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

基本信息

批准号：
10077565
负责人：
Eric E. Alani
金额：
$ 39.19万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/10077565
关键词：
Affect Alleles Aneuploidy Binding Biochemical Bypass Chromosomal Rearrangement Chromosome Segregation Cleaved cell Colon Carcinoma Complex Cruciform DNA DNA DNA Sequence DNA Sequence Alteration 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 Lead Link Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Meiosis Meiotic Recombination Methods Mismatch Repair Modeling Molecular Molecular Chaperones Molecular Conformation Mutation Pathogenicity Pathway interactions Pregnancy Protein Family Proteins Reaction Research Role Saccharomyces cerevisiae Sister Chromatid Site Specificity Syndrome System Testing Topoisomerase Work base cohesion colon cancer patients endonuclease gene repair genome wide screen helicase homologous recombination insertion/deletion mutation molecular modeling mutant nuclease premature prevent reconstitution recruit repaired single molecule tool

项目摘要

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.

项目概要 DNA 错配修复 (MMR) 系统可消除 DNA 复制过程中发生的错误掺入错误。在真核生物中，MSH 蛋白在碱基和插入/缺失的背景下识别这些错误错配并招募 MLH 复合物形成与复制因子（RPA、 RFC、PCNA) 和 Exo1 通过错配位点切除新复制的 DNA 链。通过 DNA 重新合成步骤，错配因子还可以识别链侵入步骤中形成的错配。同源重组；它们招募解旋酶复合物来解旋（拒绝）重组中间体并允许新的同源性搜索此外，MMR因子的子集在减数分裂中发挥作用来解决。重组中间体形成交叉 (CO) 在面包酵母中，大多数减数分裂 CO 形成于一种干扰依赖性途径，其中双霍利迪连接体（dHJ）被认为是通过 Msh4-Msh5 通过 STR 解旋酶/拓扑异构酶、Exo1 核酸酶和 MutLγ 的作用来解析 (Mlh1-Mlh3) 核酸内切酶的工作重点是开发分子模型来解释不同的情况。 MSH 和 MLH 因子在上述途径中发挥作用，这项工作将使我们能够了解分子机制。这些因素的缺陷是人类不孕症和遗传性结肠癌的基础，以及染色体如何我们将通过三个不同的研究主题来检验这些想法。项目 1 我们正在研究 MLH 蛋白的构象变化如何通过 ATP 结合和水解，与 MMR 和减数分裂重组中的链特异性步骤相关。（Mlh1-Pms1 和 Mlh1-Mlh3 中本质上无序的结构域发生突变，并迫使 MLH 二聚化蛋白）、生物化学（体外重构反应以确定 MLH 蛋白在 MMR 和 MMR 中的特定作用质谱）和单分子（检查沿 DNA 的扩散以及蛋白质如何绕过屏障）项目 2 的重点是了解 MutLγ 如何发挥作用来解决 ZMM 途径中的 dHJ。当前资助期内的工作与在 MMR 和减数分裂中激活 MutLγ 核酸内切酶一致通过形成 MutLγ 丝进行交叉我们将使用这些信息和生化、质量。利用我们对 mlh3 功能分离的鉴定的光谱测定法和遗传方法我们的早期工作鼓励我们首先关注 MutLγ。与 Exo1 核酸酶的相互作用，之后我们将单独和组合测试已确定的因素与 MutLγ 相互作用以裂解模型 HJ 和 dHJ 底物的能力项目 3 旨在了解如何裂解模型。我们将分析组蛋白如何发生突变，从而做出修复或拒绝重组中间体的决定。分子伴侣和脱乙酰酶，单独或组合，会影响抗重组，并会采用这项工作也将鼓励我们使用诱导系统来对这些影响进行时间和物理测量。进行全基因组筛选，以确定调节修复/拒绝决策的新因素。