Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1.

Abstract

O6-Methylguanine (O6-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O6-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O6-MeG by gp90 exo-. O6-MeG partially inhibited full-length extension by gp90 exo-. O6-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo- extends beyond T:O6-MeG 2-fold more efficiently than C:O6-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O6-MeG show fast burst phases. The pre-steady-state incorporation efficiency (kpol/Kd,dNTP) is decreased in the order of dCTP:G>dTTP:O6-MeG>dCTP:O6-MeG. The presence of O6-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg2+. Misincorporation of dTTP opposite O6-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O6-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O6-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.

DOI: 10.1016/j.dnarep.2017.06.021

Cite this paper

@article{Gu2017ErrorproneBO, title={Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1.}, author={Shiling Gu and Jingyuan Xiong and Ying Shi and Jia You and Zhenyu Zou and Xiaoying Liu and Huidong Zhang}, journal={DNA repair}, year={2017}, volume={57}, pages={35-44} }