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Biochimie. 2016 Feb;121:161-9. doi: 10.1016/j.biochi.2015.12.009. Epub 2015 Dec 15.

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

Author information

1
Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
2
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232, United States.
3
Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China. Electronic address: huidongzhang@tmmu.edu.cn.

Abstract

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.

KEYWORDS:

8-oxoG; Pre-steady-state enzyme kinetics; Yeast DNA polymerase η(core); dNTP incorporation

PMID:
26700143
PMCID:
PMC5193378
DOI:
10.1016/j.biochi.2015.12.009
[Indexed for MEDLINE]
Free PMC Article

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