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

O-Methylguanine (O-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 O-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O-MeG by gp90 exo. O-MeG partially inhibited full-length extension by gp90 exo. O-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo extends beyond T:O-MeG 2-fold more efficiently than C:O-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O-MeG show fast burst phases. The pre-steady-state incorporation efficiency (k/K) is decreased in the order of dCTP:G>dTTP:O-MeG>dCTP:O-MeG. The presence of O-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg. Misincorporation of dTTP opposite O-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O-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 O-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.