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SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

大肠杆菌错配修复的特异性

基本信息

批准号：
2391949
负责人：
MARTIN G. MARINUS
金额：
$ 22.94万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-01-01 至 1999-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2391949
关键词：
DNA binding protein DNA repair DNA replication Escherichia coli adenosinetriphosphatase bacterial DNA bacterial genetics bacterial proteins deoxyribonuclease I enzyme activity gene mutation hydrolysis mutant nucleic acid sequence protein purification suppressor mutations western blottings

项目摘要

Errors of DNA replication are potentially a major source of spontaneous and drug-induced mutation. Repair systems exist to minimize such damage. The best understood is DNA adenine methylation (Dam)-directed mismatch repair in Escherichia coli. Among the proteins involved in this repair system, three (MutH, MutL and MutS) are of particular importance. These three proteins appear to form a complex in vivo with DNA containing a replication error. MutL and MutS protein homologs have been detected in other bacteria, yeast, mouse and man indicating that the same basic repair mechanism is used. This proposal seeks to define, in E. coli, how the MutS protein (a) interacts with DNA, (b) with other Mut proteins and (c) the role of its ATPase activity by using genetic methods coupled with biochemical assays. Dominant negative mutations in the mutS gene have been selected by a newfangled procedure and all the mutations mapped so far by DNA sequencing are located in the most evolutionarily conserved amino acids. The mutants were sorted into different classes by their ability to be complemented in trans by MutS+, MutH+ and MutL+. Purified MutS protein from representative mutants will be assayed in vitro for ability to hydrolyze ATP, bind to DNA and assemble to form a complex (MutH,L,S) capable of incising DNA. These biochemical assays will then allow correlations to be made between location of the mutation in the gene and functional activity of protein domains. To confirm the assignment of protein-protein interfaces, a genetic selection will be used to isolate compensatory mutations which suppress a defective target Mut protein. This proposal represents a way not only to increase our knowledge about the origin of spontaneous birth defects and chemical teratogenesis but also to understand in general how proteins assemble into functional complexes.

DNA复制的错误可能是一个主要的来源，自发突变和药物诱导突变。维修系统的存在，尽量减少这种伤害。最好的理解是DNA腺嘌呤甲基化（Dam）指导的大肠杆菌中的错配修复。在参与该修复系统的蛋白质中，有三种（MutH， MutL和MutS）是特别重要的。这三种蛋白似乎在体内与含有复制的DNA形成复合物错误. MutL和MutS蛋白同源物已在其他细胞中检测到。细菌、酵母菌、老鼠和人类的基因组表明，使用修复机制。该建议旨在定义，在E。大肠杆菌中MutS蛋白（a）与DNA相互作用，（B）与其他Mut蛋白相互作用，以及（c）其ATP酶活性通过使用遗传方法结合生化分析mutS基因的显性负突变都是通过一种新奇的方法挑选出来的到目前为止通过DNA测序绘制的地图位于进化上保守的氨基酸。突变体被分为不同的类，他们的能力，以补充在反式 MutS+、MutH+和MutL+。来自代表性样品的纯化MutS蛋白将在体外测定突变体水解ATP、结合与DNA结合并组装形成复合物（MutH，L，S），切割DNA这些生化分析将使相关性在基因突变的位置和蛋白质结构域的功能活性。确认任务蛋白质-蛋白质界面，遗传选择将用于分离补偿突变，其抑制缺陷靶 Mut蛋白质。这个建议不仅能增加我们的知识关于自发性出生缺陷的起源和化学致畸作用，也是为了了解蛋白质组装成功能复合物。