Roles for Mismatch Repair Proteins in Maintaining Genome Stability

项目摘要 DNA错配修复（MMR）系统用于切除DNA复制期间发生的错误掺入错误。 在真核生物中，MSH蛋白在碱基-碱基和插入/缺失的背景下识别这些错误 错配并募集MLH复合物以形成与复制因子（RPA， RFC，PCNA）和Exo 1通过错配位点切除新复制的DNA链。紧随其后的 通过DNA再合成步骤。MMR因子还识别在链侵入步骤中形成的错配， 同源重组;它们募集解旋酶复合物，解旋（排斥）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起作用， 重组中间体转化为交叉（CO）。在面包酵母中，大多数减数分裂CO是在 一种干扰依赖性途径，其中双霍利迪连接（dHJ），被认为是稳定的 Msh 4-Msh 5通过STR解旋酶/拓扑异构酶、Exo 1核酸酶和MutLγ的作用而分解。 （Mlh 1-Mlh 3）内切核酸酶。我们的工作重点是开发分子模型来解释不同的 MSH和MLH因子在上述通路中起作用。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和遗传性结肠癌的基础， 基因重排会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我们正在研究如何在MLH蛋白的构象变化，介导的ATP结合， 水解与MMR和减数分裂重组中的链特异性步骤有关。我们将使用基因 （Mlh 1-Pms 1和Mlh 1-Mlh 3中固有无序结构域的突变和MLH的二聚化 蛋白）、生物化学（体外重建反应以确定MMR中MLH蛋白的特定作用， 质谱）和单分子（检查沿着DNA的扩散以及蛋白质如何绕过屏障） 接近。项目2的重点是了解MutLγ如何在ZMM途径中分解dHJ。我们 目前资助期的工作与MutLγ内切酶在MMR和减数分裂中被激活是一致的。 通过形成MutLγ细丝进行交叉。我们将利用这些信息和生物化学，质量 光谱法和遗传学方法，这些方法利用了我们对MLH 3功能分离的鉴定 突变体以鉴定MutLγ相互作用因子。我们的早期工作鼓励我们首先关注MutLγ 与Exo 1核酸酶的相互作用，之后，我们将测试单独和组合的识别因子， 与MutLγ相互作用以切割模型HJ和dHJ底物的能力。项目3旨在了解如何 做出修复或拒绝重组中间体的决定。我们将分析组蛋白的突变 分子伴侣和脱乙酰酶，单独或组合，影响抗重组，并将采用 诱导系统提供这些影响的时间和物理测量。这项工作也将鼓励我们 进行全基因组筛选，以确定调节修复/排斥决定的新因素。

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.