▪ 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 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 loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 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.

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.

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

The circadian clock is a global regulatory system that interfaces with most other regulatory systems and pathways in mammalian organisms. Investigations of the circadian clock–DNA damage response connections have revealed that nucleotide excision repair, DNA damage checkpoints, and apoptosis are appreciably influenced by the clock. Although several epidemiological studies in humans and a limited number of genetic studies in mouse model systems have indicated that clock disruption may predispose mammals to cancer, well-controlled genetic studies in mice have not supported the commonly held view that circadian clock disruption is a cancer risk factor. In fact, in the appropriate genetic background, clock disruption may instead aid in cancer regression by promoting intrinsic and extrinsic apoptosis. Finally, the clock may affect the efficacy of cancer treatment (chronochemotherapy) by modulating the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs as well as the activity of the DNA repair enzymes that repair the DNA damage caused by anticancer drugs.