SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

该项目的长期目标是确定 错配碱基修复的体内外研究及其意义 产生碱基错配的机制(S)。为此，我们有 开发了一个正向突变系统，并分离和鉴定了 野生型和错配修复缺陷菌株中产生的突变。在……里面 错配修复缺陷菌株，大多数突变是AT 到GC的过渡。令人惊讶的是，这些转变突变是聚集的 在我们的目标中仅有的两个5‘-GATC-3’序列附近的三个热点中 吉恩。当前提案的具体目标1旨在确定影响 当一个或另一个5‘-GATC-3’位点被删除时，热点形成。这个 创建新的5‘-GATC-3’位点对突变的影响也将是 评估过了。具体目标2试图确定 错配修复缺陷菌株产生突变。我们会 寻找自发增加或减少的第二部位抑制物 错配修复缺陷菌株中的突变。我们还将测试 已知参与DNA复制和/或的基因的等位基因的影响 修复突变频率。这些研究将使我们能够确定 生成热点的过程。具体目标3旨在澄清 错配修复的两个相关方面。是不匹配的效率 修复与距离最近的失配相关 5‘-GATC-3’？因为我们已经确定了许多不同种类的突变 在我们的系统中，我们可以使用各种转换来回答这个问题， 易位和移码突变。事实上，只有在我们的系统中才能 这个问题得到了回答。具体目标3的第二部分旨在 解决维修系统有多少个不匹配的底座的问题 都能认出。我们有一组确定的缺失突变，可以 解决此问题。第四个具体目标将允许确定 一株高产DAM甲基酶的菌株的突变谱。从理论上讲， 光谱应与失配修复缺陷中的光谱相同 菌株。我们还将使用为Dam和Muth基因删除的菌株来 排除或排除我们观察到的光谱是由于 这些基因的产物与5‘-GATC-3’位点结合。独一无二的 我们开发的突变系统将给我们最大的成功机会 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.