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：
7887048
负责人：
Eric E. Alani
金额：
$ 17.02万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-03 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7887048
关键词：
Address Alleles Amino Acids Apoptotic Behavior Binding Biochemical Biological Assay Cell Cycle Progression Colon Carcinoma Complex DNA DNA Damage DNA Repair DNA Repair Pathway DNA Sequence DNA biosynthesis Defect Disease Eukaryota Excision Fluorescence Microscopy Fungal Genome Genes Genetic Genetic Polymorphism Genetic Recombination Genome Genome Stability Genomic Instability Goals Grant Heteroduplex DNA Human Immunoglobulin Genes Immunoglobulin Somatic Hypermutation In Vitro Inherited Kinetics Laboratories MLH1 gene MSH2 gene MSH6 gene Malignant Neoplasms Mismatch Repair Modeling Molecular Molecular Evolution Mutagenesis Mutation PMS1 gene Pathway interactions Penetrance Phenotype Population Population Genetics Predisposition Process Proteins Recruitment Activity Research Personnel Role Saccharomyces cerevisiae Sequence Analysis Signal Transduction Signaling Protein Single-Stranded DNA Slide Spottings System Tail Technology Testing Walking Work Yeasts chromatin immunoprecipitation disease phenotype genetic analysis genome-wide helicase improved in vitro Model in vivo mutant prevent repaired research study segregation sensor single molecule tool

项目摘要

DESCRIPTION (provided by applicant): Mismatch repair (MMR) improves the fidelity of DNA replication by about 1000 fold by excising mismatches in the newly replicated strand arising from mis-incorporation and DNA slippage. MSH proteins initiate MMR by binding to DNA mismatches and then interacting with MLH proteins to recruit downstream repair factors. Mutations in MSH and MLH genes confer significant increases in mutation rate. MMR factors also prevent recombination between divergent DNA sequences, and process recombination intermediates containing nonhomologous single-stranded ends. The mechanisms by which these proteins identify mismatches and signal downstream factors during DNA replication and recombination are not well understood. In addition, the role of genetic background in determining the penetrance of MMR mutations with respect to disease phenotype has not been explored in depth. This proposal is focused on understanding how MMR proteins identify mismatches and signal downstream factors during DNA replication and repair, and the role of genetic background in determining the penetrance of MMR mutations. In Aim 1 we will analyze the behavior of single MSH and MLH complexes interacting with DNA using total internal fluorescence microscopy. These studies will take advantage of a large number of MMR mutants generated previously in the lab and are aimed at distinguishing between competing models for how MSH and MLH proteins signal downstream steps in MMR. In Aim 2 we will use SNP scanner technology to examine genome-wide mutation accumulation in MMR mutants. This work will allow us to determine the actual mutation rate in a MMR-defective strain and provide information that should help cancer researchers distinguish mutations critical for transformation to a cancer state from those that occur after transformation. Aim 3 is also focused on genome stability and presents in vitro and in vivo biochemical approaches to test interactions between MMR components and the SGS1 helicase to prevent recombination between divergent DNA sequences. Aim 4 outlines experiments aimed at understanding how genome instabilities arise from genetic incompatibility in MMR. This work will provide models to explain how genetic background contributes to disease penetrance in humans. In addition it offers new tools to identify genetic interactions in DNA repair pathways, with the overall goal of understanding cancer susceptibility and the molecular pathways that function in DNA repair.

描述（由申请人提供）：错配修复（MMR）通过切除新复制链中因错误掺入和DNA滑移而产生的错配，将DNA复制的保真度提高约1000倍。 MSH 蛋白通过与 DNA 错配结合启动 MMR，然后与 MLH 蛋白相互作用以招募下游修复因子。 MSH 和 MLH 基因的突变导致突变率显着增加。 MMR 因子还可以防止不同 DNA 序列之间的重组，并处理含有非同源序列的重组中间体单链末端。这些蛋白质识别错配和发出信号的机制 DNA 复制和重组过程中的下游因素尚不清楚。此外，遗传背景在确定 MMR 突变与疾病表型的外显率方面的作用尚未得到深入探讨。该提案的重点是了解 MMR 蛋白如何在 DNA 复制和修复过程中识别错配并向下游因子发出信号，以及遗传背景在确定 MMR 突变外显率中的作用。在目标 1 中，我们将使用全内部荧光显微镜分析单个 MSH 和 MLH 复合物与 DNA 相互作用的行为。这些研究将利用实验室之前生成的大量 MMR 突变体，旨在区分 MSH 和 MLH 蛋白如何向 MMR 中的下游步骤发出信号的竞争模型。在目标 2 中，我们将使用 SNP 扫描仪技术来检查 MMR 突变体的全基因组突变积累。这项工作将使我们能够确定 MMR 缺陷菌株的实际突变率，并提供信息，帮助癌症研究人员区分对转变为癌症状态至关重要的突变与转变后发生的突变。 Aim 3 还关注基因组稳定性并在体外和体内进行展示生化方法来测试 MMR 成分和 SGS1 解旋酶之间的相互作用，以防止不同的 DNA 序列之间的重组。目标 4 概述了旨在了解 MMR 中基因不相容如何引起基因组不稳定性的实验。这项工作将提供模型来解释遗传背景如何影响人类的疾病外显率。此外，它还提供了新的工具来识别 DNA 修复途径中的遗传相互作用，总体目标是了解癌症易感性和在 DNA 修复中发挥作用的分子途径。