Mutagenesis of Benzo[a]pyrene Diol Epoxide in Yeast: Requirement for DNA Polymerase ζ and Involvement of DNA Polymerase η

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Abstract
Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Polζ and Polη in (±)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (±)-anti-BPDE induced 85% base substitutions with predominant G → C followed by G → T transversions, 9% deletions of 1−3 nucleotides, and 6% insertions of 1−3 nucleotides. In rad30 mutant cells lacking Polη, (±)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1−3 nucleotides. In rev3 mutant cells lacking Polζ, (±)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1−3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Polζ. Consistent with the in vivo results, purified yeast Polζ performed limited translesion synthesis opposite (+)- and (−)-trans-anti-BPDE-N2-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (±)-anti-BPDE-induced mutagenesis in yeast requires Polζ and partially involves Polη and suggest that Polζ directly participates in nucleotide insertions opposite the lesion, while Polη significantly contributes to deletions and insertions of 1−3 nucleotides.