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Functional Analysis Of Mismatch Repair Protein Tmlh1 In Tetrahymena Thermophila

Posted on:2021-02-28Degree:MasterType:Thesis
Country:ChinaCandidate:R X YangFull Text:PDF
GTID:2370330626955345Subject:Biochemistry and Molecular Biology
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DNA mismatch repair?MMR?is a highly-conserved DNA repair pathway that plays major roles during DNA replication,repair and recombination.In eukaryotes the MMR process begins when a Mut S-homolog recognizes and binds to mismatched DNA base pairs.Upon binding,the Mut S-DNA complex initiates recruitment of Mut L to form a ternary complex.The eukaryotic Mut L activities function as heterodimeric complexes: Mut L?,Mut L? and Mut L?,with MLH1 serving as a common subunit.These Mut L complexes not only involves in DNA mismatch repair?MMR?,but also plays a major role in the resolution of recombination intermediates during meiosis.Tetrahymena thermophila is a unicellular organism with two nuclei,a diploid germ line micronucleus?MIC?and a polyploid somatic macronucleus?MAC?.During sexual reproduction,micronucleus undergoes meiosis and is passed on to the offspring as new MACs and new MICs.During meiosis prophase,the extreme elongation of nuclei is triggered by DSB formation.Thereby,this bouquet-like arrangement is believed to promote homologous pairing and CO.The genome of MAC developed from the postzygotic MIC,but the IES?internal eliminated sequences?were eliminated and DNA was duplicated many times.In our previous work,Mlh3,a MMR protein in Tetrahymena thermophila,regulates genome replication,Chromosome segregation and DNA elimination.However,the role of Tmlh1,the Mut L core subunit,is still unclear.In this study,the mismatch repair protein Tmlh1 was first studied in Tetrahymena thermophila,and the preliminary results are as follows.1.Identification of Tmlh1 and the construction of knockout mutantTMLH1?TTHERM00127000?,the homologous gene of MLH1 was identified from the the Tetrahymena Genome Database.TMLH1 sequence is composed of 4553 bp that results in 756 amino acid.In the microarray expression profile of Tmlh1,there is a low level expression of TMLH1 during vegetative growth,a stage specific high-level expression in conjugation 2 h-4 h,and a peak of expression at this 10 h.The different mating type germ line TMLH1 knockout strains were created.The growth and generation time of mutant cells was prolonged.The fluorescence signal of ?-H2 A.X show that the efficiency of DNA damage repair was reduced in the vegetative growth.At the conjugation stage,reduced Cell pairing rate,delayed sexual development,and the abnormal formation of micronuclei were found.Thus,the deletion of TMLH1 gene causes the defect of DNA damage repair and genomic instability,affects the process of cell vegetative growth and sexual reproduction.2.Location pattern of tmlh1 in Tetrahymena thermophilaTo study the presence and localization of Tmlh1 p In Tetrahymena,we constructed strains over expressing HA-Tmlh1.Localization of Tmlh1 p was observed by the indirect immunofluorescence localization.In the Interphase of vegetative growth,HA-Tmlh1 p was present in vegetative micronuclei,produced weak signals in macronuclei.Immunostaining for HA-Tmlh1 showed a dynamic localization pattern in the the conjugation stage of Tetrahymena.During the early mating stages,2HA-Tmlh1 localized at MICs and parent meganucleus which has transcriptional active.At the selection of pronuclei and anlagen stage,Tmlh1 is only localized to the functional nucleus and disappears from the degraded MICs and old MACs.It is important to note that Tmlh1 forms spindle-like structure and the common localization signal of Tmlh1 and ?-tubulin appears during meiosis and mitosis.In the later stage of sexual reproduction,the location signals of Tmlh1 and Pdd1 overlap.It is suggested that Tmlh1 plays various functions in different stages of Tetrahymena development.3.The endonuclease activity of Tmlh1Tmlh1p contains a globular N-terminal domain?NTD;1-416?and C-terminal domain?CTD;492-756?connected by a flexible linker arm?Linker,417-491?.The NTD contains ATP binding domain?37-125?that are conserved in Mut L homologue.The CTD consists of the S1 domain?Mut L heterodimerization site 1?,the PIP-box motif?PCNA interacting protein?and the conserved CTH motif?Mlh1 C-terminal homology?.And the S1 domain was distributed at two terminal of CTD?S1 site-N,532-557 and S1 site-C,738-754?.The c DNA sequence of TMLH1 was optimized for preferential expression in Escherichia coli and then was artificially synthesized by Biotech company.The recombinant plasmid was constructed by gene cloning and further expressed as the fusion protein with tag in the prokaryotic expression system.The purified fusion protein was obtained by affinity chromatography.Tmlh1 apparently exhibits concentration dependent nicking endonuclease activity for ds DNA in vitro,which can be promoted by Cu2 +.And it has a concentration dependent suppression of ATP/ADP on the activity.4.Identification of thepotential binding factors of Tmlh1In order to study whether Mlh3 p interact with Tmlh1 p and form functional complex Mut L? in T.thermophile,Tmlh1 and Mlh3 was tagged respectively at the N-terminu with GST?Glutathione S-Transferase?and 6His-MBP tag.We demonstrate that Tmlh1 p is a direct partner of Mlh3 p using pull down experiments.And C-terminal domain of Tmlh1 can be combined with mlh3 independently.In this study,we generated a truncated variant of Tmlh1-?C10 that lacks the CTH motif?residues 746-756?.As expected,the loss of CTH motif reduced the structural stability of Tmlh1 protein and affected the interaction with Mlh3.There are many potential interaction factors of Tmlh1 were identified by the 2HA-Tmlh1 CO-IP-MS,such as the Mut S family protein,crossover factors Rad51 and DMC1,tubulin proteins Atu1 and Gtu1,heterochromatin formation and histone methyltransferase Ezl1,chromatin structure remodeling factor Hmb1.Taken together,as a mismatch repair protein in Tetrahymena thermophila,tmlh1 was identified for the first time in this study.In addition,stable TMLH1 knockout lines were produced and diversity localization patterns of Tmlh1 were studied by immunofluorescence.Tmlh1 protein were expressed and purified in vitro.As experimental results,the endonuclease activity of Tmlh1 was found for the first time,and Tmlh1 involves in homologous recombination of DNA,rearrangement of macronuclear genome and correct separation of chromosomes.These processes are of great significance for maintaining the stability of Tetrahymena genome during vegetative growth and sexual conjugation development.Furthermore,these results provide the important theoretical bases for the further Functional evolution study of mismatch repair protein MLH1 in eukaryotes.
Keywords/Search Tags:Tmlh1, Mismatch repair, endonuclease, homologous recombination, Tetrahymena thermophile
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