Error-Free Bypass of 7,8-dihydro-8-oxo-2'-deoxyguanosineby DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Shiling Gu; Qizhen Xue; Qin Liu; Mei Xiong; Wanneng Wang; Huidong Zhang

doi:10.3390/genes8010018

Error-Free Bypass of 7,8-dihydro-8-oxo-2'-deoxyguanosineby DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Genes (Basel). 2017 Jan 6;8(1):18. doi: 10.3390/genes8010018.

Authors

Shiling Gu^{1

2}, Qizhen Xue³, Qin Liu⁴, Mei Xiong⁵, Wanneng Wang⁶, Huidong Zhang⁷

Affiliations

¹ College of Pharmacy and Bioengineering, Chongqing University of Technology, No. 29 Hongguang Street, Banan District, Chongqing 400054, China. shilinggu@foxmail.com.
² Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, No. 17 People's South Road, Chengdu 610041, China. shilinggu@foxmail.com.
³ Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, No. 17 People's South Road, Chengdu 610041, China. xueqzhen@163.com.
⁴ College of Pharmacy and Bioengineering, Chongqing University of Technology, No. 29 Hongguang Street, Banan District, Chongqing 400054, China. liuqin@2014.cqut.edu.cn.
⁵ Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, No. 17 People's South Road, Chengdu 610041, China. xhmei2016@126.com.
⁶ College of Pharmacy and Bioengineering, Chongqing University of Technology, No. 29 Hongguang Street, Banan District, Chongqing 400054, China. wannengw@cqut.cn.
⁷ Public Health Laboratory Sciences and Toxicology, West China School of Public Health, Sichuan University, No. 17 People's South Road, Chengdu 610041, China. huidong.zhang@foxmail.com.

Abstract

As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonasaeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (k_pol/K_d,_dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg²⁺, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1.

Keywords: 8-oxoG; DNA polymerase; P. aeruginosa phage PaP1; nucleotide incorporation; pre-steady-state kinetics; steady-state kinetics.