Enzymology of Mismatch Repair in Yeast

酵母错配修复的酶学

• 批准号: 7883713
• 负责人: Richard D Kolodner
• 金额: $ 5.35万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7883713
• 关键词: Address; Alleles; Biochemical; Biochemical Genetics; Biological Models; Cell Cycle Arrest; Cell Death; Characteristics; Chemicals; Collaborations; Complex; Coupled; DNA; DNA Damage; DNA Sequence; DNA biosynthesis; Defect; Dissection; Enzymatic Biochemistry; Eukaryotic Cell; Event; Excision; Gene Mutation; Gene Proteins; Genes; Genetic; Genetic Recombination; Genetic Screening; Genomic Instability; Goals; Health; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Human Genetics; In Vitro; Individual; Inherited; Lead; Malignant Neoplasms; Mediating; Mismatch Repair; Modeling; Molecular Models; Mus; Mutation; Pathway interactions; Play; Predisposition; Property; Proteins; Proteomics; Reaction; Resistance; Role; Saccharomyces cerevisiae; Signal Transduction; System; Yeasts; base; chemotherapy; in vivo; insight; interest; killings; molecular modeling; mouse model; mutant; nuclease; prevent; protein complex; protein function; reconstitution; repaired; research study; response; Address; Alleles; Biochemical; Biochemical Genetics; Biological Models; Cell Cycle Arrest; Cell Death; Characteristics; Chemicals; Collaborations; Complex; Coupled; DNA; DNA Damage; DNA Sequence; DNA biosynthesis; Defect; Dissection; Enzymatic Biochemistry; Eukaryotic Cell; Event; Excision; Gene Mutation; Gene Proteins; Genes; Genetic; Genetic Recombination; Genetic Screening; Genomic Instability; Goals; Health; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Human Genetics; In Vitro; Individual; Inherited; Lead; Malignant Neoplasms; Mediating; Mismatch Repair; Modeling; Molecular Models; Mus; Mutation; Pathway interactions; Play; Predisposition; Property; Proteins; Proteomics; Reaction; Resistance; Role; Saccharomyces cerevisiae; Signal Transduction; System; Yeasts; base; chemotherapy; in vivo; insight; interest; killings; molecular modeling; mouse model; mutant; nuclease; prevent; protein complex; protein function; reconstitution; repaired; research study; response

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.

DNA不匹配修复（MMR）在真核细胞中起许多关键作用，包括：1）抑制 在DNA复制过程中因误差错误而导致的突变以及化学损害 DNA和DNA前体； 2）在重组中间体中修复错误的基础； 3）防止 不同的DNA序列之间的重组； 4）信号表明存在DNA损伤 在细胞反应中，例如细胞周期停滞和细胞死亡。由于这些细胞作用， MMR缺陷增加了自发突变率和改变的重组事件的速率 在特征性的基因组不稳定性签名中，并导致某些人对杀戮的抵抗力增加 DNA破坏代理。了解MMR的机制将影响许多人的健康 原因：1）遗传性非旋转病结肠癌是由于MMR和许多零星的遗传缺陷所致 癌症似乎有缺陷，但是MMR缺陷的遗传后果尚未完全了解。和， 2）许多化学疗法损害DNA和MMR缺陷会导致对其中一些的耐药性 因此，代理商了解MMR可能会改善这些代理的功效，以及 绕过MMR缺陷介导的电阻。 该提案的目的是使用酿酒酵母研究生化和遗传 依赖于MMR途径的真核MUTS和MUTL幽门螺杆菌的机制。以下行 将进行实验：1）遗传研究将确定MMR基因和突变以用于 解剖MMR蛋白的生化特性； 2）单个MMR蛋白的生化研究 包括MSH2-MSH3，MSH2-MSH6，MLH1-PMS1和MLH1-MLH3复合物，RPA，PCNA，RFC，EXO1和EXO1和 其他蛋白质将继续确定这些蛋白质在MMR中的作用。 3）生化基础 对于在MMR中起作用的高阶蛋白质复合物，将确定； 4）部分和完整的MMR 将使用纯化的蛋白质在体外重新构成反应，以研究MMR的机制。 5） 协作小鼠模型研究将继续将见解从酿酒酵母的研究扩展到 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。这些研究的关键特征是使用酿酒酵母 探索人类癌症易感性和协作小鼠研究提出的问题 探索在酿酒酵母中发展的结果的广泛含义。结果，预计 这些研究将提供遗传和生化见解，可以应用于研究 人类癌敏感性的遗传学。