Kinetic Mechanism Of Pseudomonas Aeruginosa Phage PaP1 DNA Polymerase Bypass DNA Damage

Pseudomonas aeruginosa is an opportunistic pathogen that can cause various infectious diseases in humans.In recent years,clinical treatment becomes difficult due to the irrational use and even abuse of antibiotics,and lack of effective antibacterial agents.Phage is a kind of virus,which has the characteristic of host specificity.It can be used to treat the infection of multidrug resistant bacteria.Recently,a strain of Pseudomonas aeruginosa lytic phage PaP1 has been isolated,which can effectively infect and lyse Pseudomonas aeruginosa.In this process,a large number of progeny phages were generated by the rapid replication by DNA polymerase gp90.However,DNA replication is easy to be affected by DNA damage in phage,which further affect the ability of phage infection and Pseudomonas aeruginosa lysis.In order to better study the mechanism of phage infection,we performed in vitro purification of phage PaP1 DNA polymerase gp90 and studied the dynamic mechanism of DNA replication and translesion DNA synthesis by gp90 and its mutants.Objective:The construction and expression of exonuclease-deficient DNA polymerase of Pseudomonas aeruginosa phage PaP1 was performed to study the mechanism of DNA replication and its bypass of DNA damage at the molecular level to provide theoretical guidance for the development of new drugs and rational ultilization of drugs.Methods:(1)The construction,expression and purification of exonuclease-deficient DNA polymerase gp90.DNA polymerase gp90 contains exonuclease activities on ssDNA and dsDNA.The exonuclease activity must be removed in in vitro kinetic analysis.Firstly,the plasmid was constructed to express the mutants of exonuclease-deficient DNA polymerase.Then,these mutant proteins were expressed by induction with IPTG and purified by Ni column.(2)Kinetic analysis of 8-oxoG bypass by DNA polymerase.Full-length extension,single nucleotide incorporation and next-base extension bypass 8-oxoG were performed;the effect of 8-oxoG on the single nucleotide insertion and the interaction of protein with DNA were investigated by pre-steady state kinetics and SPR,respectively.(3)Kinetic analysis of O~6-MeG bypass by DNA polymerase.The study of DNA replication efficiency and fidelity by full-length extension,single nucleotide insertion and next-base extension bypass O~6-MeG were performed;the effect of O~6-MeG on single nucleotide insertion,the interaction of protein with DNA and the conformational change were studied by pre-steady-state kinetics,SPR,and SF-120,respectively.Result:(1)Glu-60,Asp-137 and Asp-234 in gp90 were mutated to alanine by point mutation Kit.DNA polymerase mutants were expressed at low temperature and purified by Nicolumn.To detect the exonuclease and polymerase activity,32 P labeling technique was used.Only gp90 D234 A eliminates the exonuclease activity but still retains polymerase activity.Then,this mutant was denoted as gp90 exo-.(2)Compared with normal template G,8-oxoG and O~6-MeG partillay inhibit the full-length extension by DNA polymerase,with the presence of some intermediate products.(3)The dNTP misincorproation frequencies were in the range of 10-4-10-5 for unmofieid G with similar kcat values of all four dNTPs but greatly increased Km values for three wrong dNTPs compared with that of dCTP.For 8-oxoG,misincorporation frequencies retained in the range of 10-4-10-5,but all the incorporation efficiencies were greatly reduced compared with unmodified G.The misincorporation of dNTP opposite 8-oxoG had higher Km but lower kcat values compared with that of dCTP incorporation opposite G.For O~6-MeG,dTTP was preferentially incorporated,67-fold more efficient than dCTP and 103-104-fold more efficient than dATP and dGTP.The misincorporation frequencies of dATP and dGTP opposite O~6-MeG were in the range of 10-2-10-3,approximately 100-fold higher than those opposite G.(4)Incorporation of dGTP opposite next base C were approximately 8-fold preferential in extension beyond C:G(primer:template)rather than A:G,50-fold preferential in extension beyond C:G rather than T:G,due to higher kcat values but unchanged Km values.For template 8-oxoG,both C:8-oxoG and A:8-oxoG could be similarly extended by gp90 exo-.For template O~6-MeG,incorporation of dGTP opposite next base C were approximately 2-fold preferential in extension beyond T:G(primer:template)rather than C:G.These results indicate that gp90 exo-preferential extends C:G base pair rather than mispair A:G pair but show same preferential in extension beyond C:8-oxoG or A:8-oxoG and lost priority in extension beyond C:O~6-MeG rather than T:O~6-MeG.(5)Among four dNTPs,incorporation of dCTP opposite G was preferential and showed a fast burst phase,indicating that dCTP incorporation is faster than the dissociation of gp90 exo-from DNA.For incorporation of dCTP,dATP or dTTP opposite G,the product and reaction time exhibited a linear relationship,without biphasic shape.The incorporation of dCTP or dATP opposite 8-oxoG was preferential relative to dTTP or dGTP and showed biphasic shapes.Incorporation of dTTP or dCTP opposite O~6-MeG was preferential relative to dATP or dGTP and showed biphasic shape.(6)The absence of dCTP and Mg2+ in buffer led to a random binding of DNA polymerase to DNA to form a binary complex.DNA polymerase binding to DNA containing G,8-oxoG and O~6-MeG exhibited similar dissociation constants,indicating that 8-oxoG and O~6-MeG does not affect the binding of gp90 exo-to DNA.The presence of dCTP and Mg2+ stabilizes the binding of polymerase to in polymerase-DNA-dCTP ternary complex.Notably,8-oxoG at template reduced 3-fold the stability of binding gp90 exo-to DNA.O~6-MeG reduced the stability by 2-fold compared with G.Mismatch can further weaken the binding ability of DNA polymerase to DNA.(7)dTTP was preferentially inserted opposite O~6-MeG.The conformational change was detected by Auto SF-120.The conformation of polymerase in polymerase-DNA-dTTP ternary complex was changed rapidly,which accelerated dNTP incporporation efficiency.