ENZYMOLOGY OF MISMATCH REPAIR IN YEAST

酵母错配修复的酶学

• 批准号: 2187571
• 负责人: Richard D Kolodner
• 金额: $ 24.89万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187571
• 关键词: DNA directed DNA polymerase; DNA repair; Saccharomyces cerevisiae; adenosinetriphosphatase; enzyme activity; exonuclease; fungal genetics; gene conversion; gene mutation; genetic mapping; genetic recombination; laboratory rabbit; mitochondrial DNA; nucleic acid biosynthesis; nucleic acid structure; nucleobase; plasmids; polymerase chain reaction; protein purification

There are 3 ways in which mismatched bases arise in DNA: 1) DNA damage gives rise to mismatched bases. 2) Misincorporation during DNA replication produces mispaired bases. 3) Genetic recombination produces regions of heteroduplex DNA containing mispaired bases. The failure to repair mispaired bases in DNA substantially increases the spontaneous mutation rate and also gives rise to altered recombination events. Recent evidence indicates mismatch repair acts to prevent, or limit, recombination between related DNA sequences containing extensive base differences or between short repeated DNA sequences. Thus mismatch repair appears to reduce aberrant recombination events. Understanding the mechanism of mismatch repair has potential impact on human health for a number of reasons. 1) There are inherited human diseases that could be caused by defects in mismatch repair. These include diseases associated with high spontaneous mutation rates, cancers, like colon cancer that are associated with destabilization of CA repeat sequences and diseases in which mutations accumulate in mitochondrial DNA resulting in premature aging related syndromes. 2) Many chemotherapy agents act by damaging DNA and understanding how mismatch repair functions could lead to development of new ways of sensitizing cells to DNA damaging agents and a greater understanding of how cells become resistant to DNA damaging agents. 3) Purification of mismatch repair proteins will provide new reagents for studying DNA structure and detecting base changes in DNA which will be useful for fine structure genetic mapping. The goal of this proposal is to understand how enzymes catalyze the repair of mismatched nucleotides in Saccharomyces cerevisiae. Associated goals are to understand how mismatch repair interacts with genetic recombination, how mismatch repair contributes to the fidelity of DNA replication and if defects in mismatch repair are responsible for inherited diseases. The following lines of experimentation will be carried out: l) The MSH2 gene (MutS homolog), which functions in gene conversion and mismatch repair, will continue to be studied to define the role of mismatch repair in genetic recombination and mutation avoidance. 2) Biochemical analysis of overproduced MSH2 protein will be continued to define its DNA binding properties and to identify interacting proteins. 3) Biochemical characterization of a S. cerevisiae in vitro mismatch repair system will he continued to identify and purify enzymes required for mismatch repair. 4) Genetic analysis of the MSH1 gene and biochemical analysis of the overproduced MSH1 protein will be continued to define the role MSH1 plays in maintaining the integrity of mitochondrial DNA. The ultimate goal of these experiments is to reconstitute a S. cerevisiae mismatch repair reaction with purified proteins and determine the mechanism of this reaction. In addition, it is anticipated that these studies will provide tools for use in the analysis of mismatch repair in higher eukaryotes.

DNA中有三种方式出现了不匹配的碱基：1）DNA损伤 引起不匹配的基础。 2）在DNA复制过程中掺杂 产生错误的基础。 3）遗传重组产生的区域 包含误导碱基的杂化DNA。无法修复 DNA中的碱基底座大大增加了自发突变 速率并引起重组事件的改变。最近的证据 表示不匹配维修行为以防止或限制重组 相关的DNA序列包含广泛的基本差异或之间 短重复的DNA序列。因此，不匹配的维修似乎减少了 异常重组事件。了解不匹配的机制 由于多种原因，维修对人类健康有潜在的影响。 1） 有一些遗传的人类疾病可能是由于缺陷引起的 不匹配维修。这些包括与自发性相关的疾病 突变率，癌症，例如与结肠癌相关的结肠癌 CA重复序列和疾病的不稳定，其中突变 积聚在线粒体DNA中，导致过早衰老相关 综合征。 2）许多化学疗法通过损害DNA和 了解不匹配维修功能如何导致发展 将细胞敏化DNA损害剂的新方法和更大的方法 了解细胞如何对DNA损伤剂具有抗性。 3） 不匹配维修蛋白的纯化将为 研究DNA结构并检测DNA中的基础变化，这将是 用于精细的结构遗传图。 该提议的目的是了解酶如何催化维修 酿酒酵母中不匹配的核苷酸。相关目标 要了解错配维修如何与遗传相互作用 重组，不匹配修复如何促进DNA的保真度 复制和不匹配维修缺陷是否负责 继承的疾病。将携带以下实验行 OUT：L）MSH2基因（MUTS同源物），该基因在基因转化中起作用 和不匹配维修，将继续研究以定义 基因重组和避免突变的不匹配修复。 2） 生产过量的MSH2蛋白的生化分析将继续 定义其DNA结合特性并识别相互作用的蛋白质。 3） 酿酒酵母在体外不匹配修复中的生化表征 系统他将继续识别和净化所需的酶 不匹配维修。 4）MSH1基因和生化的遗传分析 对过量生产的MSH1蛋白的分析将继续定义 角色MSH1在维持线粒体DNA的完整性方面发挥作用。这 这些实验的最终目标是重建酿酒酵母 与纯化的蛋白质不匹配修复反应，并确定 这种反应的机制。此外，预计这些 研究将提供用于分析不匹配维修的工具 较高的真核生物。