Roles for Mismatch Repair Proteins in Maintaining Genome Stability

描述(由申请人提供)：错配修复(MMR)通过切除DNA聚合酶误结合错误导致的错配来提高DNA复制的保真度。MSH蛋白通过与DNA错配结合来启动MMR，然后与MLH蛋白相互作用，招募下游修复因子，从而切除包含错配的新复制的链。MSH和MLH基因的突变使突变率显著增加(~1000倍)，并与遗传性结肠癌(HNPCC)有关。MMR蛋白，特别是MLH蛋白向下游修复和重组因子发出信号的机制尚不清楚。我们的重点是了解两个MLH复合体MLH1-Pms1和MLH1-Mlh3如何在DNA修复信号中发挥作用，以及MLH因素之间的遗传不亲和性如何导致突变率增加。后一项工作可以更好地理解细胞是如何进化到对生长控制和治疗产生抵抗力的。在目标1中，我们使用全内荧光显微镜分析了单个MLH络合物与DNA相互作用的行为。我们有兴趣回答两个关键问题：1.MLH和MSH蛋白如何在错配的DNA模板上与增殖细胞核抗原相互作用？2.MLH1-Pms1如何在识别下游靶点时绕过障碍？这些研究将利用实验室产生的突变，旨在为MMR的早期步骤开发准确的模型，这是使用批量方法无法完成的。目的2重点描述MLH1-M1H3复合体在MMR和减数分裂重组中的作用。我们建议研究MLH1-Mlh3在遗传和生化分析中的功能，目的是了解这种复合体如何在两个看似无关的过程中发挥作用。具体地说，将对MLH3进行丙氨酸扫描突变，并在MMR和减数分裂交叉试验中测试这些突变的效果，并将纯化野生型和突变型MLH1-Mlh3，以在散装和单分子分析中检查与MSH-错配复合体的相互作用，还将测试MLH1-Mlh3的假定内切酶结构域是否作用于各种底物，包括那些含有错配环和预测为重组中间体的结构。AIM 3使用MMR不相容作为模型来研究适应性进化和疾病进展。MLH不相容会导致突变率上升，这有可能增加适应性突变和有害突变的发生率。我们有兴趣测试MMR不亲和性最初是否会通过有益和有害的获得性突变来增加进化性，从而允许通过随后的交配/重组重新获得MMR功能来固定大种群中的适应性突变。我们将评估相容和不相容的MLH组合在非选择性和选择性条件下的适合性。这些实验，结合以前用来识别导致DNA损伤敏感性和隐性致死性表型的突变的方法，也可以为理解HNPCC和其他癌症中驱动程序突变是如何发生的提供见解。

DESCRIPTION (provided by applicant): Mismatch repair (MMR) improves the fidelity of DNA replication by excising mismatches that result from DNA polymerase misincorporation errors. Msh proteins initiate MMR by binding to DNA mismatches and then interact with Mlh proteins to recruit downstream repair factors that excise the newly replicated strand containing the mismatch. Mutations in MSH and MLH genes confer significant increases (~1000X) in mutation rate and have been implicated in hereditary forms of colon cancer (HNPCC). The mechanisms by which MMR proteins, and in particular Mlh proteins, signal downstream repair and recombination factors are not well understood. We are focused on understanding how two Mlh complexes, Mlh1-Pms1 and Mlh1-Mlh3, function in DNA repair signaling and how genetic incompatibilities between Mlh factors can lead to increased mutation rates. The latter work can provide a better understanding of how cells evolve to become resistant to growth control and therapeutics. In Aim 1 we are analyzing the behavior of single Mlh complexes interacting with DNA using total internal fluorescence microscopy. We are interested in answering two critical questions: 1. How do the Mlh and Msh proteins interact with PCNA on a mismatch DNA template? 2. How does Mlh1-Pms1 bypass obstacles while identifying downstream targets? These studies will take advantage of mutants generated in the lab and are aimed at developing accurate models for early steps in MMR that cannot be accomplished using bulk approaches. Aim 2 is focused on characterizing a role for the Mlh1-Mlh3 complex in MMR and meiotic recombination. We propose to examine the functions of Mlh1-Mlh3 in genetic and biochemical assays, with the goal of understanding how this complex acts in two seemingly unrelated processes. Specifically, will perform an alanine-scan mutagenesis of MLH3 and test the effect of these mutations in MMR and meiotic crossover assays, and willl purify wild-type and mutant Mlh1-Mlh3 to examine interactions with Msh-mismatch complexes in bulk and single-molecule assays, and also test whether the putative endonuclease domain of Mlh1-Mlh3 acts on a variety of substrates including those containing mismatch loops and structures predicted to be recombination intermediates. Aim 3 uses MMR incompatibilities as a model to study adaptive evolution and disease progression. MLH incompatibility leads to an elevated mutation rate, which has the potential to increase the rate of both adaptive and deleterious mutations. We are interested in testing if a MMR incompatibility will initially increase evolvability through acquirig mutations, both beneficial and deleterious, allowing fixation of adaptive mutations in large populations through reacquisition of MMR functions by subsequent mating/recombination. We will assess the fitness of compatible and incompatible MLH combinations in non-selective and selective conditions. Such experiments, combined with methods used previously to identify mutations responsible for DNA damage sensitivity and recessive lethality phenotypes, can also provide insights into understanding how driver mutations arise in HNPCC and other cancers.