SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

大肠杆菌错配修复的特异性

• 批准号: 3282674
• 负责人: MARTIN G. MARINUS
• 金额: $ 17.36万
• 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/3282674
• 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 在体外和体内修复错配碱基，并阐明 产生碱基错配的机制。 为此， 开发了一个正向突变系统，并分离和表征 野生型和错配修复缺陷型菌株中出现的突变。 在 错配修复缺陷型菌株，大多数突变是AT GC转换 令人惊讶的是，这些转换突变是聚集在一起的， 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， 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 ′位点缺失时，对热点形成的影响。 的 还将讨论产生新的5 ′-GATC-3 ′位点对诱变的影响， 评估。 具体目标2旨在确定实现以下目标的机制 在错配修复缺陷型菌株中产生突变。 我们将 寻找第二部位抑制因子， 错配修复缺陷型菌株中的突变。 我们还将测试 已知参与DNA复制的基因的等位基因的影响和/或 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旨在阐明 错配修复的两个相关方面。 就是失配的效率 修复与错配距离最近的 5'-GATC-3'？ 因为我们已经描述了许多不同类型的突变 在我们系统中，我们可以使用各种转换来回答这个问题， 颠换和移码突变。 事实上，只有在我们的系统中， 这个问题得到了回答。 具体目标3的第二部分力求 解决了修复系统需要多少错配碱基的问题 可以识别。 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确定 突变谱在菌株过度生产大坝甲基化酶。 理论上， 光谱应该与失配修复缺陷中光谱相同 菌株 我们还将使用dam和mutH基因缺失的菌株， 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 ′位点的结合。 独特的 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