Replication across O6-Methylguanine by Human DNA Polymerase β in Vitro INSIGHTS INTO THE FUTILE CYTOTOXIC REPAIR AND MUTAGENESIS OF O6-METHYLGUANINE

Abstract Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase β (pol β) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol β. Our results show that m6dG is a strong but not absolute block to replication by pol β. pol β exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol β preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol β extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo−KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol β has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG. This suggests that pol β, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG. Our results also suggest that replication across m6dG by pol β may contribute to m6dG-induced G → A transition mutations.