Functional Analysis Of Mismatch Repair Protein Mlh3 In Tetrahymena Thermophila

DNA mismatch repair(mismatch repair,MMR)is an evolutionarily conserved mechanism.MMR correct the mismatched base,remove the mutation and maintain the accuracy of genome replication in DNA replication.The key components of the MMR system MutS,MutL,MutH and UvrD were identified in Escherichia coli.There are six MutS homologues(MSH1,MSH2,MSH3,MSH4,MSH5,MSH6)and four MutL homologues in yeast and mammalian MMR systems.The human MMR gene family comprises four MutL paralogues capable of forming heterodimeric MutLa(MLH1/PMS2),MutL?(MLH1/PMS1)and MutLy(MLH1/MLH3).MutL? possesses endonuclease activity,participates recombination and exchange process between non-sister chromatid.Meiotic specific nuclease Spo11 causes DNA double strand breaking,micronuclear stretching and homologous chromosome pairing in Tetrahymena thermophila.Mismatch repair protein MutS promote the formation of homologous chromosome crossover.Resolvase Mus81 and helicase Sgsl promote the segregation of homologous chromosome.Knockdown of MLH1 did lead to visible abnormal meiosis.So,the function of MutL? is not clear.In this study,MLH3 was identified from T.thermophila and the function of Mlh3 was analyzed.The main results obtained as following:1.Identification and analysis of MLH3By rapid-amplification of cDNA ends(RACE),we found that 5'UTR of MLH3 is 18 bp,3'UTR of MLH3 is 211 bp,polyA tail is 48bp,open reading frame is 960 bp and encodes 319 amino acids.Mlh3 contain conserved endonuclease activity site DQHA(X)2E(X)4E.Microarray expression profile showed that MLH3 was conjugation specific expressed gene(http://tfgd.ihb.ac.cn).2.Mlh3 localize in the sexual reproductive nucleus and the newly developed macronuclei.To analyze Mlh3 localization,pXS75-MLH3 was constructed and transformed into Tetrahymena.HA-MLH3 recombinant cells strains were screened by gradient paromomycin(PM)concentration.Immunofluorescence showed HA-Mlh3 located in the micronucleus(Mic)in early sexual reproduction stage,located in new macronucleus(Mac),and disappeared in the late stages.The results showed that Mlh3 could be involved in the development of new Mic and new Mac.3.Knockout of MLH3 affect sexual reproductionTo analyze the function of MLH3,MLH3 was completely knocked out.MLH3 knockout mutant cells have no obvious abnormalities in the early sexual development stage.When 55%WT cells develop two Macs and one Mic stage,there are no two Macs and one Mic cells appeared in MLH3 knock mutant cells,only containing 20%anlagen stage cells,45%exconjuagnt with two Macs and two Mics.When 70%WT cells developed to 2Mac and 1Mic state at 48 h,we only found 1 Mac and 1 Mic cells,the mating cell pair disappeared in MLH3 knockout mutant cells.4.MLH3 involved in the replication and repair of new macronuclear genome?H2A.X immunofluorescence stainning showed that the signals was kept in the new Macs and new Mics in the MLH3 knockout cells.In contrast,the signals were disappeared in the mating WT cells.The results indicated that there are unrepaired fragment in the new Mac and Mic genome in the MLH3 knockout cells.5.Sexual progeny of MLH3 knockout cells fail to surviveTo further confirm Mlh3 function,the progeny of MLH3 knockout cells was analyzed.The results showed no progeny was survived in MLH3 knocked out cells.Flow cytometry analysis showed that MLH3 knockout cells formed apoptosis signal when WT cells developed into 2 Mac and 1 Mic stage.Taken together,a mismatch repair gene MLH3 was identified for the first time from T.thermophila.Mlh3 located in the Mic and new Mac.Mlh3 is necessary for macronuclear genome replication and sexual progeny survival.