UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpointmechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m 2 ). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.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.

Solar ultraviolet radiation is a ubiquitous environmental carcinogen responsible for 500,000 or more new cases of skin cancer in the United States each year (1, 2). Exposure of human cells to natural sunlight leads to the formation of cyclobutane pyrimidine dimers (CPDs), 1 pyrimidine(6 -4)pyrimidone dimers, and their Dewar valence isomers (3). UV-induced DNA photoproducts are currently accepted as important underlying factors in skin carcinogenesis (2, 4). Studies with UVC (254 nm), which forms predominantly CPDs (70 -80%) and 6 -4 dimers (20 -30%), have indicated that mutations and chromosomal aberrations are induced when human cells attempt to replicate the damaged DNA (5, 6). Therefore, the mechanisms whereby human cells complete the replication of template strands containing photoproducts are of considerable interest.UVC inhibits DNA replication in diploid human fibroblast strains by a variety of mechanisms, including G 1 arrest (6) and inhibition of replicon initiation in S phase cells (7). These two checkpoint responses appear to be protective processes, providing more time for DNA repair to remove photoproducts before DNA replication. UVC also induces inhibition of DNA synthesis in active replicons (7,8). The latter is thought to reflect, at least in part, the stalling of DNA replication forks at pyrimidine dimers (9 -11), perhaps due to a reduced capacity of DNA polymerases to incorporate DNA precursors into nascent strands opposite template lesions (12-15). This inhibition, however, is not absolute, and bypass replication eventually takes place, as evidenced by the generation of replicated DNA containing photoproducts and the induction of point mutations at dipyrimidine sites. UV-induced mutations in the p53 tumor suppressor gene in non-melanoma skin cancers are characterized by a high proportion of C 3 T transitions (16 -18). Base substitution mutations at thymines do not occur with high frequency in UV-damaged genes that are replicated at their natural chromosomal locations (19) or as part of shuttle vectors (20 -22).Previous studies have demonstrated that protein extracts from HeLa cells are capable of replicating past CPDs during in vitro replication of UV-damaged plasmids carrying the SV40 origin of replication (23)(24)(25)(26). Experimental evidence in support of this conclusion was found primarily by probing for the presence of sites sensitive to nicking by the CPD-specific enzyme, T4 endonuclease V (T4 endoV), in replicated (DpnI-resistant), closed circular DNA molecules (23)(24)(25)(26). In addition, UV-induced mutagenesis at dipyrimidine sites of randomly damaged plasmids (almost exclusively C 3 T) presumably reflected error-prone bypass replication (trans-lesion synthesis) of CPDs (23,24).Bypass replication of a single dimer strategically placed on one or the other anti-parallel strand of DNA has also been examined (25,26). Inference as to whether bypass replication occurred via leading or lagging strand synthesis was made on the basis of the location and orientation of the dimer, vis à vis...

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Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability.

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G 1 /S transition to prevent rereplication. The strict inhibition of licensing after the G 1 /S transition means that all origins used in S phase must have been licensed in the preceding G 1 . Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G 1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G 1 progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells.

Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression.

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase -null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase at frequencies 2-to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase . These results indicate that polymerase is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase , suggesting that oxaliplatin is nonmutagenic in human fibroblasts.