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：
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
项目类别：
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

项目摘要

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）：不匹配修复（MMR）通过切除由DNA聚合酶失物误差误差引起的不匹配的DNA复制的保真度。 MSH蛋白通过与DNA不匹配的结合，然后与MLH蛋白相互作用以募集下游修复因子，从而启动MMR，从而募集含有不匹配的新复制链的下游修复因子。 MSH和MLH基因的突变赋予突变率显着增加（约1000倍），并与遗传形式结肠癌（HNPCC）有关。 MMR蛋白，尤其是MLH蛋白，信号下游修复和重组因子的机制尚不清楚。我们专注于了解两个MLH复合物MLH1-PMS1和MLH1-MLH3如何在DNA修复信号传导中功能以及MLH因子之间的遗传不兼容如何导致突变率提高。后者的工作可以更好地了解细胞如何发展对生长控制和治疗剂的抗性。在AIM 1中，我们正在分析使用总内荧光显微镜与DNA相互作用的单个MLH复合物的行为。我们有兴趣回答两个关键问题：1。在不匹配DNA模板上，MLH和MSH蛋白如何与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，而MMR无法使用批量方法来完成。 AIM 2的重点是表征MLH1-MLH3复合物在MMR和减数分裂重组中的作用。我们建议研究MLH1-MLH3在遗传和生化测定中的功能，目的是了解该复合物如何在两个看似无关的过程中起作用。 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包括包含被预测为重组中间体的不匹配循环和结构的那些。 AIM 3使用MMR不相容性作为研究适应性进化和疾病进展的模型。 MLH不相容性导致突变率升高，这有可能提高自适应和有害突变的速率。我们有兴趣测试MMR不兼容性是否最初通过Acquirig突变（有益和有害的）提高可变性，从而通过随后的交配/重组来重新稳定大量人群中的自适应突变。我们将评估在非选择性和选择性条件下兼容和不兼容的MLH组合的适应性。这种实验与先前用于识别导致DNA损伤敏感性和隐性致死性表型突变的方法相结合，也可以提供有关了解HNPCC和其他癌症中驱动器突变如何出现的见解。