错配修复蛋白Tmlh1维持嗜热四膜虫细胞核的稳定性  

The Mismatch Repair Protein Tmlh1 Maintains Nuclear Stability in Tetrahymena thermophila

作　　者：许静 杨茹霞[1] 薄涛 王伟 XU Jing;YANG Ru-Xia;BO Tao;WANG Wei(Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Shanxi University,Taiyuan 030006,China;College of Life Sciences,Shanxi University,Taiyuan 030006,China)

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006 [2]山西大学生命科学学院,太原030006

出　　处：《中国生物化学与分子生物学报》2020年第10期1210-1219,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31872224,31471999);山西省自然科学基金(No.201901D111008)资助课题。

摘　　要：DNA错配修复(DNA mismatch repair,MMR)蛋白Mlh1和其它因子形成多种不同的复合物,在DNA复制后的MMR途径和减数分裂DNA重组中发挥重要作用。然而对Mlh1的功能并不完全清楚,进一步分析Mlh1在不同进化地位生物中的功能具有重要意义。嗜热四膜虫(Tetrahymena thermophila)含有不同的错配修复复合物,基因表达谱分析发现,MutL复合物中的TMLH1(TTHERM_00127000)在营养生长期和饥饿期低水平表达,在有性生殖减数分裂期表达水平显著上调。免疫荧光定位显示营养生长期,Tmlh1定位于生殖系小核和转录活跃的大核;有性生殖期,定位于功能性小核和亲本大核,但在凋亡的大核和小核中消失。在减数分裂和有丝分裂时期,Tmlh1和α-微管蛋白(α-tubulin)存在共定位;而有性生殖后期,Tmlh1与异染色质蛋白Pdd1共定位于DNA删除的异染色结构域。TMLH1敲除细胞增殖速率降低,DNA损伤修复抑制,导致有性生殖细胞配对率降低和微核形成。1 mmol/L甲基甲磺酸甲酯(methy methanesulfonate,MMS)处理下,ΔTMLH1细胞传代时间增加了4.53%±0.35%,而野生型细胞传代时间增加了0.60%±0.14%。TMLH1敲除突变细胞株小核上呈现强烈的γ-H2A.X的荧光信号。免疫共沉淀和蛋白质谱分析发现,Tmlh1同微管蛋白、错配修复因子MutS、同源重组修复关键因子Rad51,非同源末端修复因子Ku80因子等存在相互作用。这些结果表明,嗜热四膜虫错配修复蛋白Tmlh1通过多种途径参与DNA修复和基因组重排,从而维持四膜虫生长发育和有性生殖过程中细胞核的稳定性。The DNA mismatch repair factor Mlh1 forms various complexes with other proteins and these complexes play important roles during DNA replication,repair and meiosis.However,the function of Mlh1 is not yet clear.Tetrahymena thermophila contains different mismatch repair proteins.Microarray analyses of gene expression showed TMLH1(TTHERM_00127000)has low expression levels during vegetative growth and starvation stages,and high expression levels during the conjugation stage.Immunofluorescence staining showed Tmlh1 localized in the germline micronuclei(Mics)and somatic macronuclei(Macs)during vegetative growth.The Tmlh1 signal occurred in functional Mics and parental Macs,and the signal disappeared from the degraded Mics and parental Macs during the sexual reproduction stage.The co-localization signal of Tmlh1 andα-tubulin appeared at meiotic Mics and mitotic Mics.In the late stage of sexual reproduction,the co-localization signal of Tmlh1 and Pdd1 occurred around DNA eliminating structure.In the TMLH1-knockout strains,the proliferation rate and DNA damage repair reduced in the vegetative growing stage,and abnormal Mics were found at the conjugation stage.Under 1 mmol/L methyl methanesulphonate(MMS)treatment,ΔTMLH1 cells proliferation time increased by 4.53%±0.35%,while wild-type cells only increased by 0.60%±0.14%.The increasedγ-H2A.X signal was observed around the Mics in TMLH1-knockout mutants.The co-immunoprecipitation and mass spectrometry analysis identified different interaction factors of Tmlh1:including tubulin proteins,the MutS family protein,homologous recombination factors Rad51,and nonhomologous end-joining pathway factor Ku80.These results suggested that Tmlh1 is involved in DNA repair and rearrangement of the genome by various pathways and maintained nuclear stability during vegetative growth and sexual conjugation development in Tetrahymena.

关 键 词：错配修复 纺锤体 异染色质体 嗜热四膜虫 

分 类 号：Q956[生物学—动物学]