Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase δ

Abstract

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase δ (pol δ) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol δ preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased Km). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol δ preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol δ replication fidelity at 8-oxoG depends upon contributions from Km,Kdd, and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.