Analysis Of The Kinetic Mechanism Bypasses An Abasic Site By DNA Polymerase Of Pseudomonas Aeruginosa Phage Pap1

Objective:DNA polymerase of Pseudomonas aeruginosa phage PaP1 was performed to study the mechanism of DNA replication and its bypass of DNA damage abasic site at the molecular level.It helps us to understand the mechanisms by which this damage causes DNA lesions and mutation in DNA replication,thus laying the foundation for the treatment of human diseases.Aimed at revealing the relationship between DNA damage synthesis and DNA repair,providing a scientific basis for studying the mechanism by which environmental toxicants causes DNA abasic lesions.Methods:The construction,expression and purification of exonuclease-deficient DNA polymerase Gp90.Kinetic analysis of abasic site bypass by DNA polymerase Gp90 exo^-.Full-length extension,single nucleotide incorporation and next-base extension bypass abasic site were performed;the effect of abasic site on the single nucleotide insertion and the interaction of protein with DNA were investigated by pre-steady state kinetics and SPR,respectively.Results:?1?Wild-type Gp90 produced fully extended and degraded products on the unmodified template.Only the digested products were observed in two abasic site templates.Exonuclease-defective Gp90 exo^-could readily extend the primer on the unmodified template but was strongly blocked by the abasic site in both different templates.Only a28-mer product was produced,corresponding to one nucleotide incorporated opposite the abasic site.?2?dCTP was preferentially incorporated opposite G,and the misincorporation frequencies of the other three incorrect dNTPs were in the range of 10^-4-10^-5.For abasic site templates,dATP was preferentially incorporated opposite the abasic site in both templates,two orders of magnitude more efficiently than other three dNTPs.?3?Steady-state kinetic parameters(K_m,k_cat)were measured for next-base extension beyond G or abasic site by Gp90 exo^-.C or G at the 3'terminus of primer was paired or mispaired with G-template.dGTP incorporation opposite the 5'-next template C was approximately390-fold more efficient in extension beyond C:G pair than G:G mispair.The efficiency of misincorporation of dATP opposite the 5'next template C was similar for extension beyond a C:G pair and a G:G mispair.dGTP was preferentially incorporated with the AP₁template in which C is the nucleotide 5'to the abasic site.Similarly,dTTP was preferentially incorporated with the AP₂ template in which A is 5'to the abasic site.?4?Among four dNTPs,dCTP was preferentially incorporated opposite G and showed a fast burst phase with a burst rate k_p of 158 s^-1.The incorporation of the three incorrect dNTPs exhibited a linear phase but not the biphasic shape,indicating that only dCTP incorporation was faster than the dissociation of Gp90 exo^-from DNA.Incorporation of dATP opposite the abasic site was preferential relative to the other three dNTPs and showed a biphasic shape with a k_p of 0.48 or 0.44 s^-1.Incorporations of the other three dNTPs opposite abasic site showed linear phases.?5?In the absence of d NTP and Mg²⁺,the dissociation constants(K_d,DNA)were 64 nM for unmodified DNA and 79 nM or 85 nM for the abasic site,indicating that abasic site slightly weakened the binding affinity of the polymerase to DNA.In the presence of dNTP and Mg²⁺,the dissociation constants(K_d,DNA)were 16-19 nM for unmodified DNA,43-54 nM for AP₁-template,and 50-64 nM for AP₂-template.The presence of dNTP stabilized the binding of polymerase to DNA in ternary complex relative to in the binary complex.So,abasic site reduced the binding affinity of Gp90 exo^-to DNA in ternary complex.Conclusion:DNA replication is blocked by an abasic site,which results in a decrease in the insertion and elongation rate of individual dNTP.DNA polymerase and DNA binding are also weakened by an abasic site.Gp90 exo^-preferentially inserts dATP to bypass an abasic site not through a-1frameshift deletion or a loop-out mechanism.