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 9-1-1 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.

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader͞DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC͞9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA. E ukaryotic cells exposed to genotoxic agents activate the DNA damage checkpoint signaling pathway, which arrests cellcycle progression and in so doing prevents cell death or mutations. Recent work has revealed that in mammalian cells, the ATM and ATR proteins, which belong to the phosphatidylinositide kinase-like kinase family, and the Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complexes, which have structural similarities to the replication clamp loader and replication clamp RFC and proliferating cell nuclear antigen (PCNA), respectively, are involved in damage recognition, which activates the checkpoint response (reviewed in refs. 1-4). Studies with budding and fission yeasts have shown that the orthologs of these proteins perform similar functions. However, biochemical data on the specific roles of the phosphatidylinositide kinase-like kinase family members and the Rad17-RFC and 9-1-1 complexes are scarce, and hence the damage sensing step of the checkpoint response remains illdefined. We previously reported that ATR directly recognizes and is activated by damaged DNA (5). In this article, we investigate the interactions of Rad17-RFC and the 9-1-1 checkpoint complexes with DNA to gain some insight into their roles as damage sensors.Rad17-RFC is one of the three known RFC-like complexes in mammalian cells. In this form of RFC, the p140 subunit is replaced by the 75-kDa Rad17 protein, which has homology to all RFC subunits (6). Yeast genetic studies indicate that the orthologs of human Rad17 function exclusively in the DNA damage checkpoint response (7,8). The 9-1-1 checkpoint complex is a heterotrimer of Rad9, Rad1, and Hus1 proteins, which were predicted to have structural homology to PCNA (9-13). Previously, we showed that Rad17 associates with the four small RFC subunits to make an RFC-like complex, which by electron microscopy exhibits an RFC-like structure (14). Similarly, we found that Rad9, Rad1, and Hus1 f...

Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at ∼5 pm and nadir at ∼5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.C hronochemotherapy is the administration of chemotherapeutic drugs at specific times of the day so as to optimize efficacy and minimize side effects of the drug (1, 2). Cisplatin is one of the three most commonly used chemotherapeutic drugs (3) for which chronotherapy is thought to have some beneficial effects. Factors that modulate the efficacy of cisplatin therapy include drug uptake and efflux, DNA adduct formation, DNA repair, and cellular proliferation (4, 5). Cisplatin produces DNA intra-and interstrand diadducts and DNA-protein crosslinks (4), and it is well established that the intrastrand diadducts Pt-(GpG), Pt-(ApG), and Pt-(GpXpG), that constitute up to 90% of the total DNA lesions, are the main cause of its cytotoxicity and hence its therapeutic effects. These lesions are removed exclusively by nucleotide excision repair (excision repair) in mammalian cells and hence the status of excision repair is an important factor in the success of chemotheraphy with cisplatin (4, 6).In humans and mice, excision repair is carried out by the coordinated action of six core repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1, which remove the damage in the form of 24-32 nt-long oligomers; the resulting gap is filled by DNA polymerases and ligated (7-9).Recently, we found that the rate of excision repair of a UV photoproduct in the mouse brain exhibits a daily rhythm (10). Furthermore, it appears that this rhythmic pattern is due to the circadian (circa ¼ about, dies ¼ day) oscillation of the XPA (xeroderma pigmentosum A) protein that is one of the ratelimiting factors in excision repair. Even though in that study the damaged-DNA substrate was a UV photoproduct, we suggested that the findings were relevant to the repair of cisplatin because nucleotide excision repair is the only repair system capable of removing bulky DNA lesions produced by UV or by UVmimetic agents such as cisplatin (11). Although cisplatin is used for treating certain brain cancers, the blood-brain barrier is a serious impediment for its gen...

a b s t r a c tMammalian cells possess a cell-autonomous molecular clock which controls the timing of many biochemical reactions and hence the cellular response to environmental stimuli including genotoxic stress. The clock consists of an autoregulatory transcription-translation feedback loop made up of four genes/proteins, BMal1, Clock, Cryptochrome, and Period. The circadian clock has an intrinsic period of about 24 h, and it dictates the rates of many biochemical reactions as a function of the time of the day. Recently, it has become apparent that the circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. These new insights are expected to guide development of novel mechanism-based chemotherapeutic regimens.