Abstract
BackgroundDNA 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.MethodsTo 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.ResultsThe 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.ConclusionsTaken 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.
Highlights
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)
Effect of reduced hRev7 protein on the survival of human fibroblasts exposed to benzo[a]pyrene diol epoxide (BPDE) and on the frequency of BPDE-induced mutations Using small interfering RNA (siRNA), we previously generated two derivatives of the human fibroblast strain 9N.58 with significantly reduced levels of hRev7 protein [22]
The level of hRev7 was reconstituted in cell strain 2-2 (2-2 + R7) by transfecting that cell strain with a vector expressing hRev7 mRNA with a nucleotide sequence that was altered in such a way as to make it insensitive to siRNA-induced degradation (Figure 1)
Summary
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). Human cells undergo countless rounds of DNA replication, which must be very accurate to preserve critical genetic information To maintain such a significant level of accuracy, the classical replicative polymerases have evolved highly selective active sites that only accommodate nucleotides when they are correctly paired to the DNA template. Many of these DNA polymerases possess 3’®5’ proofreading exonuclease activity, which removes nucleotides that are incorrectly incorporated during replication, allowing an additional attempt at accurate DNA synthesis. Because of their stringency, the classical replicative polymerases cannot tolerate fluctuations in the DNA structure, including those that result from DNA damage. If the high fidelity replicative polymerase complex encounters a DNA lesion that blocks elongation, potentially fatal stalling or arrest of replication can occur
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