title: Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2'-deoxyguanosine by the catalytic core of yeast DNA polymerase η.
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.
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Qizhen
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Mengyu
authors.initials: M
authors.lastname: Zhong
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Binyan
authors.initials: B
authors.lastname: Liu
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Wei
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232, United States.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China. Electronic address: huidongzhang@tmmu.edu.cn.
authors.forename: Huidong
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D020134:Catalytic Domain; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011742:Pyrimidine Nucleotides / Q000378:metabolism
chemical_list: D011742:Pyrimidine Nucleotides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine
keywords: 8-oxoG; Pre-steady-state enzyme kinetics; Yeast DNA polymerase η(core); dNTP incorporation