title: Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta.
abstract: Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼ 50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol η(core) (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP >> dCTP and dTTP. Pol η(core) showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol η(core) bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol η(core) relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Juntang
authors.initials: J
authors.lastname: Yang
authors.affiliation: Institute of Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China.
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
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: 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: Hao
authors.initials: H
authors.lastname: Zeng
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: D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000235:genetics* / Q000378:metabolism; D006147:Guanine; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053719:Tandem Mass Spectrometry; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; D006147:Guanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
keywords: Abasic sites; Pre-steady-state; Yeast DNA polymerase η(core); dNTP incorporation; kinetic