SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

大肠杆菌错配修复的特异性

• 批准号: 2176931
• 负责人: MARTIN G. MARINUS
• 金额: $ 20.95万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176931
• 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

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 复制错误是潜在的主要来源 自发突变和药物诱导突变。修复系统的存在是为了 尽量减少此类损害。最容易理解的是 DNA 腺嘌呤 大肠杆菌中甲基化 (Dam) 定向的错配修复。 在参与该修复系统的蛋白质中，有 3 种（MutH、 MutL 和 MutS) 特别重要。这三种蛋白质 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、小鼠和人表明基本相同 使用修复机制。 该提案旨在定义，在大肠杆菌中，MutS 蛋白 (a) 与 DNA 相互作用，(b) 与其他 Mut 蛋白相互作用，以及 (c) 通过使用遗传方法结合其 ATP 酶活性 生化测定。 mutS 基因的显性失活突变 已通过新奇的程序选择并且所有突变 迄今为止，通过DNA测序绘制的图谱位于最 进化上保守的氨基酸。将突变体分类为 不同类别的能力可以通过反式补充 MutS+、MutH+ 和 MutL+。从代表性的纯化 MutS 蛋白 突变体将在体外进行水解 ATP、结合 DNA 并组装形成复合物 (MutH,L,S)，能够 切割DNA。这些生化测定将允许相关性 在基因突变的位置和 蛋白质结构域的功能活性。确认分配 蛋白质-蛋白质界面的遗传选择将用于 分离抑制有缺陷的靶标的补偿性突变 突变蛋白。 该提案不仅是增加我们知识的一种方式 关于自发性出生缺陷的起源和化学 致畸的同时还要了解蛋白质是如何产生的 组装成功能复合体。