Biochemical And In Vivo Functional Analysis Of The DNA Mismatch Repair Endonuclease EndoMS In Sulfolobus Islandicus

Mismatch repair(MMR)is one of the major pathways of DNA repair.It is responsible for the repair of mismatches caused by incorrect insertion during DNA replication or inner and external factors.MMR is conserved in many kinds of bacteria and eukaryotes and plays an important role in maintaining the genome stability.The first step of the classic mismatch repair is the recognition of mismatch by MutS protein followed by the recruitment of MutL protein to form a complex,which is responsible for the stimulation of endonuclease activity of MutH.Afterwards,MutH cleaves the mismatch strands and generates a nick at the 5'or 3' direction of the mismatch.UvrD helicase then removes the MutH and unwinds the DNA to the mismatched site.The nuclease,ExoI degrades the DNA fragment containing the mismatches.The gap is filled by DNA polymerase ? holoenzyme and the strand is ligated by a DNA ligase.Finally,DNA methylase methylates the nascent strand to form a mature DNA strand.The conserved MutS/MutL protein and its homologs exist in most bacteria,eukaryotes and few kinds of archaea,but in most archaea,actinomycetes including Mycobacterium tuberculosis lack the MutS/MutL protein and its homologs,so the mismatch repair mechanism in these organisms is still not well understood.Recently,a novel mismatch repair protein EndoMS was characterized in Thermococcus kodakaraensis,the homologue of NucS from Pyrococcus furiosus.In vitro biochemical experiment showed that EndoMS in T.kodakaraensis only recognize G-T,G-G,T-T,T-C,A-G five kind of mismatches and can cut the mismatched substrate in a way similar to restriction enzyme,generating a double-stranded DNA break.However,still there are many questions unresolved.For example,how cells can identify and repair other kinds of mismatches and,how to finish the repair after cutting?Here we used biochemical and genetic methods to study the properties,function and mechanism of EndoMS in the crenarchaeon Sulfolobus islandicus.First,the gene coding SisEndoMS protein was cloned and expressed.DNA substrate containing 32P labelled mismatch strand was prepared and incubated with the protein.We found that the protein has specificity and can only cut three kind of mismatches G-G,G-T,and T-T by using Mg2+ as a cofactor.After ensuring the protein has endonuclease activity,we did phenotypic analysis of the strain overexpressing SisEndoMS and the knockout strain.We constructed the overexpressed strain and analyzed the growth,found that after the growth of the over-expression strain was obviously retarded.At the same time,the cell cycle and cell morphology of overexpressed strain were tested by flow cytometry and microscopy.The results showed that the number of cells with diploid DNA content was significantly higher in overexpressed strain than that of the control.The average cell size also increased.These results showed that the over-expression protein could prevent the chromosome separation after DNA replication,thus affecting cell division.On the other hand,we used the marker replacement strategy to knockout SisEndoMS gene and analyzes the phenotype of the knockout strain.It is found that,the knockout strain grew faster than the wild type in the liquid medium.But on the solid plate,the knockout strain form fewer colonies than the wild type,which indicates SisEndoMS plays an important role in the repair of S.islandicus.Due to the incomplete repair system,the knockout strain could not correct the error caused by the external factors,resulting the faster replication and division.But on the solid plate,where the stress increased,the knockout strain failed to repair effectively after the deletion of the repair system,resulting a poor growth.In order to know under which condition can the protein recognize all the kind of mismatches to further understand its function in vivo and to extend the application of it,we performed site-directed mutagenesis to make the SisEndoMS to recognize more types of mismatches.Finally,we got a mutant protein SisEndoMSN71T,which can recognize and cleave all eight kind mismatches in the presence of 2 mM Mn2+.