DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G(1)-, G(2)- or G(3)-positions of the NarI site (5′-G(1)G(2)CG(3)CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G(3) construct were mutants, largely G→T, compared to 18% and 24% from the G(1) and G(2) constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G(1) and G(3) constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G(2) construct. In vitro TLS using yeast pol ζ showed that it can extend G(3)*:A pair more efficiently than G(3)*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.