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 ␦ (pol ␦) was examined by steady-state and pre-steadystate rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol ␦ 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 ␦ 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 presteady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol ␦ 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.High fidelity DNA replication is critical to the preservation of genomic stability and the avoidance of mutations that can disrupt the regulation of complex biological systems. Cells contain several DNA polymerases and complex DNA repair systems to preserve genomic integrity (1, 2). Accurate replication is disrupted by the presence of covalent DNA-chemical adducts, which can be misread and lead to mutations and cancer (3). Understanding the miscoding events induced by modified DNA is important in understanding risks of environmental chemicals, as well as aspects of chemotherapeutic treatment. Misincorporation is primarily a kinetic phenomenon and not simply thermodynamic. Work with several DNA adducts and artificial DNA bases clearly indicates that both the identity of incorporated bases and their frequency of substitution are functions of which polymerase is used as a catalyst (4 -10). Our own work on how polymerases influence misincorporation has been focused on 8-oxoG 1 (9,(11)(12)(13)(14). 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 (15-17).Polymerases derived from prokaryotic systems have been used extensively as models for mechanistic studies because of their availability, the general lack of need for complex accessory proteins, and the availability of structural and mechanistic information (18). The question arises as to how relevant findings made with the...

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

“…Human DNA pol η incorporates dATP and dCTP opposite the lesion with comparable efficiencies, but proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) enhance the correct 8-OH-Gua bypass [25][26][27][28][29][30]. The mammalian replicative DNA pols α and δ are error-prone against 8-OH-Gua [27,28,[42][43][44][45][46][47]. Thus, the knock-down of DNA pol η might force the "error-prone" replicative DNA pols to bypass 8-OH-Gua.…”

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

“…A signature of oxidative stress-induced substitutions is a G-C to T-A transversion, which results when dAMP is incorporated opposite 8-oxo-G in its syn conformation in the template strand [39]. This G-C to T-A transversion accounts for the majority of base substitutions in the msh6E339A mutant yeast strain (Tables 3 and 4), despite the fact that it is only one of among eight possible single base substitutions.…”

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

“…DNA polymerases incorporate 8-OH-dGTP opposite C and A in vitro [7,[16][17][18][19][20][21][22][23]. In terms of the accumulation of 8-OH-Gua in DNA, the contribution of 8-OH-dGTP from the nucleotide pool and the direct oxidation of G bases in DNA has been reported to be almost equal [24].…”