STUDIES OF DNA REPAIR USING RHIZOBIUM MELILOTI

这里提出的实验解决了以下基本问题： DNA修复和诱变的分子机制， i）我自己的实验室在E. coli和我们熟悉的R.苜蓿遗传学和ii）许多 创新互动的可能性，在我们的内部发展 集团在过去几年。 拟议的研究路线采取 高分辨率实验的全部优点， E. coli和R.草木樨，并提供详细的见解的可能性 关于机制。 为了获得一个进化上发散的类似物， E. coli O 8-甲基鸟嘌呤-DNA甲基转移酶，我们将克隆和测序 R.苜蓿O 8-甲基鸟嘌呤-DNA甲基转移酶， 我们在上一个融资期的特点。 我们将分析 这个基因在各种遗传学研究中， 删除以定义蛋白质的最小功能单位， R.苜蓿O 8-甲基鸟嘌呤-DNA 甲基转移酶和相应的E.大肠杆菌蛋白，并携带 选择定点突变研究来探测 某些关键的保守氨基酸 这些研究将利用 由布鲁斯·登普尔和利昂娜·萨姆森的实验室构建的突变体， Bill Thilly实验室开发的“HiFi”PCR技术。 我们将采取 我的实验室和利昂娜·萨姆森和布鲁斯的实验室 我们观察到的一个新现象表明， 错配修复可以影响由 烷基化剂 我们将根据我们在前一次会议上的观察结果采取后续行动。 通过分离R. 苜蓿草，一种缺乏依赖于dam的甲基化的生物， 在E.大肠杆菌错配修复。 最后，与约翰· Essigman，我们将探索umuD+umuC+依赖的基本机制 通过烷基化和氧化剂进行诱变。 我们将通过 探讨UmuC和激活的UmuD对DNA行为的影响 单加合物模板上的聚合酶， 烷基化剂或氧化剂。

The experiments proposed here address fundamental issues concerning molecular mechanisms of DNA repair and mutagenesis by taking advantage of i) my own lab's extensive experience with DNA repair and mutagenesis in E. coli and our familiarity with R. meliloti genetics and ii) some of the many possibilities for innovative interactions that have developed within our group over the past several years. The proposed lines of research take full advantage of the high resolution experimentation that is possible with E. coli and R. meliloti and offer possibilities for detailed insights concerning mechanisms. To obtain an evolutionarily diverged analog of the E. coli O8-methylguanine-DNA methyltransferases, we will clone and sequence the gene for the R. meliloti O8-methlguanine-DNA methyltransferases, that we characterized during the previous funding period. We will then analyze this gene in a variety of genetic studies that will include constructing deletions to define the minimal functional unit of the protein, creating chimeric proteins between the R. meliloti O8-methylguanine-DNA methyltransferase and the corresponding E. coli protein(s), and carrying out selected site-directed mutagenesis studies to probe the function of certain key conserved amino acids. These studies will take advantage of mutants constructed by Bruce Demple's and Leona Samson's lab and of the "HiFi" PCR technology developed in Bill Thilly's lab. We will take advantage of developments from my lab and those of Leona Samson and Bruce Demple to explore a novel phenomenon we have observed which indicates that mismatch repair can influence the amount of mutagenesis caused by alkylating agents. We will follow up on our observations from the previous funding period by isolating mismatch repair deficient mutants of R. meliloti, an organisms that lacks the dam-dependent methylation so important in E. coli mismatch repair. Finally, in collaboration with John Essigman, we will explore the basic mechanism of umuD+umuC+ -dependent mutagenesis by alkylating and oxidizing agents. We will do this by exploring the effect of UmuC and activated UmuD on the behavior of DNA polymerases on monoadducted templates carrying lesions introduced by alkylating or oxidizing agents.