SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

本项目的长期目标是确定特异性 在体外和体内修复错配碱基，并阐明 产生碱基错配的机制。 到 为此，我们开发了正向突变系统， 在野生型中产生的分离和表征的突变， 错配修复缺陷菌株。 错配修复 缺陷型菌株，大多数突变是AT到GC 过渡。 令人惊讶的是，这些转换突变是聚集在一起的， 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， 靶基因 本提案的具体目标1旨在 确定当一个或另一个 5'-GATC-3 '位点被删除。 诱变效应 还将评估新的5 ′-GATC-3 ′位点。 具体目标2 以确定突变产生的机制， 错配修复缺陷型菌株。 我们将寻找第二个地点 增加或减少自发性的抑制因子 错配修复缺陷型菌株中的突变。 我们还将测试 已知与DNA有关的基因的等位基因的作用 复制和/或修复突变频率。 这些研究 将使我们能够确定热点的过程， 生成的. 具体目标3试图阐明两个相关方面 错配修复 是错配修复的效率 与距离最近的5'- GATC-3 "？ 由于我们已经描述了许多不同类型的 突变，我们可以用一个 各种转换、颠换和移码突变。 在 事实上，这个问题只有在我们的制度中才能得到解答。 的 具体目标3的第二部分寻求解决以下问题： 修复系统可以修复多少错配的碱基 认识。 我们有一组明确的缺失突变， 解决这个问题。 第四个具体目标将允许 在过量生产的菌株中确定突变谱 dam甲基化酶。 理论上，光谱应该与 在错配修复缺陷菌株中。 我们还将使用 删除dam和mutH基因的菌株，以排除 我们观察到的光谱可能是由于 将这些基因的产物连接到5'-GATC-3 '位点。 独特的突变 我们开发的系统将提供最大的机会， 成功来回答这些问题。

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.