Preliminary Study On Expression,Purification And Function Of Archaeal DNA Polymerase D And Replication-related Proteins

Archaea resource is one of the hot topics in theoretical and applied research.Under the synergy of DNA polymerase and related proteins,Archaea can ensure the accurateness in transmission of genetic information,which is extremely important for the continuation of life.These proteins have not only theoretical value,but also potential market prospects.In archaea,there are many proteins playing important role in DNA replication.DNA polymerase D(Pol D)plays can continuously synthesize new DNA chains with the parent chains as templates.Endo Q,Endo MS,UNG and other proteins are responsible for eliminating all kinds of erroneous bases generated by replication and ensuring the accuracy of DNA replication.Pol D and DNA replication accuracy-related proteins Endo Q,Endo MS,and UNG are expected to be used to improve the sensitivity and fidelity of polymerase chain reaction(PCR),and further enhance the efficiency of gene cloning.Therefore,this paper carried out the following works around those proteins:(1)Heterologous expression of Pol D,Endo Q,Endo MS from thermophilus Archaea Thermococcus sp.4557 and UNG from Acinetobacter baumannii in E.coli as host.The two subunits of Pol D polymerase,DP1 and DP2,and Endo Q,Endo MS and UNG proteins were purified by Ni-NTA affinity chromatography followed by hydrophobic chromatography.Finally,DP1 protein 22.5 m L(1.49 mg/m L),DP2 protein 11 m L(0.56 mg/m L),Endo Q protein 12.5 m L(1.31 mg/m L),Endo MS protein10 m L(0.92 mg/m L),UNG protein 6.4 m L(1.62 mg/m L)were obtained to provide materials for subsequent experiments.(2)The functions of two subunits of Pol D polymerase were studied with fluorescent labeled DNA probes combined with PAGE electrophoresis experiments.The results showed that the large subunit DP2 has DNA extension activity in vitro,and the small subunit DP1 has 3'?5' exonuclease activity.When the amount of DP2 in the system is 0.1 pmol,the expansion can still be completed.If DP1 is too much,the DNA templates or products will be completely degraded.The presence of DP2 can significantly promote the exonuclease activity of DP1 while the replication factor GINS51 has an inhibitory effect on DP1 exonuclease activity.These results supplement the relevant information of Pol D in the archaeal DNA replication process.(3)The purified DNA replication repair factors,namely Endo MS,Endo Q,UNG and other proteins were applied in PCR reaction catalyzed by common Taq DNA polymerase.After amplification,those PCR products were sequenced,blasting with template sequence to find mutations and to count the mutation rate.The results showed that the intervention of Endo Q,Endo MS,and UNG reduced the overall mutation rate of PCR amplified products to 95.6%,95.3%,and 90.1% respectively,which improved the fidelity of PCR reaction.Those proteins may even play a similar role in a PCR system catalyzed by high-fidelity DNA polymerase.Thus the design above could be expected to become an alternative way to reduce the cost of high-fidelity PCR amplification.In summary,this work enriches the knowledge of Pol D subunits DP1 and DP2,Endo Q,Endo MS,UNG and other related proteins,providing a reference for improving the sensitivity and fidelity of PCR reactions.