REGULATION OF MISMATCH REPAIR IN A MODEL E COLI SYSTEM

描述：该项目的长期目标是了解 突变S、突变L和突变HDNA修复基因的调控及其可能的作用 这种调节对DNA修复的影响，自发和诱导的 在模型大肠杆菌系统中的诱变和染色体稳定性。在……里面 此外，来自这些调控研究的结构和分子工具将 应用于遗传方法以解决尚未解决的关键问题 失配修复。这项工作很重要，因为错配修复的一种形式 由大肠杆菌MutS和MutL的同源基因介导 生物体，包括人类，在这些生物中它扮演着基本的角色 继承、变异、进化、重组和发展 癌症。以下五个具体目标将在四年内完成 句号。(I)将使用生化和遗传方法来澄清 TH Hfq RNA负调控MutS和MuTH表达的机制 伴侣和rpos西格玛因子在指数增长和 静止期(不生长)细胞。(Ii)遗传和分子生物学 将对MUT的结构和规则进行分析，L和 大肠杆菌K-12中的突变株和S突变株、L突变株和天然突变株H突变株(“速变”) 大肠杆菌和沙门氏菌的分离株可能调节其 甲基定向错配(MDM)修复能力与大肠杆菌不同 K-12。(3)将使用分子生物学和生理学方法 为了研究诱变剂对MDM修复饱和的机制，如 2-氨基嘌呤，以及复制校对中的缺陷。这项研究将 解决DNA修复中长期存在的核心问题 MD通过过表达MutL或MuTH修复饱和，而不是MutS。(四) 活体、体外异源双链修复试验和体内同源异源 重组测量将用于评估MUT S的含义， MUT L和MUT H对细胞的致突变和修复能力的调节 指数级生长、稳定期、饥饿和热休克细胞。 (V)将开发强大的遗传学方法来了解体内的 MutL蛋白的功能和相互作用，最终MutS，Muth， 和UvrD，在MDM修复中，从MutL作为一个 与MutS和Muth的媒人或接口。这项工作将提供 关于一条重要的修复途径的基本新知识 显然与人类结肠癌和散发性癌症有关。

DESCRIPTION: The long-term goal of this project is to understand the regulatio of the mut S, mutL, and mut H DNA repair genes and the possible implications o this regulation to DNA repair, spontaneous and induced mutagenesis, and chromosome stability in a model E. coli system. In addition, constructs and molecular tools from these regulation studies will be applied to genetic approaches to address key unresolved issues in mismatch repair. This work is important, because a form of mismatch repair mediated by homologues of E. coli MutS and MutL is present in most organisms, including humans, where it plays fundamental roles in inheritance, variation, evolution, recombination, and the development of cancers. The following five Specific Aims will be completed in four-year period. (I) Biochemical and genetic approaches will be used to elucidate the mechanisms of negative control of MutS and MutH expression by th Hfq RNA chaperone and RpoS sigma factor in exponentially growing and stationary-phase (nongrowing) cells. (II) Genetic and molecular biological analyses will be carried out on the structure and regulation of mut L and mut in E. coli K-12 and mut S, mut L, and mut H in natural ("quick-change") isolates of E. coli and Salmonella that may regulate their methyl-directed-mismatch (MDM) repair capacity differently from E. coli K-12. (III) Molecular biological and physiological approaches will be used to investigate the mechanism of saturation of MDM repair by mutagens, such as 2-aminopurine, and defects in replicative proof-reading. This study will address the long-standing, central problem in DNA repair of the reversal of MD repair saturation by overexpression of MutL or MutH, but not MutS. (IV) in viv and in vitro heteroduplex repair assays and in vivo homeologous recombination measurements will be used to assess the implications of mut S, mut L and mut H regulation to mutagenesis and repair capacity in exponentially growing, stationary-phase, starved, and heat-shocked cells. (V) Powerful genetic method will be developed to understand the in vivo functions and interactions of the MutL protein, and eventually MutS, MutH, and UvrD, in MDM repair, starting wit the hypothesis that MutL acts as a matchmaker or interface with MutS and MutH. This work will provide fundamental new knowledge about an important repair pathway that has been clearly implicated in human colon and sporadic cancers.