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: 8523903
• 负责人: Eric E. Alani
• 金额: $ 35.39万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8523903
• 关键词: Address; Affect; Alanine; Alleles; American; Apoptotic; Behavior; Binding; Biochemical; Biological Assay; Biological Models; Bypass; Cell Cycle Progression; Cells; Chromatin; Colon Carcinoma; Colorectal Cancer; Complex; Conditioned Culture Media; Cruciform DNA; DNA; DNA Damage; DNA Repair; DNA Sequence; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Defect; Disease; Disease Progression; Eukaryota; Evolution; Fluorescence Microscopy; Frameshift Mutation; Genes; Genetic; Genetic Crossing Over; Genetic Recombination; Genome Stability; Glucose; Goals; Grant; Growth; Heteroduplex DNA; Human; Humulus; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Inherited; Lead; Link; MLH1 gene; Malignant Neoplasms; Meiosis; Meiotic Recombination; Metals; Methods; Mismatch Repair; Modeling; Mutagenesis; Mutation; Nucleosomes; PMS1 gene; Partner in relationship; Pathway interactions; Penetrance; Phenotype; Population; Process; Proteins; Recruitment Activity; Resistance; Role; Saccharomyces cerevisiae; Sampling; Scanning; Signal Transduction; Signaling Protein; Sodium Chloride; Stress; Structure; Testing; Therapeutic; Travel; Vertebral column; Work; Yeasts; endonuclease; fitness; genome sequencing; improved; insight; interest; mutant; novel diagnostics; prevent; recombinational repair; repaired; research study; sample fixation; segregation; sensor; single molecule; tool

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.

描述（由申请人提供）：错配修复（MMR）通过切除DNA聚合酶错误结合错误导致的错配来提高DNA复制的保真度。Msh蛋白通过结合错配DNA启动MMR，然后与Mlh蛋白相互作用募集下游修复因子，切除含有错配DNA的新复制链。MSH和MLH基因的突变导致突变率显著增加（约1000倍），并与遗传性结肠癌（HNPCC）有关。MMR蛋白，特别是Mlh蛋白发出下游修复和重组因子信号的机制尚不清楚。我们专注于了解两个Mlh复合物Mlh1-Pms1和Mlh1-Mlh3在DNA修复信号传导中的作用，以及Mlh因子之间的遗传不相容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抗性的。在目标1中，我们使用全内部荧光显微镜分析单个Mlh复合物与DNA相互作用的行为。我们感兴趣的是回答两个关键问题：1。Mlh和Msh蛋白如何与错配DNA模板上的PCNA相互作用？2. Mlh1-Pms1在识别下游目标时如何绕过障碍？这些研究将利用在实验室中产生的突变体，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。Aim 2的重点是表征Mlh1-Mlh3复合体在MMR和减数分裂重组中的作用。我们建议在遗传和生化分析中检测Mlh1-Mlh3的功能，目的是了解该复合物在两个看似不相关的过程中如何起作用。具体来说，将对MLH3进行丙氨酸扫描诱变，并在MMR和减数分裂交叉实验中测试这些突变的影响，并将纯化野生型和突变型Mlh1-Mlh3，以在散装和单分子实验中检测与msh -错配复合物的相互作用，并测试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。目的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不相容导致突变率升高，这有可能增加适应性和有害突变的发生率。我们感兴趣的是测试MMR不相容是否会通过获得突变（包括有益的和有害的）最初增加进化性，从而通过随后的交配/重组重新获得MMR功能，从而在大群体中固定适应性突变。我们将在非选择性和选择性条件下评估相容和不相容MLH组合的适合度。这样的实验，结合以前用于鉴定DNA损伤敏感性和隐性致死性表型的突变的方法，也可以为理解驱动突变如何在HNPCC和其他癌症中产生提供见解。