SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

大肠杆菌错配修复的特异性

• 批准号: 3282673
• 负责人: MARTIN G. MARINUS
• 金额: $ 16.69万
• 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 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282673
• 关键词: DNA; DNA methylation; DNA repair; Escherichia coli; bacterial genetics; bacteriophage lambda; gene mutation; genetic manipulation; genetic mapping; genetic recombination; genetic strain; mutant; nucleic acid sequence

The long-term goal of this project is to determine the specificity of repair of mismatched bases in vitro and in vivo and to elucidate the mechanism(s) by which base mismatches are generated. To this end we have developed a forward mutation system, and isolated and characterized mutations arising in wild type and mismatch repair defective strains. In the mismatch repair defective strains, the majority of the mutations are AT to GC transitions. Surprisingly, these transition mutations are clustered in three hotspots near the only two 5'-GATC-3' sequences in our target gene. Specific aim 1 of the current proposal seeks to determine the effect on hotspot formation when one or the other 5'-GATC-3' site is deleted. The effect on mutagenesis of creating a new 5'-GATC-3' site will also be assessed. Specific aim 2 seeks to determine the mechanism by which mutations are generated in the mismatch repair deficient strains. We will seek second-site suppressors which either increase or decrease spontaneous mutation in a mismatch repair defective strain. We will also test the effect of alleles of genes known to be involved in DNA replication and/or repair on mutation frequency. These studies will allow us to identify the process by which hotspots are generated. Specific aim 3 seeks to elucidate two related aspects of mismatch repair. Is the efficiency of mismatch repair correlated with distance of the mismatch from the nearest 5'- GATC-3'? Since we have characterized many different kinds of mutations in our system, we can answer this question using a variety of transition, transversion and frameshift mutations. In fact, only in our system can this question be answered. The second part of Specific aim 3 seeks to resolve the question of what number of mismatched bases the repair system can recognize. We have a defined set of deletion mutations which can resolve this issue. The fourth Specific aim will allow determination of mutation spectrum in a strain overproducing the dam methylase. In theory, the spectrum should be identical to that in mismatch repair defective strains. We will also use a strain deleted for the dam and mutH genes to rule in or out the possibility that the spectrum we observe is due to binding of the products of these genes to 5'-GATC-3' sites. The unique mutation system we have developed will give the greatest chance of success to answer these questions.

该项目的长期目标是确定具体 体外和体内错配碱基的修复并阐明 产生碱基不匹配的机制。 到 为此我们开发了一个正向突变系统，并且 野生型和 错配修复缺陷菌株。 在错配修复中 有缺陷的菌株，大多数突变是AT到GC 过渡。 令人惊讶的是，这些过渡突变聚集在一起 在我们的研究中仅有的两个 5'-GATC-3' 序列附近的三个热点中 目标基因。 当前提案的具体目标 1 旨在 确定其中之一对热点形成的影响 5'-GATC-3'位点被删除。 对创造诱变的影响 还将评估新的 5'-GATC-3' 位点。 具体目标2寻求 以确定突变产生的机制 错配修复缺陷菌株。 我们将寻找第二个地点 抑制器可以增加或减少自发性 错配修复缺陷菌株的突变。 我们也会测试 已知参与 DNA 的基因等位基因的影响 突变频率的复制和/或修复。 这些研究 将使我们能够识别热点的过程 生成的。 具体目标 3 旨在阐明两个相关方面 的错配修复。 是错配修复的效率 与距最近 5'- 的错配距离相关 GATC-3'？ 由于我们已经表征了许多不同类型的 我们的系统发生了突变，我们可以用 各种过渡、颠换和移码突变。 在 事实上，只有在我们的系统中才能回答这个问题。 这 具体目标 3 的第二部分旨在解决以下问题： 修复系统可以修复多少个不匹配的碱基 认出。 我们有一组定义的缺失突变，可以 解决这个问题。 第四个具体目标将允许 过量生产菌株中突变谱的测定 坝甲基化酶。 理论上，频谱应该与 错配修复有缺陷的菌株。 我们还将使用一个 删除 dam 和 mutH 基因的菌株以排除或排除 我们观察到的光谱可能是由于结合 这些基因的产物到 5'-GATC-3' 位点。 独特的突变 我们开发的系统将给予最大的机会 成功回答这些问题。