The Research In The Function Of PaP1 DNA Polymerase And The Kinetic Analysis Of Bypassing DNA Damage By Yeast Polymerase Eta

All of prokaryote,eucaryon and virus obtain DNA polymerase,which is one of the most important tool enzymes in DNA replication.Bacteriophage belongs to virus,containing DNA polymerase.During the process of infecting the host bacteria by bacteriophage,bacteria proliferation and the DNA replication in bacteriaophage itself,DNA polymerase almost participates in every step,contributing a crucial role.However,the knowledge of DNA polymerase in bacteriophage is limited,most mechanism of DNA polymerization in bacteriophage is still not clear,there are so many potential functions of DNA polymerase can be explored in the future.While,DNA polymerase also?cupies an important position in the normal operation of the organism and the life extension,comparing with the more complicated eukaryotic organism.When the organism was affected by the environment stimulation,or generated DNA mutation,the DNA polymerase can assist the organism in DNA replication,translesion and repair,which provides guarantee for the genetic material in accurate DNA replication.There are many diseases related to DNA damage,DNA damage can cause the permanent changes in DNA sequence,resulting in diseases,genetic characteristics variation,afunction,malformation and even death.The organism itself can have spontaneous mutation,leading mispairing and base altering in DNA replication.Besides,contacting with physical and chemical damage factors in nature,the DNA sequence and the base also can be changed and?cur mutation.The damage factors obtain ultraviolet,ionizing radiation,alkylation reagents and so on.DNA damage can lead to diseases that threat human health seriously,we can develop the research in the mechanism of DNA translesion,replication and repair,and it will help to lay the foundation for the treatment of related diseases.Pseudomonas aeruginosa phage PaP1 is a lytic and virulent phage,which was isolated recently.As a new bacteriophage,the function of its DNA polymerase-gene 90 protein(Gp90)and the mechanism in DNA replication by Gp90 have not been studied.This group has isolated and purified PaP1,constructed vector,expressed and purified the DNA polymerase Gp90 of PaP1,besides,used bioinformatics technology to predict the function of DNA polymerase,following function verification via experiments.Meanwhile,this group also expressed and purified the core of yeast DNA polymerase Pol ?core,studied the kinetic analysis of bypassing DNA damage.The main contents and results were summarized in following aspects:1.The function identification of PaP1 DNA polymerase Gp90: we amplified the target gene 90,connected it on the expression vector,transformed the plasmid into Escherichia coli(E.coli),expressed the target protein Gp90 in large scale,lysed the bacteria,collected supernatant after centrifugation,and loaded onto Ni Sephatose column,then got the high activity DNA polymerase Gp90.We predicted the function of Gp90 via bioinformatics analysis,compared the polymerase domain and exonuclease domain with other DNA polymerases in the same family.We traced the variation of reactions using 32 P labeled DNA.In order to verify the polymerase activity,we have used some related experiments,such as full-length extension assay,M13 ss DNA extension assay and one-base incorporation assay.We used single stranded DNA(ss DNA)and double stranded DNA(ds DNA)in exonuclease activity assays.Gp90 was compared with T7 Pol/trx in all polymerase and exonuclease activity verification experiments.The results indicated that the DNA polymerase Gp90 of PaP1 belongs to A-family DNA polymerase demonstrating ss DNA and ds DNA 3'-5' exonuclease activity.The sequenceof Gp90 is homologous but not identical to that of other A-family DNA polymerases,such as T7 DNA polymerases(Pol)and DNA Pol I.The processivity of Gp90 in DNAreplication and its efficiency in single-deoxyribonucleoside triphosphate(d NTP)incorporationare similar to those of T7 Pol with processive thioredoxin(T7 Pol/trx).Gp90 can degrade ss DNA and ds DNA in 3'-5' direction at a similar rate,which is considerably lower than that of T7 Pol/trx.The optimized conditions for polymerization were a temperature of 37? and a buffer consistingof 40 m M Tris–HCl(p H 8.0),30 m M MgCl2,and 200 m M NaCl.2.Kinetic analysis of bypass of O6-methylguanine by the catalytic core of yeast DNA polymerase eta: we chose the core of yeast DNA polymerase Pol ?core as catalyzing enzyme in all research.We also used 32 P labeled DNA in experiments to observe the changes in reaction.We have studied the kinetics of bypassing O6-MeG in the following aspects: the full length extension assay with four d NTP,steady-state one-base incorporation and next-base extension kinetic assay,steady-state single d NTP incorporation,pre-steady-state single deoxycytidine triphosphate(dCTP)incorporation and LC-MS/MS analysis of primer extension products.Comparing with the full-length Pol?,Pol ?core(residues 1-513)lacks the C-terminal C2H2 motif,eliminating the effects on d NTP incorporation.When encountering O6-MeG,the misincorporation frequencies were 10-4 for G and 0.055-0.446 for O6-MeG.Even though O6-MeG is one of the DNA damage,it does not affect the extension efficiency.Pol?core showed no fast burst phase for any incorporation opposite G or O6-MeG.Primer extension was greatly blocked by O6-MeG and about 67% deoxythymidine triphosphate(dTTP),31% dCTP and 2% deoxyadenosine triphosphate(d ATP)were incorporated opposite O6-MeG.