Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2'-deoxyguanosine by the catalytic core of yeast DNA polymerase η.

Xue Q, Zhong M, Liu B, Tang Y, Wei Z, Guengerich FP, Zhang H
Biochimie. 2016 121: 161-9

PMID: 26700143 · PMCID: PMC5193378 · DOI:10.1016/j.biochi.2015.12.009

Reactive oxygen species damage DNA bases to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), which results in G:C to T:A transversions. To better understand mechanisms of dNTP incorporation opposite 8-oxoG, we performed pre-steady-state kinetic analysis of nucleotide incorporation using the catalytic core of yeast DNA polymerase η (Pol ηcore, residues 1-513) instead of full-length Pol η, eliminating potential effects of the C-terminal C2H2 sequence motif on dNTP incorporation. Kinetic analysis showed that Pol ηcore preferred to incorporate dCTP opposite 8-oxoG. A lack of a pre-steady-state kinetic burst for Pol ηcore suggested that dCTP incorporation is slower than the dissociation of the polymerase from DNA. The extension products beyond the 8-oxoG were determined by LC-MS/MS and showed that 57% of the products corresponded to the correct incorporation (C) and 43% corresponded to dATP misincorporation. More dATP was incorporated opposite 8-oxoG with a mixture of dNTPs than predicted using only a single dNTP. The kinetic analysis of 8-oxoG bypass by yeast DNA Pol ηcore provides further understanding of the mechanism of mutation at this oxidation lesion with yeast DNA polymerase η.

Copyright © 2015. Published by Elsevier B.V.

MeSH Terms (6)

8-Hydroxy-2'-Deoxyguanosine Catalytic Domain Deoxyguanosine DNA-Directed DNA Polymerase Kinetics Pyrimidine Nucleotides

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