Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase δ: STEADY-STATE AND PRE-STEADY-STATE KINETIC ANALYSIS

Heidi J Einolf,F Peter Guengerich

doi:10.1074/jbc.m006696200

Abstract

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) 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 delta 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 K(m)). 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 delta 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 delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.

Highlights

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase ␦ was examined by steady-state and pre-steadystate rapid quench kinetic analyses
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
We used purified calf thymus DNA pol ␦, considered to be the main leading strand DNA polymerase [1, 2], in a series of steady-state and pre-steadystate kinetic experiments and concluded that the major features of the catalytic mechanism were very similar to those established in the prokaryotic models [21]

Summary

Introduction

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase ␦ (pol ␦) was examined by steady-state and pre-steadystate rapid quench kinetic analyses. 8-OxoG is a relatively simple adduct in that the only chemical attached to the DNA is one atom of oxygen, and it was selected as a model because of its relatively high mutagenicity and lack of polymerase blockage This lesion is generally regarded as being the most abundant of those induced by oxidative damage [15,16,17]. We used purified calf thymus DNA pol ␦, considered to be the main leading strand DNA polymerase [1, 2], in a series of steady-state and pre-steadystate kinetic experiments and concluded that the major features of the catalytic mechanism were very similar to those established in the prokaryotic models [21]. The increase in the amount of extended products was 2.5-fold for 8-oxoG and

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