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.
Highlights
International, by National Institutes of Health Grants ES09558 and ES12917
We have investigated the molecular basis of the intra-S-phase checkpoint elicited by benzo[a]pyrene dihydrodiol epoxide (BPDE)-adducted DNA
Similar to previous studies from other laboratories that used different genotoxins, we found that high doses of BPDE reduce Cdc25A levels, inhibit Cdk2 activity, and perturb association between Cdc7 and Dbf4
Summary
International, by National Institutes of Health Grants ES09558 and ES12917 Low concentrations of BPDE (Ͻ100 nM) inhibit initiation of DNA synthesis transiently, yet do not affect elongation of existing replicons [8]. The transient inhibition of DNA synthesis induced by Ͻ100 nM BPDE is an S-phase checkpoint that inhibits firing of origins of replication in response to DNA damage acquired during S-phase. Higher concentrations of BPDE (200 – 600 nM) inhibit initiation and elongation steps of DNA replication, resulting in an irreversible block to DNA synthesis and loss of viability [8]. The inhibition of DNA synthesis induced by 600 nM BPDE is not due to checkpoint signaling, but instead results from global blocks to the progression of DNA polymerases by BPDE adducts [8]
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