The Chk1-mediated S-phase Checkpoint Targets Initiation Factor Cdc45 via a Cdc25A/Cdk2-independent Mechanism

Abstract

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

“…30 Average labeled fiber lengths obtained in these experiments were 34.9 kb (n = 497) and 35.59 kb (n = 380) for siCon-and correlates with inhibition of DNA synthesis when the S-phase checkpoint is active. 26 Taken together, our data show association of FANCD2 with Cdc45 in the chromatin concomitant with DNA replication. These results are consistent with a potential role for FANCD2 in promoting efficient chromatin loading of Cdc45 and facilitating initiation and/or elongation stages of DNA synthesis.…”

“…Rat anti-Cdc45 monoclonal antibody has been described previously. 26 Antibodies against Cdc6 (sc-9964), Cdk2 (sc-163), Cdk4 (sc-260), Chk1 (sc-7898), Cyclin A (sc-596), FANCL (sc-66887).…”

“…2,6,21 H1299 human lung carcinoma cells were chosen for these initial experiments, because we and others have characterized cell cycle events and DNA replication parameters extensively in this line. [22][23][24][25][26] We achieved efficient ablation of FANCD2 expression in exponentially-growing H1299 cells as shown by immunoblotting (Fig. 1A).…”

“…The association of Cdc45 with chromatin and pre-RC components is negatively regulated by the Chk1-mediated S-phase checkpoint in a CDK2-independent manner. 26 Interestingly, the association of FANCD2 with Cdc45 on chromatin was also reduced by approximately 32% following genotoxin (BPDE)-treatment under conditions of S-phase checkpoint activation (Fig. 4B).…”

“…However, a 50% inhibition of DNA synthesis resulting from reduced elongation in genotoxin-treated cells is readily detectable by velocity sedimentation. 26 Therefore, any putative changes in rates of fork progression due to FANCD2-deficiency are likely to be very modest. Alternatively, it is possible that FANCD2-deficiency only affects elongation of a subset of replicons and does not affect the elongation stage of DNA synthesis globally.…”