The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE)

BACKGROUND: DNA polymerase zeta (Polζ) is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS). The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet((254 nm) )(UV) radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008) 597-604). The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), the reactive form of the widespread environmental carcinogen benzo[a]pyrene. METHODS: To determine whether hRev7 also plays a role in protecting human cells from the cytotoxicity and mutagenesis induced by benzo[a]pyrene diol epoxide (BPDE), cell strains with reduced hRev7 were compared to their parental strain and a vector control strain for the effect of BPDE on cell survival, induction of mutations, and the ability to progress through the cell cycle. RESULTS: The results show that cell strains with reduced hRev7 are more sensitive to the cytotoxic effect of BPDE than the control strains, and progress through S-phase at a slower rate than the control cells following BPDE treatment, indicating that hRev7, and likely hPolζ, is required for efficient bypass of BPDE-induced DNA lesions. However, neither the frequency nor kinds of mutations induced by BPDE in cells with reduced hRev7 differ significantly from those induced in the control strains, suggesting that hPolζ is not essential for inserting nucleotides opposite BPDE-induced DNA damage. CONCLUSIONS: Taken together, our results which show that hRev7 is required for TLS past BPDE-induced DNA lesions but that it is not essential for inserting nucleotides opposite such lesions suggest a role for hPolζ in the extension step of translesion synthesis.