kim, W.H., Han, B.G., Vijg, J., Park, S.C., 2002. Aging alters the apoptotic response to genotoxic stress. Nat. Med. 8, 3–4. Takai, H., Smogorzewska, A., de Lange, T., 2003. DNA damage foci at dysfunctional telomeres. Curr. Biol. 13, 1549–1556. Tang, W., Willers, H., Powell, S.N., 1999. p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in gamma-irradiated mouse fibroblasts. Cancer Res. 59, 2562–2565. Tucker, J.D., Spruill, M.D., Ramsey, M.J., Director, A.D., Nath, J., 1999. Frequency of spontaneous chromosome aberrations in mice: effects of age. Mutat. Res. 425, 135–141. Tyner, S.D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., Lu, X., Soron, G., Cooper, B., Brayton, C., Hee Park, S., Thompson, T., Karsenty, G., Bradley, A., Donehower, L.A., 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45–53. Vijg, J., 2000. Somatic mutations and aging: a re-evaluation. Mutat. Res. 447, 117–135. Vijg, J., 2004. Impact of genome instability on transcription regulation of aging and senescence. Mech. Ageing Dev. 125, 747–753. Vijg, J., Dolle, M.E.T., 2002. Large genome rearrangements as a primary cause of aging. Mech. Ageing Dev. 123, 907–915. Vijg, J., Dolle, M.E.T., Martus, H.-J., Boerrigter, M.E.T.I., 1997. Transgenic mouse models for studing mutations in vivo: applications in aging research. Mech. Ageing Dev. 98, 189–202. Vogel, H., Lim, D.S., Karsenty, J., Finegold, M., Hasty, P., 1999. Deletion of Ku 86 cases erly onset of senescence in mice. Proc. Natl. Acad. Sci. U.S.A. 96, 10770–10775. von Zglinicki, T., Saretzki, G., Ladhoff, J., d’Adda di Fagagna, F., Jackson, S.P., 2005. Human cell senescence as a DNA damage response. Mech. Ageing Dev. 126 (1), 111–117.
628
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628
Webley, K., Bond, J., Jones, C., Blaydes, J., Craig, A., Hupp, T., WynfordThomas, D., 2000. Posttranslational modification of p53 in replicative senescence overlapping but distinct from those induced by DNA damage. Mol. Cell. Biol. 20, 2803–2808. Weeda, G., Donker, I., de Wit, J., Morreau, H., Janssens, R., Vissers, C.J., Nigg, A., van Steeg, H., Bootsma, D., Hoeijmakers, J.H., 1997. Disription of mouse ERCC1 results in novel repair syndrome with growth failure, nulear abnormalities and senescence. Curr. Biol. 7, 427–439.
Xia, S.J., Shammas, M.A., Shmookler Reis, R.J., 1997. Elevated recombination in immortal human cells is mediated by hsRAD51 recombinase. Mol. Cell. Biol. 17, 7151–7158. Yannone, S.M., Roy, S., Chan, D.W., Murphy, M.B., Huang, S., Campisi, J., Chen, D.J., 2001. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J. Biol. Chem. 276, 38242– 38248. Zhou, B.B.S., Elledge, S.J., 2000. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.
Mini-review
Making ends meet in old age: DSB repair and aging Vera Gorbunova *, Andrei Seluanov Department of Biology, University of Rochester, River Campus Box 270211, Rochester, NY 14627, USA Received 22 December 2004; received in revised form 14 February 2005; accepted 14 February 2005 Available online 24 March 2005
Abstract Accumulation of somatic mutations has long been considered as a major cause of aging and age-related diseases such as cancer. Genomic rearrangements, which arise from aberrant repair of DNA breaks, are the most characteristic component of the mutation spectra in aging cells and tissues. The studies conducted in the past few years provide further support for the role of DNA double-strand break (DSB) repair in aging and cellular senescence. Evidence was obtained that in addition to accumulation of mutations the efficiency and fidelity of repair declines with age. We propose that DNA damage and age-related decline of DNA repair form a vicious cycle leading to amplification of damage and progression of aging, and discuss a hypothesis on how the interplay between the two pathways of DSB repair, homologous recombination and nonhomologous end joining, may contribute to the aging process. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: DNA repair; Aging; Double-strand DNA breaks; Homologous recombination; Nonhomologous DNA end joining
1. Accumulation of mutations The vital information about cellular structure and function is encoded in the DNA. Cellular DNA is constantly subjected to damage, which comes from the environment and as a result of cellular metabolism. A disruption of one essential DNA sequence may kill the cell. To deal with the damage cells have evolved elaborate DNA repair machinery. DNA repair is not perfect and frequently results in a mutation. In contrast to a DNA lesion, mutation becomes a permanent part of a DNA sequence and is transmitted to the following generations of cells. Having some level of mutagenesis is important as mutations are the driving force of evolution, however, except for those rare mutations that confer selective advantage, mutations are almost always deleterious. So, what happens to the DNA during aging? Accumulation of mutations has been studied extensively in human and mouse using various genetic systems. HPRT gene has been the most popular, since it provided a convenient selectable system to study mutations in cultured cells isolated * Corresponding author. Tel.: +1 585 275 7740; fax: +1 585 275 2070. E-mail address: [email protected] (V. Gorbunova).
from individuals of different age (Dempsey et al., 1993; Finette et al., 1994; Jones et al., 1995; Morley, 1998; Morley et al., 1982). These studies indicated accumulation of mutations with age in both human and mouse. A different set of assay systems based on the use of mice with transgenic reporter genes allowed the study of mutation frequency without additional in vitro cell culture. The assay systems are based on the chromosomally integrated lacZ (Vijg et al., 1997) or lacI (Kohler et al., 1991; Stuart and Glickman, 2000; Stuart et al., 2000) reporter gene cloned into a shuttle vector. Mutations arising within the reporter gene can be rescued from different mouse tissues, transferred to E. coli and sequenced. Using these mice, it was demonstrated that point mutations accumulate with age (Dolle et al., 1997; Dolle et al., 2000; Stuart and Glickman, 2000; Stuart et al., 2000; Vijg, 2000) and furthermore, the mutation rate is higher in old animals (Stuart and Glickman, 2000). An important conclusion from these studies is that the accumulation of mutations during lifespan will eventually reduce fitness and may be responsible for the aging phenotype. Furthermore, the increase of mutation rate suggests that the DNA repair systems become less efficient with age, which further contributes to the loss of fitness.
0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.02.008
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2. Genome rearrangements – a signature of old cells Increased incidence of cancer with age (DePinho, 2000) is the best-known evidence of age-related genomic instability. Chromosomal abnormalities are a hallmark of most tumors, and genomic instability is believed to be a prerequisite for tumorogenesis (Nowell, 1976). Genome stability during normal aging has been studied by cytogenetic methods (Curtis and Crowley, 1963; Martin et al., 1985; Ramsey et al., 1995; Tucker et al., 1999). An increase in the frequency of chromosomal aberrations such as translocations, insertions, dicentrics and acentric fragments was observed in human lymphocytes (Ramsey et al., 1995), and an increase in the frequency of translocations was observed in mouse lympocytes (Tucker et al., 1999) and in mouse kidney cells (Martin et al., 1985). Interestingly, nearly all the increase in chromosomal aberrations was observed above the age of 50 in human and above the age of 60 weeks in mice. The presence of a threshold at a similar relative point in the lifespan of two different species suggests that biological processes associated with aging, rather than accumulated damage is responsible for agerelated genomic instability. Southern blot analysis of HLA-A locus in human lymphocytes revealed similar accumulation of deletions and mitotic recombination events in older individuals (Grist et al., 1992). Interestingly, genomic instability has also been reported in old yeast cells (McMurray and Gottschling, 2003), suggesting that age-related genomic instability is a general phenomenon from unicellular to higher organisms. Transgenic mice carrying lacZ gene developed by Vijg and colleagues (Dolle et al., 1997) provide unique opportunity in addition to point mutations to monitor genomic rearrangements, and also to analyze the events on the sequence level. This has been instrumental to uncover the role of genome rearrangements in the aging process (Vijg and Dolle, 2002). Analysis of mutation spectra in these mice has shown that genome rearrangements were a characteristic component of the mutation spectra in old animals. Some of the rearrangements were very large, up to 66 Mb, with one breakpoint in the reporter gene and the other in the mouse flanking sequence (Dolle et al., 1997). Approximately, half of the breakpoints mapped to the chromosome carrying the reporter gene and another half of the events involved other chromosomes. Therefore, a complete repertoire of genome rearrangements was observed including deletions, inversions, and translocations. No regions of extended homology were found at the breakpoints, suggesting that the rearrangements may have resulted from erroneous non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). From these studies, it is possible to speculate that the fidelity of DSB repair deteriorates with aging, and also that perturbations of the end-joining pathway make the most contributions to this process.
3. Accumulation of DSB foci in senescent cells and in old mice Several studies using variations of the comet assay have shown that the number of DSBs increases in senescent cells (Chevanne et al., 2003) and in tissues of old mice (Singh et al., 2001). Recently, these results received a graphic confirmation, as it became possible to visualize the sites of DSBs in situ by monitoring nuclear foci of phosphorylated histone H2AX (g-H2AX). Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gamma H2AX foci (Rogakou et al., 1999). The foci then recruit DSB repair and signaling factors to the broken site (Paull et al., 2000). It was shown that g-H2AX foci form at telomeres uncapped through inhibition of telomere binding protein TRF2 (Takai et al., 2003), since inhibition of TRF2 induces senescence in primary fibroblasts (Smogorzewska and de Lange, 2002), these results suggest that DSB signaling is involved in induction of senescence. It was further demonstrated that replicatively senescent normal cells (Bakkenist et al., 2004; d’Adda di Fagagna et al., 2003; Sedelnikova et al., 2004) and aging mice (Sedelnikova et al., 2004) accumulate g-H2AX foci, which co-localize with DSB repair factors. Inactivation of DNA damage checkpoint kinases in senescent cells restored cell cycle progression into S phase (d’Adda di Fagagna et al., 2003), which is strong evidence that senescence is induced by DSB DNA damage checkpoint. There has been some controversy as to the location of the DSB foci, since d’Adda di Fagagna et al. (2003) demonstrated association of the foci with shortened telomeres, while the later (Sedelnikova et al., 2004) study did not find significant association with telomeres. It is likely, that accumulation of DSB at both telomeric and nontelomeric sites contributes to induction of senescence, and regardless of the location of the DSBs, the recent studies have proven that cellular senescence can be considered as cellular response to DNA damage (von Zglinicki et al., 2005).
4. Pathways of DSB repair DSBs in DNA are repaired by two major mechanisms: homologous recombination (HR) and non-homologous end joining. During HR-mediated repair of DSB, the sister chromatid is used as a template to copy the missing information into the broken locus. Repair by HR is mediated by hRAD51 protein with the help of other members of RAD52 epistasis group, single-strand binding protein RPA, BRCA1, BRCA2, and MRE11–RAD50–NBS1 nuclease complex (for detailed review see Helleday, 2003; Jackson, 2002). Since sister chromatids are identical to each other, DNA damage can be repaired faithfully with no genetic
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consequence. In contrast, the NHEJ pathway simply fuses two broken ends with little or no regard for sequence homology. NHEJ starts with binding of Ku heterodimer (Ku70–Ku80) to the broken DNA ends (Lieber, 1999). Ku facilitates recruitment of Artemis–DNA–PKcs complex, which is thought to trim single stranded overhangs (Lieber et al., 2003). Next, the ends are covalently joined by XRCC4–DNA ligase IV complex (Lieber et al., 2003). A few nucleotides at each end of the DNA break are lost in most instances during NHEJ event (Critchlow and Jackson, 1998; Lieber et al., 2003). NHEJ may also be associated with larger deletions or insertions of filler DNA (Gorbunova and Levy, 1997; Liang et al., 1998; Lin and Waldman, 2001a; Lin and Waldman, 2001b; Sargent et al., 1997). A crucial component of the DNA DSB signaling cascade in mammalian cells is the protein kinase, ATM (for reviews see Abraham, 2001; Shiloh, 2001). ATM is recruited to and activated at sites of DNA DSBs (Andegeko et al., 2001). Once activated, ATM then phosphorylates various downstream substrates, including p53, the checkpoint kinase CHK2, BRCA1 and NBS1 (for detailed reviews see refs. Abraham, 2001; Andegeko et al., 2001; Bartek et al., 2001; Khanna and Jackson, 2001; Shiloh, 2001; Zhou and Elledge, 2000). Mutations in DSB repair genes are often lethal. Remarkably, many of the viable mutations in DSB repair factors, which are discussed in the next section, lead to accelerated aging and premature senescence in culture, which further supports the role of DSB repair in aging.
5. Mutations in DSB repair factors cause premature senescence The premature aging syndrome, Werner syndrome, is caused by a mutation in a RecQ DNA helicase/nuclease (WRN) named after the disease. Werner patients display an increased risk for atherosclerosis, cancer, osteoporosis and type two diabetes, as well as early graying and loss of hair, atrophy of the skin and cataracts (Epstein et al., 1966; Goto, 1997). Cultured somatic cells from Werner patients have shortened replicative lifespan, display increased rates of genome rearrangements (Fukuchi et al., 1989) and extensive deletions during NHEJ (Oshima et al., 2002). Recent studies of WRN-interacting proteins further implicated WRN in DSB repair (reviewed in Bohr, 2002; Chen and Oshima, 2002; Comai and Li, 2004; Fry, 2002; Hickson, 2003; Opresko et al., 2003). WRN forms a tight complex with Ku heterodimer (Karmakar et al., 2002b; Li and Comai, 2000; Orren et al., 2001), and serves as a substrate for DNA–PKcs (Karmakar et al., 2002a; Yannone et al., 2001), which are the central components of NHEJ. WRN also interacts with the components of HR pathway. It binds RAD52 (Baynton et al., 2003) and colocalizes with RAD51 and RPA following DNA damage (Sakamoto et al., 2001). It was also shown that WRN associates with MRE11 complex via binding to NBS1 (Cheng et al., 2004).
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Another example of a mutation in DSB repair gene causing premature aging is Ku86. Ku86 knock out mice (Difilippantonio et al., 2000; Vogel et al., 1999) are completely defective in NHEJ and display various symptoms of accelerated aging including osteopososis, atropic skin, liver pathology, sepsis, cancer and shortened life-span (Vogel et al., 1999). DNA–PKcs is another key component of NHEJ pathway (Lieber et al., 2003). DNA–PKcsdeficient mice have shortened life-span, and show early onset of lymphomas, which is a common malignancy in old mice, loss of bone density, and kyphosis (abnormally increased curvature of the spine) (Espejel et al., 2004). ERCC1 is a structure-specific endonuclease that functions in HR repair of DSBs caused by DNA crosslinks (Niedernhofer et al., 2004). Disruption of mouse ERCC1 results in DNA repair deficiency, greatly reduced life span, abnormalities in liver, kidney and spleen, and lack of subcutaneous fat (Weeda et al., 1997). Similarly to other mutations in DNA repair genes, ERCC1 deficiency caused early onset of cellular senescence in primary fibroblasts (Weeda et al., 1997). Mutations of Atm gene in human result in the genetic disorder ataxia-telangiectasia (A-T). The hallmarks of A-T are neurodegeneration, immunodeficiency, genomic instability, cancer predisposition, and premature aging (Shiloh and Kastan, 2001). Fibroblasts from A-T patients are hypersensitive to DSBs, and have shortened replicative life-span (Shiloh et al., 1982). p53 protein provides further link between DSB repair and senescence. p53 is a major regulator of DNA damage response (Ko and Prives, 1996; Levine, 1997), it also plays a central role in induction of senescence (Itahana et al., 2001), and has been implicated in regulating HR (Bertrand et al., 1997; Dudenhoffer et al., 1999; Mekeel et al., 1997; Xia et al., 1997), and NHEJ (Akyuz et al., 2002; Bill et al., 1997; Lin et al., 2003; Okorokov et al., 2002; Tang et al., 1999). Heterozygous mice that express N-terminally truncated form of p53 have shortened life-span and display a number of premature aging phenotypes, possibly due to abnormal activation of p53 (Dumble et al., 2004; Maier et al., 2004; Tyner et al., 2002). Although telomerase is not a DSB repair factor per se, telomerase inactivation in mice leads to genomic instability (Rudolph et al., 1999) mediated by aberrant DSB repair of uncapped telomeres. What is the mechanism by which mutations in DSB repair genes or DSB DNA damage in general cause premature senescence? Defective genome maintenance may have multiple consequences. Deficient DNA repair is likely to result in genomic rearrangements, which may cause cancer if an oncogene or a tumor suppressor is involved, or cumulatively may impair transcription of essential genes and result in senescence (Vijg, 2004). DSBs may also induce the DNA damage response, which in turn may lead to senescence (von Zglinicki et al., 2005). Alternatively, DSBs may induce apoptosis. Elevated apoptosis will result in exhaustion of the stem cell pool, and lead to loss of organ
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cellularity and senescence. It is likely that all three pathways, impair transcription, cellular senescence, and apoptosis contribute to premature aging induced by DNA damage.
6. Why do old cells display genomic instability? We discussed the mechanisms that lead to premature senescence in DSB repair mutants, but what leads to increased mutation rate, chromosomal aberrations and genomic instability in normal aging? Impaired apoptosis is likely to contribute to age-related genomic instability. We have shown that presenescent human fibroblasts are unable to stabilize p53 in response to DNA damage, and consequently, do not undergo apoptosis when subjected to genotoxic stress (Seluanov et al., 2001). Down-regulation of DNA-damage induced apoptosis was also observed in vivo in old rats, where it was linked to alterations in MAP kinase signaling (Suh, 2002; Suh et al., 2002). p53 acquires a specific phosphorylation pattern in senescent cells, which may affect both its trans-activation and DNA repair activities (Webley et al., 2000). Altered DNA damage response is likely to lead to the changes in DSB repair in senescent cells. Another possibility, which has not received enough attention, is that DSB repair itself may decline with age. We have recently analyzed senescence-related changes in NHEJ in normal human fibroblasts (Seluanov et al., 2004). The efficiency of NHEJ was quantified in young, pre-senescent, and senescent fibroblasts using fluorescent reporter substrate. The efficiency of NHEJ was reduced up to 4.5 fold in presenescent and senescent relative to young cells. Decline of NHEJ efficiency has also been reported in rat brain during in vivo aging (Ren and de Ortiz, 2002). Strikingly, we observed that end joining in old cells was associated with extended deletions. Formation of extended deletions can be explained by competition between exonucleases and NHEJ activities at the sites of DSBs. The drop in the efficiency of end joining may allow more time for exonuclease degradation of the ends, leading to larger deletions. These results indicate that end joining becomes less efficient and more error-prone in senescent cells (Seluanov et al., 2004). Diminished efficiency and fidelity of NHEJ in old cells may explain the previously observed accumulation of DSBs (Bakkenist et al., 2004; Chevanne et al., 2003; d’Adda di Fagagna et al., 2003; Sedelnikova et al., 2004; Singh et al., 2001) and genomic rearrangements (Curtis and Crowley, 1963; Dolle et al., 1997; Grist et al., 1992; Ramsey et al., 1995; Tucker et al., 1999) in senescent cells and mice. It is tempting to propose that error-prone DSB repair and errorprone NHEJ, in particular, are responsible for genomic rearrangements and increased incidence of cancer with age. Formation of extended deletions at the sites of DSBs may lead to loss of genetic material and cell transformation. In addition to larger deletions at the site of a break, the persistence of DSBs due to lower efficiency of NHEJ could
Fig. 1. Relation between DNA damage, repair, genomic instability and aging. DNA damage causes mutations and genomic rearrangements and triggers cellular responses such as DNA damage response and apoptosis. Mutations and genomic rearrangements impair transcription of essential genes, induction of DNA damage response leads to cellular senescence, and increased apoptosis may result in exhaustion of the stem cell reserves. All these events result in functional decline, ultimately leading to aging. Agerelated changes, in turn, make DNA repair less efficient and more errorprone. The vicious cycle continues leading to amplification of damage and progression of aging.
lead to joining of inappropriate ends, giving rise to genomic rearrangements. In the model shown in Fig. 1, DNA damage elicits multiple cellular responses that ultimately lead to aging. However, aging itself may result in compromised DNA repair, which will cause even more damage, as depicted by the long reversed arrow (Fig. 1). This may be a selfamplifying process, and it is not clear yet what triggers the vicious cycle. There could be a putative regulatory process that shuts down genome maintenance systems past a certain age, or alternatively accumulation of a certain level of spontaneous damage may affect the function of DNA repair.
7. Interplay between HR and NHEJ in aging As we discussed above, the two pathways of DSB repair, HR and NHEJ differ in their fidelity. HR is usually precise, since the missing information is copied from genetically identical sister chromatid, while NHEJ may be associated
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with deletions or insertions of filler DNA. We (Gorbunova and Levy, 1997) and others (Liang et al., 1998; Lin and Waldman, 2001a; Lin and Waldman, 2001b; Sargent et al., 1997) have shown that filler DNA can be large and complex, with DNA sequences from different genomic locations being copied into the break site, leading to genomic rearrangements. Genomic rearrangements observed in aging cells are consistent with the products of NHEJ (Vijg and Dolle, 2002), suggesting that this repair pathway is responsible for age-related genomic instability. Both HR and NHEJ pathways are utilized to repair DSBs in mammalian cells. The exact mechanism that determines which pathway will be used at any given time is still unclear. It has recently been shown in yeast and in mammalian cells that the choice of DSB repair pathway depends on cell cycle stage (Ferreira and Cooper, 2004; Kruger et al., 2004; Rothkamm et al., 2003; Saleh-Gohari and Helleday, 2004). HR requires the repair template, and although the homologous chromosome is a potential repair template for HR, it is used three orders of magnitude less frequently than the sister chromatid (Johnson and Jasin, 2001). Therefore, it is safe for the cell to use HR when the sister chromatid is present. Inappropriate HR between repeated sequences may lead to gross genomic rearrangements. In a recent study, HR occurred at only low frequency in G1/G0 cells despite the presence of a homologous template on the same chromosome, which lead to the proposal that either recombination proteins are absent outside the 4N phase, or there may be factors that suppress HR in G1/G0 cells (Saleh-Gohari and Helleday, 2004). WRN protein interacts with components of both HR (Baynton et al., 2003; Cheng et al., 2004; Sakamoto et al., 2001) and NHEJ pathways (Karmakar et al., 2002a; Karmakar et al., 2002b; Li and Comai, 2000; Orren et al., 2001; Yannone et al., 2001), and the products of DSB repair are altered in Werner syndrome cells (Monnat and Saintigny, 2004; Prince et al., 2001). This led to an elegant hypothesis that WRN may play a role in the control DSB repair by suppressing NHEJ, and facilitating recruitment of error-free HR machinery during the S and G2 phases of cell cycle (Comai and Li, 2004). Thus, it is possible to speculate that activation of NHEJ contributes to premature senescence in WS cells. We propose, that a similar process may take place during normal aging. As postmitotic and senescent cells accumulate in the aging body, the mode of repair will shift from HR toward NHEJ. Since, the absolute efficiency of NHEJ declines with age (Seluanov et al., 2004), and NHEJ becomes the only available DSB repair pathway in senescent cells, this will result in greater accumulation of damage. Errors in HR and utilization of NHEJ would result in accumulation of genomic rearrangements, which lead to functional decline and age-associated diseases (Fig. 2). In agreement with this hypothesis, is the observation that HR is more efficient in embryonic than in adult cells (Arbones et al., 1994). Further studies of the DSB repair pathways with respect to aging are needed to test this model. It will be
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Fig. 2. Hypothetical model explaining how alterations of the ratio between HR and NHEJ lead to genomic instability during aging. Aging organisms accumulate nondividing cells. This may result in a higher proportion of DSBs repaired via error-prone NHEJ pathway, since the precise HR repair pathway only operates in dividing cells where the sister chromatid is available as repair template. Utilization of NHEJ pathway increases the frequency of deletions and genomic rearrangements leading to functional decline. WRN protein may play a role in suppressing the error-prone NHEJ and activating HR.
interesting to see how the ratio between HR and NHEJ changes during aging and cellular senescence, and whether stimulation of an error-free HR pathway may prevent agerelated genomic instability.
Acknowledgements We thank Dr. Thomas Eickbush for critically reading the manuscript. Work in the author’s laboratory is supported by the Ellison Medical Foundation and a start-up fund from the University of Rochester.
References Abraham, R.T., 2001. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177–2196. Akyuz, N., Boehden, G.S., Susse, S., Rimek, A., Preuss, U., Scheidtmann, K.H., Wiesmuller, L., 2002. DNA substrate dependence of p53mediated regulation of double-strand break repair. Mol. Cell. Biol. 22, 6303–6317. Andegeko, Y., Moyal, L., Mittelman, L., Tsarfaty, I., Shiloh, Y., Rotman, G., 2001. Nuclear retention of ATM at sites of DNA double strand breaks. J. Biol. Chem. 276, 38224–38230. Arbones, M.L., Austin, H.A., Capon, D.J., Greenburg, G., 1994. Gene targeting in normal somatic cells: inactivation of the interferon-gamma receptor in myoblasts. Nat. Genet. 6, 90–97. Bakkenist, C.J., Drissi, R., Wu, J., Kastan, M.B., Dome, J.S., 2004. Disappearance of the telomere dysfunction-induced stress response in fully senescent cells. Cancer Res. 64, 3748–3752. Bartek, J., Falck, J., Lukas, J., 2001. CHK2 kinase—a busy messenger. Nat. Rev., Mol. Cell. Biol. 2, 877–886. 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. Bertrand, P., Rouillard, D., Boulet, A., Levalois, C., Soussi, T., Lopez, B.S., 1997. Increase of spontaneous intrachromosomal homologous recom-
626
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628
bination in mammalian cells expressing mutant p53 protein. Oncogene 14, 1117–1122. Bill, C.A., Yu, Y., Miselis, N.R., Little, J.B., Nickoloff, J.A., 1997. A role for p53 in DNA end rejoining by human cell extracts. Mutat. Res. 385, 21– 29. Bohr, V.A., 2002. Human premature aging syndromes and genomic instability. Mech. Ageing Dev. 123, 987–993. Chen, L., Oshima, J., 2002. Werner syndrome. J. Biomed. Biotechnol. 2, 46–54. 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. Chevanne, M., Caldini, R., Tombaccini, D., Mocali, A., Gori, G., Paoletti, F., 2003. Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians. Biogerontology 4, 97–104. Comai, L., Li, B., 2004. The Werner syndrome protein at the crossroads of DNA repair and apoptosis. Mech. Ageing Dev. 125, 521–528. Critchlow, S.E., Jackson, S.P., 1998. DNA end-joining: from yeast to man. Trends Biochem. Sci. 23, 394–398. Curtis, H., Crowley, C., 1963. Chromosome aberrations in the liver cells in relation to the somatic mutation theory of aging. Radiat. Res. 19, 337– 344. d’Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N.P., Jackson, S.P., 2003. A DNA damage checkpoint response in telomere-initiated senescence. Nature 426, 194–198. Dempsey, J.L., Pfeiffer, M., Morley, A.A., 1993. Effect of dietary restriction on in vivo somatic mutation in mice. Mutat. Res. 291, 141–145. DePinho, R.A., 2000. The age of cancer. Nature 408, 248–254. Difilippantonio, M.J., Zhu, J., Chen, H.T., Meffre, E., Nussenzweig, M.C., Max, E.E., Ried, T., Nussenzweig, A., 2000. DNA repair protein Ku80 suppresses chromosomal aberratons and malignant transformation. Nature 404, 510–514. Dolle, M.E.T., Giese, H., Hopkins, C.L., Martus, H.-J., Hausdorf, J.M., Vijg, J., 1997. Rapid accumulation of genome rearrangements in liver but not in brain of old mice. Nat. Genet. 17, 431–434. Dolle, M.E.T., Snyder, W.K., Gossen, J.A., Lohman, P.H.M., Vijg, J., 2000. Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine. Proc. Natl. Acad. Sci. U.S.A. 97, 8403–8408. Dudenhoffer, C., Kurth, M., Janus, F., Depport, W., Wiesmuller, L., 1999. p53 domains involved in the control of recombination. Oncogene 18, 5773–5784. Dumble, M., Gatza, C., Tyner, S., Venkatachalam, S., Donehower, L.A., 2004. Insights into aging obtained from p53 mutant mouse models. Ann. N. Y. Acad. Sci. 1019, 171–177. Epstein, C.J., Martin, G.M., Schultz, A.L., Motulsky, A.G., 1966. Werner’s syndrome a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine (Baltimore) 45, 177–221. Espejel, S., Martin, M., Klatt, P., Martin-Caballero, J., Flores, J.M., Blasco, M.A., 2004. Shorter telomeres, accelerated ageing and increased lymphoma in DNA–PKcs-deficient mice. EMBO Rep. 5, 503–509. Ferreira, M.G., Cooper, J.P., 2004. Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle. Genes Dev. 18, 2249–2254. Finette, B.A., Sullivan, L.M., O’Neill, J.P., Nicklas, J.A., Vacek, P.M., Albertini, R.J., 1994. Determination of hprt mutant frequencies in Tlymphocytes from a healthy pediatric population: statistical comparison between newborn, children and adult mutant frequencies, cloning efficiency and age. Mutat. Res. 308, 223–231. Fry, M., 2002. The Werner syndrome helicase-nuclease—one protein, many mysteries. Sci. Aging Knowl. Environ. 2002, re2. Fukuchi, K., Martin, G.M., Monnat, R.J., 1989. Mutator phenotype of Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci. U.S.A. 86, 5893–5897.
Gorbunova, V., Levy, A.A., 1997. Nonhomologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res. 25, 4650–4657. Goto, M., 1997. Hierarchical deterioration of body systems in Werner’s syndrome: implications for normal ageing. Mech. Ageing Dev. 98, 239– 254. Grist, S.A., McCarron, M., Kutlaca, A., Turner, D.R., Morley, A.A., 1992. In vivo human somatic mutation: frequency and spectrum with age. Mutat. Res. 266, 189–196. Helleday, T., 2003. Pathways for mitotic homologous recombination in mammalian cells. Mutat. Res. 532, 103–115. Hickson, I.D., 2003. RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3, 169–178. Itahana, K., Dimri, G., Campisi, J., 2001. Regulation of cellular senescence by p53. Eur. J. Biochem. 268, 2784–2791. Jackson, S.P., 2002. Sensing and repairing DNA double-strand breaks. Carcinogenesis 23, 687–696. Johnson, R.D., Jasin, M., 2001. Double-strand-break-induced homologous recombination in mammalian cells. Biochem. Soc. Trans. 29, 196–201. Jones, I.M., Thomas, C.B., Tucker, B., Thompson, C.L., Pleshanov, P., Vorobtsova, I., Moore II, D.H., 1995. Impact of age and environment on somatic mutation at the hprt gene of T lymphocytes in humans. Mutat. Res. 338, 129–139. Karmakar, P., Piotrowski, J., Brosh Jr., R.M., Sommers, J.A., Miller, S.P., Cheng, W.H., Snowden, C.M., Ramsden, D.A., Bohr, V.A., 2002a. Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation. J. Biol. Chem. 277, 18291–18302. Karmakar, P., Snowden, C.M., Ramsden, D.A., Bohr, V.A., 2002b. Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus. Nucleic Acids Res. 30, 3583–3591. Khanna, K.K., Jackson, S.P., 2001. DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254. Ko, L.J., Prives, C., 1996. p53: puzzle and paradigm. Genes Dev. 10, 1054– 1072. Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Sorge, J.A., Putman, D.L., Short, J.M., 1991. Spectra of spontaneous and mutagen-induced mutations in the LacI gene in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 88, 7958–7962. Kruger, I., Rothkamm, K., Lobrich, M., 2004. Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells. Nucleic Acids Res. 32, 2677–2684. Levine, A.J., 1997. p53, the cellular gatekeeper for growth and division. Cell 88, 323–331. Li, B., Comai, L., 2000. Functional interaction between Ku and the werner syndrome protein in DNA end processing. J. Biol. Chem. 275, 28349– 28352. Liang, F., Han, M., Romanienko, P.J., Jasin, M., 1998. Homology-directed repair is a major double strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 95, 5172–5177. Lieber, M.R., 1999. The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes. Genes Cells 4, 77–85. Lieber, M.R., Ma, Y., Pannicke, U., Schwarz, K., 2003. Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev., Mol. Cell. Biol. 4, 712–720. Lin, Y., Waldman, A.S., 2001a. Capture of DNA sequences at double strand breaks in mammalian chromosomes. Genetics 195, 1665–1674. Lin, Y., Waldman, A.S., 2001b. Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA. Nucleics Acids Res. 29, 3975–3981. Lin, Y., Waldman, B.C., Waldman, A.S., 2003. Suppression of highfidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha, a chemical inhibitor of p53. DNA Repair (Amst.) 2, 1–11.
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628 Maier, B., Gluba, W., Bernier, B., Turner, T., Mohammad, K., Guise, T., Sutherland, A., Thorner, M., Scrable, H., 2004. Modulation of mammalian life span by the short isoform of p53. Genes Dev. 18, 306–319. Martin, G.M., Smith, A.C., Ketterer, D.J., Ogburn, C.E., Disteche, C.M., 1985. Increased chromosomal aberrations in first metaphases of cells isolated from the kidneys of aged mice. Isr. J. Med. Sci. 21, 296–301. McMurray, M.A., Gottschling, D.E., 2003. An age-induced switch to a hyper-recombinational state. Science 301, 1908–1911. Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.-M., DeFrank, J.S., Powell, S.N., 1997. Inactivation of p53 results in high rates of homologous recombination. Oncogene 14, 1847–1857. Monnat, R.J., Jr., Saintigny, Y., 2004. Werner syndrome protein—unwinding function to explain disease. Sci. Aging Knowl. Environ. 2004, re3. Morley, A., 1998. Somatic mutation and aging. Ann. N. Y. Acad. Sci. 854, 20–22. Morley, A.A., Cox, S., Holliday, R., 1982. Human lymphocytes resistant to 6-thioguanine increase with age. Mech. Ageing Dev. 19, 21–26. Niedernhofer, L.J., Odijk, H., Budzowska, M., van Drunen, E., Maas, A., Theil, A.F., de Wit, J., Jaspers, N.G., Beverloo, H.B., Hoeijmakers, J.H., Kanaar, R., 2004. The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks. Mol. Cell. Biol. 24, 5776–5787. Nowell, P.C., 1976. The clonal evolution of tumor cell populations. Science 194, 23–28. Okorokov, A.L., Warnock, L., Milner, J., 2002. Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential mechanism of DNA double-strand break repair modulation. Carcinogenesis 23, 549–557. Opresko, P.L., Cheng, W.H., von Kobbe, C., Harrigan, J.A., Bohr, V.A., 2003. Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process. Carcinogenesis 24, 791–802. Orren, D.K., Machwe, A., Karmakar, P., Piotrowski, J., Cooper, M.P., Bohr, V.A., 2001. A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA. Nucleic Acids Res. 29, 1926– 1934. Oshima, J., Huang, S., Pae, C., Campisi, J., Schiestl, R.H., 2002. Lack of WRN results in extensive deletion at nonhomologous joining ends. Cancer Res. 62, 547–551. Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., Bonner, W.M., 2000. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886– 895. Prince, P.R., Emond, M.J., Monnat Jr., R.J., 2001. Loss of Werner syndrome protein function promotes aberrant mitotic recombination. Genes Dev. 15, 933–938. Ramsey, M.J., Moore, D.H., Briner, J.F., Lee, D.A., Olsen, L.A., Senft, J.R., Tucker, J.D., 1995. The effects of age and lifestyle factors on the accumulation of cytogenetic damage as measured by chromosome painting. Mutat. Res. 338, 95–106. Ren, K., de Ortiz, S.P., 2002. Non-homologous DNA end joining in the mature rat brain. J. Neurochem. 80, 949–959. Rogakou, E.P., Boon, C., Redon, C., Bonner, W.M., 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916. Rothkamm, K., Kruger, I., Thompson, L.H., Lobrich, M., 2003. Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol. Cell. Biol. 23, 5706–5715. Rudolph, K.L., Chang, S., Lee, H.W., Blasco, M., Gottlieb, G.J., Greider, C., DePinho, R.A., 1999. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701–712. Sakamoto, S., Nishikawa, K., Heo, S.J., Goto, M., Furuichi, Y., Shimamoto, A., 2001. Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. Genes Cells 6, 421–430.
627
Saleh-Gohari, N., Helleday, T., 2004. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 32, 3683–3688. Sargent, R.G., Branneman, M.A., Wilson, J.H., 1997. Repair of site-specific double-strand breaks in mammalian chromosome by homologous and illegitimate recombination. Mol. Cell. Biol. 17, 267–277. Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner, W.M., Barrett, J.C., 2004. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170. Seluanov, A., Gorbunova, V., Falcovitz, A., Sigal, A., Milyavsky, M., Zurer, I., Shohat, G., Goldfinger, N., Rotter, V., 2001. Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53. Mol. Cell. Biol. 21, 1552–1564. Seluanov, A., Mittelman, D., Pereira-Smith, O.M., Wilson, J.H., Gorbunova, V., 2004. DNA end joining becomes less efficient and more errorprone during cellular senescence. Proc. Natl. Acad. Sci. U.S.A. 101, 7624–7629. Shiloh, Y., 2001. ATM and ATR: networking callular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77. Shiloh, Y., Kastan, M.B., 2001. ATM: genome stability, neuronal development, and cancer cross paths. Adv. Cancer Res. 83, 209–254. Shiloh, Y., Tabor, E., Becker, Y., 1982. Colony-forming ability of ataxiatelangiectasia skin fibroblasts is an indicator of their early senescence and increased demand for growth factors. Exp. Cell Res. 140, 191–199. Singh, N.P., Ogburn, C.E., Wolf, N.S., van Belle, G., Martin, G.M., 2001. DNA double-strand breaks in mouse kidney cells with age. Biogerontology 2, 261–270. Smogorzewska, A., de Lange, T., 2002. Different telomere damage signaling pathways in human and mouse cells. EMBO J. 21, 4338–4348. Stuart, G.R., Glickman, B.W., 2000. Through a glass, darkly: reflections of mutation from lacI transgenic mice. Genetics 155, 1359–1367. Stuart, G.R., Oda, Y., de Boer, J.G., Glickman, B.W., 2000. Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice. Genetics 154, 1291–1300. Suh, Y., 2002. Cell signaling in aging and apoptosis. Mech. Ageing Dev. 123, 881–890. Suh, Y., Lee, K.A., Kim, W.H., Han, B.G., Vijg, J., Park, S.C., 2002. Aging alters the apoptotic response to genotoxic stress. Nat. Med. 8, 3–4. Takai, H., Smogorzewska, A., de Lange, T., 2003. DNA damage foci at dysfunctional telomeres. Curr. Biol. 13, 1549–1556. Tang, W., Willers, H., Powell, S.N., 1999. p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in gamma-irradiated mouse fibroblasts. Cancer Res. 59, 2562–2565. Tucker, J.D., Spruill, M.D., Ramsey, M.J., Director, A.D., Nath, J., 1999. Frequency of spontaneous chromosome aberrations in mice: effects of age. Mutat. Res. 425, 135–141. Tyner, S.D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., Lu, X., Soron, G., Cooper, B., Brayton, C., Hee Park, S., Thompson, T., Karsenty, G., Bradley, A., Donehower, L.A., 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45–53. Vijg, J., 2000. Somatic mutations and aging: a re-evaluation. Mutat. Res. 447, 117–135. Vijg, J., 2004. Impact of genome instability on transcription regulation of aging and senescence. Mech. Ageing Dev. 125, 747–753. Vijg, J., Dolle, M.E.T., 2002. Large genome rearrangements as a primary cause of aging. Mech. Ageing Dev. 123, 907–915. Vijg, J., Dolle, M.E.T., Martus, H.-J., Boerrigter, M.E.T.I., 1997. Transgenic mouse models for studing mutations in vivo: applications in aging research. Mech. Ageing Dev. 98, 189–202. Vogel, H., Lim, D.S., Karsenty, J., Finegold, M., Hasty, P., 1999. Deletion of Ku 86 cases erly onset of senescence in mice. Proc. Natl. Acad. Sci. U.S.A. 96, 10770–10775. von Zglinicki, T., Saretzki, G., Ladhoff, J., d’Adda di Fagagna, F., Jackson, S.P., 2005. Human cell senescence as a DNA damage response. Mech. Ageing Dev. 126 (1), 111–117.
628
V. Gorbunova, A. Seluanov / Mechanisms of Ageing and Development 126 (2005) 621–628
Webley, K., Bond, J., Jones, C., Blaydes, J., Craig, A., Hupp, T., WynfordThomas, D., 2000. Posttranslational modification of p53 in replicative senescence overlapping but distinct from those induced by DNA damage. Mol. Cell. Biol. 20, 2803–2808. Weeda, G., Donker, I., de Wit, J., Morreau, H., Janssens, R., Vissers, C.J., Nigg, A., van Steeg, H., Bootsma, D., Hoeijmakers, J.H., 1997. Disription of mouse ERCC1 results in novel repair syndrome with growth failure, nulear abnormalities and senescence. Curr. Biol. 7, 427–439.
Xia, S.J., Shammas, M.A., Shmookler Reis, R.J., 1997. Elevated recombination in immortal human cells is mediated by hsRAD51 recombinase. Mol. Cell. Biol. 17, 7151–7158. Yannone, S.M., Roy, S., Chan, D.W., Murphy, M.B., Huang, S., Campisi, J., Chen, D.J., 2001. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J. Biol. Chem. 276, 38242– 38248. Zhou, B.B.S., Elledge, S.J., 2000. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.