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

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 Pol(zeta) and Pol(eta) 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 Pol(eta), (+/-)-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 Pol(zeta), (+/-)-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 Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.

Comparison of p53 Mutations Induced by PAH o-Quinones with Those Caused by anti-Benzo[a]pyrene Diol Epoxide in Vitro: Role of Reactive Oxygen and Biological Selection

“…One study found that (±)-anti-BPDE also caused G>T transversions in the same yeast assay that we use. However, another study found a preference for G>C transversions in a yeast mutagenesis assay with (±)-anti-BPDE (40). The preference for G to C over G to T transversions may be governed by the trans-lesional by pass polymerase Polζ which has been shown to preferentially incorporate G opposite an (+)-anti-BPDE-N 2 -dGuo adduct in yeast strains proficient in mutagenesis (40).…”

DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

“…85–88 Synthesis opposite the bulky N 2 -dG adducts is slow, 67 although pol η may play a role in the mutagenic bypass of N 2 -BP-dG. 68,89,90 These studies do not provide any mechanistic rationale for the ability of pol η to insert dTTP and dATP opposite O 2 -alkyl-dT. Our in vitro kinetic data do agree with the mammalian cell culture studies in which pol η is crucial to the bypass.…”

Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

“…The rate of G>T transversions seems to be concentration-dependant [31]. Interestingly, using a yeast assay with various strains deficient in nucleotide excision repair, it was demonstrated that DNA polymerases η and ζ were required in combination for the translesion repair across BPDE adducts, leading to G>T transversions [35]. Pol ζ alone was also able to generate large deletions in this assay.…”