We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18 −/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18 −/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

Cdc7 phosphorylates Rad18 to integrate S phase progression with postreplication DNA repair, ensuring genome stability.

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.

MicroRNAs (miRNAs) are deregulated in cancer and have been shown to exhibit both oncogenic and tumor suppressive functions. Although the functional effects of several miRNAs have been elucidated, those of many remain to be discovered. In silico analysis identified microRNA-206 (miR-206) binding sites in the 3′-untranslated regions (3′-UTR) of both the mouse and human CCND1 gene. Cyclin D1 is a recognized oncogene involved in direct phosphorylation of the retinoblastoma (Rb) protein and promoting cell cycle transition from G1 to S. miR-206 specifically binds to the CCND1 3′-UTR and mediates reduction of both cyclin D1 protein and mRNA. Expression of miR-206 induced a G1 arrest and a decrease in cell proliferation in breast cancer cells. Ectopic expression of miRNA-resistant cyclin D1 was able to reverse the miR-206-induced decrease in cell proliferation. Therefore, we identified miR-206 as an activator of cell cycle arrest resulting in a decrease in cell proliferation that is dependent on the inhibition of cyclin D1. Interestingly, prostatic cancer (PCa) cells express low levels of miR-206 resulting in deregulated cyclin D1 expression compared with non-transformed primary prostatic epithelial cells (PrEC). Finally, we demonstrate that cyclin D1 is regulated by miR-206 in PrEC but not in PCa cells and this is due to the absence of a CCND1 3'-UTR in these cells. This suggests that miR-206-based anti-cyclin D1 targeted therapy would be beneficial in cancers where cyclin D1 is overexpressed and contains a 3′-UTR.