MOLECULAR MECHANISMS OF MAMMALIAN DNA REPAIR AND THE DNA DAMAGE CHECKPOINTS

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1 Annu. Rev. Biochem :39 85 doi: /annurev.biochem Copyright 2004 by Annual Reviews. All rights reserved First published online as a Review in Advance on January 20, 2004 MOLECULAR MECHANISMS OF MAMMALIAN DNA REPAIR AND THE DNA DAMAGE CHECKPOINTS Aziz Sancar, 1 Laura A. Lindsey-Boltz, 1 Keziban Ünsal-Kaçmaz, 1 and Stuart Linn 2 1 Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina ; aziz_sancar@med.unc.edu, llindsey@med.unc.edu, kezi_unsal@med.unc.edu 2 Division of Biochemistry and Molecular Biology, University of California, Berkeley, California ; slinn@socrates.berkeley.edu Key Words damage recognition, excision repair, PIKK family, Rad17-RFC/ complex, signal transduction f Abstract DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-s checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed /04/ $

2 40 SANCAR et al. CONTENTS INTRODUCTION SUBSTRATES AND SIGNALS FOR DNA REPAIR AND DNA DAMAGE CHECKPOINTS Replication, Recombination, and Repair Intermediates DNA Base Damages DNA Backbone Damages Cross-links DNA DAMAGE RECOGNITION Direct Damage Recognition Multistep Damage Recognition Recognition by Proxy Recognition of DNA Repair Intermediates DNA REPAIR MECHANISMS Direct Repair Base Excision Repair Nucleotide Excision Repair Double-Strand Break Repair and Recombinational Repair Cross-link Repair DNA DAMAGE CHECKPOINTS What Are the DNA Damage Checkpoints? Molecular Components of the DNA Damage Checkpoints The G1/S Checkpoint The Intra-S-Phase Checkpoint The G2/M Checkpoint The Replication Checkpoint (S/M Checkpoint) CONCLUSIONS INTRODUCTION The primary structure of DNA is constantly subjected to alteration by cellular metabolites and exogenous DNA-damaging agents. These alterations may cause simple base changes or they may cause more complex changes including deletions, fusions, translocations, or aneuploidy. Such alterations may ultimately lead to cellular death of unicellular organisms or degenerative changes and aging of multicellular organisms. DNA damages can perturb the cellular steady-state quasi-equilibrium and activate or amplify certain biochemical pathways that regulate cell growth and division and pathways that help to coordinate DNA replication with damage removal. The four types of pathways elicited by DNA damage known, or presumed, to ameliorate harmful damage effects are DNA repair, DNA damage checkpoints, transcriptional response, and apoptosis (1) (Figure 1). Defects in any of these pathways may cause genomic instability (2). This review focuses upon the mechanisms of DNA repair and the DNA damage checkpoints.

3 DNA REPAIR AND THE DNA DAMAGE CHECKPOINTS 41 Figure 1 DNA damage response reactions in mammalian cells. The four responses (DNA repair, transcriptional response, DNA damage checkpoints, and apoptosis) may function independently, but frequently a protein primarily involved in one response may participate in other responses. SUBSTRATES AND SIGNALS FOR DNA REPAIR AND DNA DAMAGE CHECKPOINTS Proteins that bind to a specific sequence or structure in DNA, in contrast to enzymes with simple substrates, must recognize their target in the vast excess of related structures. Damage sensors not only bind undamaged DNA in search of damage, but they also contact undamaged DNA during specific binding. Therefore, they generally have nonnegligible affinity for undamaged DNA. Since the amount of undamaged DNA vastly exceeds that of damaged DNA, DNA damage sensors spend far more time associated with undamaged DNA than with damaged DNA. Yet, these sensors carry out their specific functions in the presence of high concentrations of nonspecific DNA, because damage recognition is usually a multistep reaction. There is thus a low probability that all of the steps will occur subsequent to the initial contact with undamaged DNA. However, even with multistep recognition, the discrimination between undamaged and damaged DNA is not absolute, so that neither DNA repair nor the DNA damage checkpoints should be envisioned as operated by molecular switches. Rather, both processes are operative at all times, but the magnitudes of the repair or checkpoint reactions are amplified by the presence of DNA damage. DNA damages include covalent changes in DNA structure and noncovalent anomalous structures, including base-pair mismatches, loops, and bubbles arising from a string of mismatches. The first are processed by DNA repair and recombination pathways, whereas the latter are processed by mismatch repair pathways (3, 4). This review focuses mostly on cellular responses to covalent

4 62 SANCAR et al. TABLE 2 DNA damage checkpoint proteins Protein function Mammals S. pombe S. cerevisiae Sensors RFC-like Rad17 Rad17 Rad24 PCNA-like Rad9 Rad9 Ddc1 Rad1 Rad1 Rad17 Hus1 Hus1 Mec3 PI3-Kinases (PIKK) ATM Tel1 Tel1 ATR Rad3 Mec1 PIKK binding partner ATRIP Rad26 Ddc2/Lcd1/Pie1 Mediators MDC1 53BP1 TopBP1 Cut5 Dpb11 Claspin Mrc1 Mrc1 BRCA1 Crb2/Rph9 Rad9 Transducers Kinase Chk1 Chk1 Chk1 Chk2 Cds1 Rad53 Molecular Components of the DNA Damage Checkpoints The DNA damage checkpoint, like other signal transduction pathways, conceptually has three components: sensors, signal transducers, and effectors (Table 2). Extensive genetic analysis in yeast and more recently in human cells, combined with rather limited biochemical studies, have helped to identify proteins involved in damage sensing, signal transduction, and effector steps of the DNA damage checkpoints (Figure 8). However, there is not an absolute demarcation between the various components of the checkpoint. For example, the damage sensor, ATM, also functions as a signal transducer. Moreover, a fourth class of checkpoint proteins, called mediators, has been identified. This class includes BRCA1, Claspin, 53BP1, and MDC1 and is conceptually placed between sensors and signal transducers. However, as in the case of ATM functioning as both a sensor and a transducer, these mediator proteins also appear to participate in more than one step of the checkpoint response. Although the G1/S, intra-s, and the G2/M checkpoints are distinct, the damage sensor molecules that activate the various checkpoints appear to either be shared by all three pathways or to play a primary sensor role in one pathway and a back-up role in the others. Similarly, the signal-transducing molecules, which are protein kinases and phosphatases, are shared by the different checkpoints to

5 DNA REPAIR AND THE DNA DAMAGE CHECKPOINTS 63 Figure 8 Components of the DNA damage checkpoints in human cells. The damage is detected by sensors that, with the aid of mediators, transduce the signal to transducers. The transducers, in turn, activate or inactivate other proteins (effectors) that directly participate in inhibiting the G1/S transition, S-phase progression, or the G2/M transition. varying degrees (1, 132). The effector components (proteins that inhibit phase transition) of the checkpoints are, naturally, what gives the checkpoints their unique identities. However, various sensors, mediators, and signal transducers

6 68 SANCAR et al. The G1/S Checkpoint The G1/S checkpoint prevents cells from entering the S phase in the presence of DNA damage by inhibiting the initiation of replication (Figure 9). Under suitable conditions, cells in the G1 phase of the cell cycle become committed to enter the S phase at a stage called the restriction point in mammalian cells and start in budding yeast (205). The restriction point precedes the actual start of DNA synthesis by about 2 h in human cells. If there is DNA damage, however, entry into S phase is prevented regardless of whether the cells have passed the restriction point. Current evidence suggests the following sequence of events for the G1/S checkpoint in human cells. If the DNA damage is double-strand breaks caused by ionizing radiation or radiomimetic agents, ATM is activated and phosphorylates many target molecules, notably p53 and Chk2. These phosphorylations result in the activation of two signal transduction pathways, one to initiate and one to maintain the G1/S arrest (204). The reaction that initiates the G1/S arrest is phosphorylation of Chk2, which in turn phosphorylates Cdc25A phosphatase, causing its inactivation by nuclear exclusion and ubiquitin-mediated proteolytic degradation (206, 207). Lack of active Cdc25A results in the accumulation of the phosphorylated (inactive) form of Cdk2, which is incapable of phosphorylating Cdc45 to initiate replication at the sites of preformed ORC-ORI complexes. If the DNA damage is by UV light or UV-mimetic agents, the signal is sensed by ATR, Rad17-RFC, and the complex, leading to phosphorylation of Chk1 by ATR. The activated Chk1 then phosphorylates Cdc25A, leading to G1 arrest. Whether the initial arrest is caused by the ATM-Chk2-Cdc25A pathway or the ATR-Chk1-Cdc25A pathway, this rapid response is followed by the p53- mediated maintenance of G1/S arrest, which becomes fully operational several hours after the detection of DNA damage (204). In the maintenance stage, ATM or ATR phosphorylates Ser15 of p53 directly and Ser20 through activation of Chk2 or Chk1, respectively (142, 146, ). The phosphorylation of p53 3 Figure 9 The G1/S checkpoint. DNA damage is sensed by ATM after double-strand breaks or by ATR, Rad17-RFC, and the complex after UV-damage. ATM/ATR phosphorylates Rad17, Rad9, p53, and Chk1/Chk2 that in turn phosphorylates Cdc25A, causing its inactivation by nuclear exclusion and ubiquitin-mediated degradation. Phosphorylated and inactivated Cdk2 accumulates and cannot phosphorylate Cdc45 to initiate replication. Maintenance of the G1/S arrest is achieved by p53, which is phosphorylated on Ser15 by ATM/ATR and on Ser20 by Chk1/Chk2. Phosphorylated p53 induces p21 WAF-1/Cip1 transcription, and p21 WAF-1/Cip1 binds to the Cdk4/CycD complex, thus preventing it from phosphorylating Rb, which is necessary for the release of the E2F transcription factor and subsequent transcription of S-phase genes. p21 WAF-1/Cip1 also binds to and inactivates the Cdk2/CycE complex, thus securing the maintenance of the G1/S checkpoint.

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8 DNA REPAIR AND THE DNA DAMAGE CHECKPOINTS 77 accumulation of Y15-phosphorylated Cdc2 and mitotic arrest. Initiation of the G2/M checkpoint may not be as simple as presented above. MAP kinases p38 (272) and p38 (273) have been implicated in the G2/M checkpoint responses to ionizing radiation and UV, respectively. Clearly, the interfacing of two major cellular signal transduction pathways, the MAP kinase- and the checkpoint response pathways, deserves more study. The Replication Checkpoint (S/M Checkpoint) The replication checkpoint (also referred to as the S/M checkpoint) is the process by which mitosis is inhibited while DNA replication is ongoing or blocked (128). In both the G2/M and replication checkpoints, the ATR-Chk1-Cdc25 signal transduction pathway is utilized to inhibit mitosis, although the initiating signals for the two checkpoints are different. Ongoing replication or replication forks blocked by DNA damage or nucleotide starvation initiate the replication checkpoint. Evidence from in vitro studies with Xenopus egg extracts indicates that the initiating signal is a component of the replication fork, and experiments with various replication inhibitors suggested that the signal might actually be the RNA primer of Okazaki fragments (274). However, more recent experiments have raised questions about the true identity of the replication checkpoint signal, as the inhibitors used to assign it to RNA primers inhibited the entire replisome assembly (275). The replication checkpoint has been observed in all model systems, including yeast (110) and mammalian cells (266). Surprisingly, however, recent evidence indicates that in mice the replication checkpoint activated by hydroxyurea and aphidicolin is independent of ATM and ATR, but the UV- and ionizing radiationinduced replication checkpoint is dependent on these damage sensor/signal transducer kinases (266), raising the possibility that replication forks and DNA damage during S phase may inhibit mitosis by different signaling mechanisms. CONCLUSIONS DNA damage activates several distinct biochemical pathways. First, DNA repair enzymes of varying complexities recognize and eliminate the damage. Second, DNA damage activates DNA damage checkpoints, which arrest cell cycle progression. Under most circumstances, checkpoints aid in cellular survival. Third, DNA damage activates transcription of certain genes (transcriptional response). The role of the transcriptional response in cell survival is unknown. Finally, apoptosis in metazoans is the programmed cell death that is activated by either cell death ligands or DNA damage, and serves to eliminate superfluous or deregulated and dangerous cells. The four DNA damage response pathways described above can and do function independently under certain circumstances. However, under most con-

9 78 SANCAR et al. ditions there is extensive interaction between these response reactions. Clearly, the individual players in the checkpoint responses serve not only to delay the cell cycle, but also to mediate DNA repair, directly and indirectly. Exactly how this occurs and, in the greater sense, how these agents assess DNA damage both quantitatively and qualitatively so as to choose between mediating DNA repair or apoptosis are outstanding areas for study. These aspects of the DNA damage response pathways must be considered in future research for a better understanding of the cell s response to genotoxicants and for the development of strategies aimed at targeting these pathways for cancer prevention and chemotherapy. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants GM32833 (to A.S.), GM20830 (to L.A.L.-B.), and GM59424 (to S.L.), and by the Miller Institute for Basic Research in Science (A.S.). The Annual Review of Biochemistry is online at LITERATURE CITED 1. Zhou BB, Elledge SJ Nature 408: Kolodner RD, Putnam CD, Myung K Science 297: Modrich P, Lahue R Annu. Rev. Biochem. 65: Kolodner RD, Marsischky GT Curr. Opin. Genet. Dev. 9: Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ Nature 404: Cox MM Mutat. Res. 510: Minko IG, Zou Y, Lloyd RS Proc. Natl. Acad. Sci. USA 99: Sancar A Biochemistry 33: Deisenhofer J Mutat. Res. 460: Todo T Mutat. Res. 434: Sancar A Chem. Rev. 103: Park H, Zhang KJ, Ren YJ, Nadji S, Sinha N, et al Proc. Natl. Acad. Sci. USA 99: Orren DK, Selby CP, Hearst JE, Sancar A J. Biol. Chem. 267: Sancar A, Hearst JE Science 259: Johnson PF, McKnight SL Annu. Rev. Biochem. 58: Chen L Curr. Opin. Struct. Biol. 9: Naar AM, Lemon BD, Tjian R Annu. Rev. Biochem. 70: Sancar A, Franklin KA, Sancar GB Proc. Natl. Acad. Sci. USA 81: Tornaletti S, Hanawalt PC Biochimie 81: Friedberg EC Annu. Rev. Biochem. 65: Selby CP, Sancar A Science 260: Mellon I, Spivak G, Hanawalt PC Cell 51: Mellon I, Hanawalt PC Nature 342: Cox MM Annu. Rev. Genet. 35: Zhou J, Ahn J, Wilson SH, Prives C EMBO J. 20: Todo T, Takemori H, Ryo H, Ihara M, Matsunaga T, et al Nature 361: