Enzymology of Mismatch Repair in Yeast

DNA错配修复（MMR）在真核细胞中发挥着重要作用，包括：1）抑制 在DNA复制过程中由于错误插入错误以及化学损伤导致的突变， DNA和DNA前体; 2）修复重组中间体中的错配碱基; 3）防止 不同DNA序列之间的重组;以及4）发出DNA损伤的存在的信号， 在细胞反应中，如细胞周期停滞和细胞死亡。由于这些细胞的作用， MMR缺陷增加了自发突变率和改变的重组事件的速率， 在一个特征性的基因组不稳定性签名，以及导致增加的抵抗杀死一些 DNA破坏剂。了解MMR的机制将在许多方面影响人类健康。 原因：1）遗传性非息肉病性结肠癌是由于遗传缺陷的MMR和许多散发性 癌症似乎存在MMR缺陷，但MMR缺陷的遗传后果尚未完全了解;并且， 2)许多化疗药物损伤DNA，MMR缺陷可导致对其中一些药物的耐药性 因此，了解MMR的药物可以改善这些药物的疗效， 规避MMR缺陷介导的抗性。 本提案的目的是利用酿酒酵母研究生物化学和遗传 真核MutS-和MutL-同源物依赖的MMR途径的机制。以下行 将进行以下实验：1）遗传研究将确定MMR基因和突变，用于 剖析MMR蛋白的生化特性; 2）单个MMR蛋白的生化研究 包括Msh 2-Msh 3、Msh 2-Msh 6、Mlh 1-Pms 1和Mlh 1-Mlh 3复合物、RPA、PCNA、RFC、Exo 1和 其他蛋白质将继续确定这些蛋白质在MMR中的作用; 3）生物化学基础 确定在MMR中起作用的高级蛋白质复合物; 4）部分和完全MMR 将使用纯化的蛋白质在体外重建反应，以研究MMR的机制;以及5） 合作小鼠模型研究将继续扩展从S.酿酒厂， 哺乳动物系统这些实验的最终目标是用纯化的蛋白质重建MMR 并确定这些反应的机制。这些研究的一个关键特征是使用S。酿酒酵母 探索人类癌症易感性遗传学提出的问题，以及合作的小鼠研究， 探索S.啤酒。因此，预计 这些研究将提供遗传和生物化学的见解，可应用于研究 人类癌症易感性的遗传学。

DNA mismatch repair (MMR) plays a number of critical roles in eukaryotic cells including: 1) suppression of mutations that result from misincorportation errors during DNA replication as well as chemical damage to DNA and DNA precursors; 2) repair of mispaired bases in recombination intermediates; 3) preventing recombination between divergent DNA sequences; and 4) signaling the presence of DNA damage resulting in cellular responses such as cell cycle arrest and cell death. As a consequence of these cellular roles, MMR defects increase the spontaneous mutation rate and the rate of altered recombination events resulting in a characteristic genome instability signature as well as resulting in increased resistance to killing by some DNA damaging agents. Understanding the mechanism of MMR will impact human health for a number of reasons: 1) Hereditary non-polyposis colon cancer is due to inherited defects in MMR and many sporadic cancers appear MMR defective, yet the genetic consequences of MMR defects are not fully understood; and, 2) many chemotherapy agents damage DNA and MMR defects can result in resistance to some of these agents so understanding MMR could lead to improvements in the efficacy of these agents as well as ways to circumvent MMR defect-mediated resistance. The goal of this proposal is to use Saccharomyces cerevisiae to study the biochemical and genetic mechanisms of the eukaryotic MutS- and MutL-horriologue dependent MMR pathways. The following lines of experimentation will be carried out: 1) genetic studies will identify MMR genes and mutations for use in dissecting the biochemical properties of MMR proteins; 2) biochemical studies of individual MMR proteins including the Msh2-Msh3, Msh2-Msh6, Mlh1-Pms1 and Mlh1-Mlh3 complexes, RPA, PCNA, RFC, Exo1, and other proteins will be continued to determine the roles these proteins play in MMR; 3) the biochemical basis for the higher order protein complexes that function in MMR will be determined; 4) partial and complete MMR reactions will be reconstituted in vitro using purified proteins to study the mechanisms of MMR; and 5) collaborative mouse model studies will be continued to extend insights from studies with S. cerevisiae to mammalian systems. The ultimate goal of these experiments is to reconstitute MMR with purified proteins and determine the mechanisms of these reactions. A key feature of these studies is the use of S. cerevisiae to explore questions raised by the genetics of human cancer susceptibility, and collaborative mouse studies to explore the broader implications of results developed in S. cerevisiae. As a consequence, it is anticipated that these studies will provide genetic and biochemical insights that can be applied to the study of the genetics of human cancer susceptibility.