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复制过程中发生的错配错误。 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 错配和招募MLH复合体以形成与复制因子(RPA， RFC、增殖细胞核抗原)和Exo1通过错配位点切除新复制的DNA链。接下来的是 通过DNA重新合成步骤。MMR因子还识别在链侵袭步骤中形成的错配 同源重组；他们招募解旋酶复合体来解开(拒绝)重组中间产物 并允许进行新的同源搜索。此外，MMR因子的子集在减数分裂中起作用，以解决 将中间体重组为交叉(CoS)。在面包酵母中，大多数减数分裂的CoS形成于 一种干扰依赖的途径，其中双Holliday连接(DHJ)被认为是稳定的 Msh4-Msh5是通过STR解旋酶/拓扑异构酶、外显子1核酸酶和MutLγ的作用来分解的 (MLH1-MLH3)内切酶。我们的工作重点是开发分子模型来解释不同的 MSH和MLH因子在上述途径中起作用。这项工作将使我们能够理解分子 这些因素的缺陷是人类不孕不育和遗传性结肠癌的基础，以及染色体 重新安排可能会导致疾病。我们将通过三个不同的研究主题来检验这些想法。在……里面 项目1我们正在研究MLH蛋白的构象变化是如何由ATP结合和 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 MLH1-Pms1和MLH1-Mlh3固有无序结构域的突变与MLH的强制二聚化 蛋白质)，生化(体外重组反应，以确定MLH蛋白在MMR和 质谱学)和单分子(研究DNA扩散和蛋白质如何绕过屏障) 接近了。项目2的重点是了解MutLγ如何在ZMM途径中分解dHJ。我们的 当前授权期的工作与Mmr和减数分裂中MutLγ内切酶的激活一致 通过形成MutLγ细丝进行交叉。我们将利用这些信息和生化、质量 光谱学，以及利用我们对mlh3功能分离的鉴定的遗传学方法 突变体以确定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核酸酶的相互作用，之后我们将单独和组合测试已识别的因子 能够与MutLγ相互作用，切割模型HJ和DHJ底物。项目3旨在了解如何 决定修复或拒绝重组中间体。我们将分析组蛋白的突变是如何 伴侣和脱乙酰酶单独和联合影响反重组，并将使用一个 提供这些影响的时间和物理测量的诱导性系统。这项工作也将鼓舞我们 进行全基因组筛查，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.