In vitro bypass of the major malondialdehyde-and base propenal-derived DNA adduct by human Y-family DNA polymerases κ, τ, and Rev1

Abstract

3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a] purin-10(3H)-one (M 1dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M 1dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M 1dG → A and M 1dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M 1dG in vivo. Previous reports described the bypass of M 1dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M 1dG by the human Y-family DNA polymerases κ, τ, and Rev1. M 1dG was incorporated into template-primers containing either dC or dT residues 5′ to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol τ, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M 1dG:dC > M 1dG:dG > M 1dG:dT ∼ M 1dG:dA, but neither hPol τ nor Rev1 extended M 1dG-containing template-primers. Liquid chromatography-mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3′-GXC-5′ template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M 1dG in the 3′-GXT-5′ template sequence. The results indicate that DNA hPol κ or the combined action of hPol τ or Rev1 and hPol κ bypass M 1dG residues in DNA and generate products that are consistent with some of the mutations induced by M 1dG in mammalian cells. © 2010 American Chemical Society.