Roles for Mismatch Repair Proteins in Maintaining Genome Stability

错配修复蛋白在维持基因组稳定性中的作用

• 批准号: 10727007
• 负责人: Eric E. Alani
• 金额: $ 8.4万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10727007
• 关键词: Affect; Alleles; Aneuploidy; Binding; Biochemical; Bypass; Chromosomal Rearrangement; Chromosome Segregation; Colon Carcinoma; Complex; Cruciform DNA; DNA; DNA Sequence; DNA biosynthesis; Deacetylase; Defect; Diffusion; Dimerization; Disease; Ensure; Eukaryota; Failure; Filament; Genetic; Genetic Crossing Over; Genetic Recombination; Genome Stability; Grant; Hereditary Nonpolyposis Colorectal Neoplasms; Histones; Homologous Gene; Human; Hydrolysis; In Vitro; Infertility; Inherited; Invaded; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Meiosis; Meiotic Recombination; Methods; Mismatch Repair; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Pathogenicity; Pathway interactions; Patients; Pregnancy; Protein Family; Proteins; Reaction; Research; Role; Saccharomyces cerevisiae; Sister Chromatid; Site; Specificity; Syndrome; System; Testing; Topoisomerase; Work; base; cohesion; endonuclease; gene repair; genome wide screen; helicase; homologous recombination; insertion/deletion mutation; molecular modeling; mutant; nuclease; premature; prevent; reconstitution; recruit; repaired; single molecule; tool

Project Summary DNA mismatch repair (MMR) systems act to excise misincorporation errors that occur during DNA replication. In eukaryotes MSH proteins recognize these errors in the context of base-base and insertion/deletion mismatches and recruit MLH complexes to form ternary complexes that work with replication factors (RPA, RFC, PCNA) and Exo1 to excise the newly replicated DNA strand through the mismatch site. This is followed by DNA re-synthesis steps. MMR factors also recognize mismatches that form during strand invasion steps in homologous recombination; they recruit a helicase complex that unwinds (rejects) recombination intermediates and allows a new homology search. In addition, subsets of MMR factors act in meiosis to resolve recombination intermediates into crossovers (COs). In baker’s yeast the majority of meiotic COs are formed in an interference-dependent pathway in which double Holliday junctions (dHJs), thought to be stabilized by Msh4-Msh5, are resolved through the actions of STR helicase/topoisomerase, Exo1 nuclease, and the MutLγ (Mlh1-Mlh3) endonuclease. Our work is focused on developing molecular models to explain how the different MSH and MLH factors act in the above pathways. This work will enable us to understand how molecular defects in these factors underlie human infertility and hereditary forms of colon cancer, and how chromosomal rearrangements can lead to disease. We will test these ideas through three distinct research themes. In Project 1 we are studying how conformational changes in MLH proteins, mediated by ATP binding and hydrolysis, are linked to strand specificity steps in MMR and meiotic recombination. We will use genetic (mutations in intrinsically disordered domains in Mlh1-Pms1 and Mlh1-Mlh3 and force dimerization of MLH proteins), biochemical (in vitro reconstitution reactions to determine specific roles for MLH proteins in MMR and mass-spectrometry) and single-molecule (examine diffusion along DNA and how proteins bypass barriers) approaches. Project 2 is focused on understanding how MutLγ acts to resolve dHJs in the ZMM pathway. Our work in the current grant period is consistent with MutLγ endonuclease being activated in MMR and meiotic crossing over through the formation of a MutLγ filament. We will use this information and biochemical, mass spectrometry, and genetic methods that take advantage of our identification of mlh3 separation of function mutants to identify MutLγ interacting factors. Our early work encourages us to initially focus on MutLγ interactions with the Exo1 nuclease, after which we will test identified factors alone and in combination for their ability to interact with MutLγ to cleave model HJ and dHJ substrates. Project 3 is aimed at understanding how the decision is made to repair or reject recombination intermediates. We will analyze how mutations in histone chaperones and deacetylases, separately and in combination, affect anti-recombination, and will employ an inducible system to provide a temporal and physical measure of these effects. This work will also encourage us to pursue a genome-wide screen to identify new factors that regulate the repair/rejection decision.

项目摘要 DNA不匹配修复（MMR）系统的作用是经历DNA复制过程中发生的错误结构误差。 在真核生物中，MSH蛋白在基础和插入/删除的背景下识别这些错误 不匹配和招募MLH复合物以形成与复制因子一起使用的三元复合物（RPA， RFC，PCNA）和EXO1通过不匹配位点切除新复制的DNA链。这是随后的 由DNA重新合成步骤。 MMR因素还识别出在链入侵步骤中形成的不匹配 同源重组；他们招募了一个放松（拒绝）重组中间体的解旋酶复合物 并允许新的同源性搜索。此外，MMR因子的子集在减数分裂中作用 重组中间成跨界（COS）。在贝克的酵母中，大多数减数分裂COS是在 双重holliday连接（DHJ）的干扰依赖性途径，被认为稳定 MSH4-MSH5通过Str解旋酶/拓扑酶，EXO1核酸酶和mutlγ的作用解决 （MLH1-MLH3）核酸内切酶。我们的工作重点是开发分子模型，以解释如何不同 MSH和MLH因子在上述途径中作用。这项工作将使我们能够了解分子 这些因素的缺陷是人类不育症和遗传形式的结肠癌的缺陷，以及染色体如何 重排可能导致疾病。我们将通过三个不同的研究主题来测试这些想法。在 项目1我们正在研究如何通过ATP结合和 水解与MMR和减数分裂重组的链特异性步骤有关。我们将使用通用 （MLH1-PMS1和MLH1-MLH3中本质上受干扰的结构域的突变以及MLH的力二聚化 蛋白质），生化（体外重构反应，以确定MMR和MMR中MLH蛋白的特定作用 质谱法）和单分子（检查沿DNA的扩散以及蛋白质旁路屏障的扩散） 方法。项目2的重点是了解MUTLγ如何在ZMM途径中解决DHJ。我们的 当前赠款期间的工作与MMR和减数分裂激活的MUTLγ内切核酸酶一致 通过形成mutlγ细丝越过。我们将使用此信息和生化，质量 光谱法和利用我们鉴定功能分离的遗传方法 突变体鉴定MUTLγ相互作用因子。我们的早期工作鼓励我们最初专注于mutlγ 与exo1核酸酶相互作用，之后我们将单独测试确定的因素，并结合使用它们 能够与MUTLγ相互作用以清除HJ和DHJ底物模型。项目3旨在了解如何 该决定是修复或拒绝重组中间体的决定。我们将分析组蛋白突变如何 伴侣和脱乙酰基酶，分别和组合，影响抗混合物，并将采用 可诱导的系统，可提供对这些影响的暂时和物理测量。这项工作也会鼓励我们 购买全基因组屏幕以确定调节维修/拒绝决策的新因素。