SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

DNA复制错误是潜在的一个主要来源 自发的和药物诱导的突变。修复系统的存在是为了 将这种损害降至最低。人们最了解的是DNA腺嘌呤 在大肠杆菌中甲基化(DAM)导向的错配修复。 在参与该修复系统的蛋白质中，有三个(Muth， MutL和MutS)尤其重要。这三种蛋白质 似乎在体内与含有复制的DNA形成了一个复合体 错误。MutL和MutS蛋白同源物在其他 细菌，酵母菌，老鼠和人表明相同的基本 使用了修复机制。 这项提议试图在大肠杆菌中定义MutS蛋白(A)如何 与DNA相互作用，(B)与其他Mut蛋白和(C)的作用 结合遗传方法研究其ATPase活性 生化分析。MutS基因的显性负性突变 是通过一种新的程序选择的，所有的突变 到目前为止通过DNA测序绘制的地图位于大多数 进化上保守的氨基酸。这些突变体被分类成 不同的职业在翻译中得到补充的能力不同 MutS、Muth和MutL。具有代表性的MutS蛋白的提纯 突变株将在体外检测其水解三磷酸腺苷、结合 脱氧核糖核酸和组装形成一个复合体(穆斯、L、S)，能够 切割DNA。然后，这些生化分析将允许 在基因突变的位置和 蛋白质结构域的功能活性。确认分配的步骤 在蛋白质-蛋白质界面中，遗传选择将被用于 分离抑制缺陷靶标的补偿性突变 MUT蛋白质。 这一建议不仅代表了一种增加我们知识的方式 关于自发性出生缺陷的起源和化学物质 但也要了解一般情况下蛋白质是如何 组装成功能复合体。

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.