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Κυριακή 11 Ιουνίου 2017

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

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Publication date: Available online 10 June 2017
Source:DNA Repair
Author(s): Shiling Gu, Jingyuan Xiong, Ying Shi, Jia You, Zhenyu Zou, Xiaoying Liu, Huidong Zhang
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.



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