Endonuclease Activity Analysis Of Mismatch Repair Protein Mlh3 From Tetrahymena Thermophila

Mismatch repair(MMR)is an evolutionarily conservative damage repair system that removes mismatches between bases and bases,insertions and deletions caused by DNA replication.In prokaryotes,MutS recognizes and binds mismatched sites,recruits MutL to form complexes,activates the endonuclease activity of MutH,and cleaves GATC sequences without methylated DNA sequences near the mismatched sites.The exonuclease removes unmethylated single-stranded DNA from the GATC site to the mismatch site with the help of UvrD helicase and SSB proteins.Then DNA polymerase and ligase complete the repair.In Saccharomyces cerevisiae and mammalian cells,MutS homologs(MSHs)recognize and bind mismatches between bases by forming heterodimers as well as insert and delete mutation regions.The MSH complex interacts with the MutL homolog(MLH)to create gaps in the new strand,recruiting other factors to facilitate deletion and complete the synthesis of the new strand.The mismatch repair protein Mlh3 is a MutL homologous protein and is highly conserved in various organisms.The C-terminus has a metal-binding motif DQHA(X)2E(X)4E.This motif can bind to metal ions to promote its endonuclease activity.In many organisms,Mlh3 not only participates in the process of mismatch repair,but also participates in meiotic homologous recombination.In Tetrahymena thermophila meiosis prophase,the micronucleus(MIC)is transcriptionally active and extremely elongated.Chromosome homologous recombination initiates after the formation of programmed DNA double-strand breaks(DSB).DNA mismatch repair(MMR)system maintains genome stability and promotes homologous recombination during meiosis.Mismatch repair protein Mlh3 is required for conjugation development in T.thermophila.However,its function was less clear.In this study,we studied the mismatch repair protein Mlh3 of Tetrahymena thermophila and obtained the following main results: 1.MLH3 sequence alignment and protein structure simulationThe DNAMAN software was used to compare the homology of MLH3 genes ofdifferent model organisms.Compared with other model organisms,there was no conserved ATP binding motif at the N-terminus of the MLH3 of Tetrahymena thermophila.However,there are DQHA(X)2E(E)4E motifs and three other((A/G)Q,ACX,CP/NHGRP)conserved motifs that together form a metal binding site and an endonuclease active site.Further simulation of the Mlh3 of Tetrahymena thermophila was performed using the Swiss-Model homology modeling.It was found that the DQHA(X)2E(X)4E motif and the C(X)HGR(X)motif are spatially close to each other,indicating that the two motifs together determine the ion binding and endonuclease activity of Mlh3.2.Artificial synthesis and prokaryotic expression purification of MLH3The Tetrahymena thermophila Mlh3 was optimized for E.coli preference for the expressed gene and then MLH3 gene was artificially synthesized.To analyze the function of Mlh3,a recombinant expression plasmid pGEX-MLH3 was constructed.By site-directed mutagenesis of the aspartic acid-encoding CAC in the Mlh3 motif DQHA(X)2E(X)4E as an asparagine in AAC,the glutamic acid-encoding GAA is GCA encoding alanine,resulting in a recombinant plasmid pGEX-MLH3D117 N and pGEX-MLH3D117E123 A.A truncated MLH3 truncation mutant containing a 84 base deletion C-terminal of CVHGRS was further obtained by PCR amplification to obtain a recombinant plasmid pGEX-MLH3D117NE123A?28.MLH3 and its mutated genes were constructed on GST-tagged prokaryotic expression vectors,and GST-tagged proteins GST-Mlh3,GST-Mlh3,GST-Mlh3-D117 N,GST-Mlh3-D117N-E123 and GST-Mlh3-D117N-E123A?28 were recombinantly expressed by E.coli.Those proteins were purified by GST affinity chromatography to obtain GST-Mlh3 protein and mutant protein,and its expression and purification were correct by 12% SDS-PAGE and Western-blotting.3.Mlh3 has a metal ion-dependent endonuclease activityThe Mlh3 protein was incubated with supercoiled DNA.The cleavage efficiency of Mlh3 on superhelical DNA was detected by nucleic acid gel electrophoresis.It was found that Mlh3 of Tetrahymena thermophila has endonuclease activity,and metal ions can promote its endonuclease activity.When ATP was added,it was found that ATP could further promote its endonuclease activity.It was shown that ATP and metal ions synergistically promoted the endonuclease activity of Mlh3.When GST-Mlh3 protein was used to act on supercoiled DNA with sizes of 3kb,5kb,8kb,and 12 kb,with the increase of supercoiled DNA fragments,the efficiency of DNA cleavage by GST-Mlh3 was changed from a gap to a complete cleavage.GST-Mlh3 protein was applied to the superhelix DNA of 3 kb 5 kb,8 kb,12 kb,it was found that,With the increase of superhelix DNA fragment,the cleavage effect of GST-Mlh3 on DNA changed from producing a gap to complete cutting into linear DNA,which indicated that Mlh3 had more obvious cutting effect on the larger segment of superhelix DNA.4.Mlh3 endonuclease activity site analysisThe GST-Mlh3-D117 N,GST-Mlh3-D117N-E123 A acted on supercoiled DNA and found that compared with GST-Mlh3,the activity of GST-Mlh3-D117 N,GST-Mlh3-D117N-E123 A endonuclease was reduced.Further,the C-terminal H(X)HGR(X)domain was truncated,and the purified GST-Mlh3-D117N-E123A?28 was acted on supercoiled DNA and found that it's endogenous enzyme activity is further reduced.It was shown that these two motifs together determine the ion binding and endonuclease activity of Mlh3.5.Identification of multiple factors interacting with Mlh3 by HA-Tag immunoprecipitationThrough the co-immunoprecipitation assay of the meiosis-phase protein of HA-Mlh3 cell line,12% SDS-PAGE electrophoresis,silver staining analysis and Western-blotting detection,the target protein sample was analyzed by mass spectrometry.The results showed that there are many potential factors interacting with Mlh3,including MutS domain III family proteins,meiotic recombinant protein Dmc1,meiosis DSB repair-associated protein Mnd1,and chromosome structure maintenance related proteins Smc2,Smc4 and other proteins.The DMC1 and MND1 genes of Tetrahymena thermophila were optimized as E.coli preference expression genes.The synthesized MND1 and DMC1 genes were further constructed to obtain recombinant plasmids.The His-SUMO-Mnd1 and His-SUMO-Dmc1 proteins were obtained through prokaryotic expression and purification.The Pull-down of GST-Mlh3 and His-SUMO-Mnd1 and His-SUMO-Dmc1 proteins were detected by 12% SDS-PAGE and Western-blotting.It was found that Mlh3 interacts with Mnd1 and Dmc1 in vitro.In this study,MLH3 was synthesized and expressed in Escherichia coli,and GST-Mlh3 protein was purified.Enzyme activity analysis showed that Mlh3 had metal-ion-dependent endonuclease activity.The HA-Mlh3 co-immunoprecipitation identified multiple possible Mlh3 interacting factors in the meiosis process of Tetrahymena thermophila.It was show that Mlh3 plays a role in meiotic mismatch repair through metal ion-dependent nucleic acid endonuclease activity and interaction with other factors.This study provides important data for the further study of the mechanism of Mlh3 in the process of meiosis in Tetrahymena thermophila.