An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage

Accurate replication and segregation of the human genome depends on interactions between cell cycle checkpoints and pathways of DNA repair. Cell cycle checkpoints are biochemical surveillance pathways that slow or arrest progression through the cell cycle, pending completion of essential events and/or repair of DNA damage. DNA damage checkpoints minimize the probability of replicating and segregating damaged DNA and therefore reduce the frequencies of mutations and chromosomal aberrations that are induced by genotoxic stress. Defects in cell cycle checkpoint function and DNA repair result in genetic instability, which may fuel cancer development (30, 38, 54). DNA damage checkpoints operate in the G1, S, and G2 phases of the cell cycle, transducing signals from sensors of DNA damage to effector proteins that mediate the appropriate response (1, 21). Essential transducers of DNA damage checkpoint responses are the protein kinases ATM (ataxia telangiectasia [AT] mutated) and ATR (ATM and Rad3 related). Cells from AT patients are hypersensitive to the genotoxic effects of DNA damage induced by ionizing radiation (IR) and are defective in DNA damage checkpoint responses to IR (1, 21, 38). ATR, on the other hand, seems to be required for checkpoint responses to replication blocks caused by hydroxyurea (HU) and UV-induced DNA damage (13, 61, 64). Although the roles of ATM and ATR in DNA damage checkpoint responses in G1 and G2 are comparatively well characterized, the S checkpoint remains a topic of intense investigation (1). The S checkpoint is activated in human cells in response to diverse forms of DNA damage (33-35, 53). The IR-induced S checkpoint response is inhibited or attenuated by mutations in ATM, Nbs1, and Mre11 (11, 53). Nbs1 and Mre11 are the gene products mutated in the familial genetic instability disorders Nijmegen breakage syndrome (NBS) and AT-like disorder (AT-LD), respectively (55). AT, NBS, and AT-LD are clinically distinct cancer-predisposing disorders, but they are phenotypically similar at the cellular level (55). Cells from these patients are hypersensitive to cell killing following exposure to IR, display increased risk of induced chromosomal aberrations, and fail to repress DNA synthesis in the presence of IR-induced DNA damage, a phenomenon termed radioresistant DNA synthesis (RDS) (11, 53). ATM has been shown to phosphorylate Nbs1 on several serine residues in response to IR. Changing any of these serines to alanine produces mutant Nbs1 proteins that fail to correct RDS in NBS cells (26, 42, 65, 67). The IR-induced S checkpoint has recently been reported to include two parallel ATM-dependent pathways (23). It appears that one pathway involves ATM signaling through Chk2, ultimately resulting in the ubiquitin-mediated proteolysis of Cdc25A (22). Loss of Cdc25A phosphatase activity results in a dramatic inhibition of cyclin E/Cdk2 activity and Cdc45 binding to origins of DNA replication (23). The second pathway is dependent on ATM signaling to the Nbs1/Mre11/Rad50 complex, which when activated localizes to sites of DNA double-strand breaks following exposure to IR (45, 49), and by unknown signaling pathways it also effects inhibition of replicon initiation (23). Although the role of ATM in the IR-induced S checkpoint is indisputable, the checkpoint pathway that mediates the inhibition of replicon initiation following UVC-induced DNA damage is less clear. Normal S-phase cells respond to UVC-induced DNA damage by reducing the rate of DNA synthesis (34, 35). This inhibition is manifested at the level of chain elongation and replicon initiation (34, 35) and is the result of both passive and active cellular responses to DNA lesions. Passive inhibition of DNA replication is attributed to the physical obstruction of the DNA replication apparatus at sites of DNA damage. An example is the inability of the replicative DNA polymerases α and δ to copy through UVC-induced template lesions such as cyclobutane pyrimidine dimers and 6-4 photoproducts (46, 47). Active inhibition is a trans effect mediated through checkpoint signals that emanate from sites of DNA damage and ultimately inhibit the initiation of distant replicons (56). This S checkpoint response imposes transient delays in S-phase progression and provides more time for DNA repair to remove lesions from unreplicated chromatin. A previous study suggested that AT cells display a reduced S checkpoint response to UVC (52). AT cells, while not hypersensitive to inactivation of colony formation by UVC, are nevertheless hypersensitive to S-dependent UV-induced chromosomal aberrations (20, 40). Less is known about the function of ATR, since it is an essential gene and its deletion results in embryonic lethality in mice (6, 16). However, overexpression of kinase-inactive ATR (ATRki) renders cells hypersensitive to cell killing by DNA damaging agents, including IR, HU, and UV (13, 64). Moreover, phosphorylation of checkpoint-associated proteins p53 and Brca1 has been shown to be ATM dependent following exposure to IR, but ATR dependent following exposure to HU and UV (3, 10, 15, 41, 60, 61). Cellular responses to UVC-induced DNA damage are dose dependent. The inhibition of replicon initiation in response to UVC is best observed after exposure to 1 J/m2, a dose which produces little reduction in cell colony-forming efficiency and very little inhibition of DNA chain elongation or mitotic entry. Cytotoxic doses between 5 and 10 J/m2 UVC, which have been shown to saturate nucleotide excision repair (NER) (39), significantly inhibit DNA chain elongation and mitotic entry (7, 36) and typically reduce colony formation by 50 to 70% (5). UVC doses of >40 J/m2, which have been used to study cellular stress responses to UVC, inhibit DNA chain elongation severely and inactivate colony formation by >95% of normal human fibroblasts (5). This study examined the role of checkpoint proteins in the response of cells to 1 J/m2 UVC, thereby negating potential contributions associated with saturation of NER, induction of stress responses, and cytotoxicity. The results strongly implicate ATR and Chk1 as important signaling molecules for the UVC-induced inhibition of replicon initiation in human cells.