SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

修复DNA中不匹配的碱基可能是生活中的普遍现象 然而，人们对这种修复的特殊性知之甚少。在……里面 除了在重组、合成后错配修复中的作用外 大肠杆菌被认为可以纠正复制错误。我们已经提议 这个校正系统是消除潜势的主要系统 移码突变。本提案旨在确定 利用大肠杆菌和噬菌体Lambda修复的特异性。二 将使用各种方法。首先，将产生lambda中的ci突变 来自错配修复能力不足的菌株。这些菌株显示出高水平 由于复制错误导致的碱基对不匹配导致的突变率 不会被更正。Ci突变将通过基因图谱和 DNA序列分析。含有野生型和突变型Ci的异源双链 将构建等位基因，并对其进行修复评分 大肠埃希菌。这将定义两个可能的不匹配中的哪一个(或两者都是) 导致碱基对变化是很重要的。除了野生型 和错配修复缺陷突变体，我们也将确定突变 聚合酶III缺陷突变株的特异性 相关的错误校对。在这样的菌株中发生了移码突变 不应该很显眼。 第二种方法包括使用已知碱基构建异源双链 所有可能的组合中的配对替换突变，然后是 转染法和修复法评分。此外，还衍生了异双链 从移码、缺失或插入突变也将被构建 并进行了维修测试。这些信息可能会增加我们对 遗传多样性的机制和自发突变的本质 这可能会影响活细胞中的任何过程。

The repair of mismatched bases in DNA is probably universal in living organisms yet little is known about the specificity of such repair. In addition to its role in recombination, post-synthetic mismatch repair in Echerichia coli is thought to correct replication errors. We have proposed that this correction system is the major one for removal of potential frameshift mutations. The present proposal seeks to determine the specificity of repair using Escherichia coli and bacteriophage lambda. Two approaches will be used. First, cI mutations in lambda will be generated from strains deficient in mismatch repair. These strains show high mutation rates because base pair mismatches arising as replication errors are not corrected. The cI mutations will be defined by genetic mapping and DNA sequence analysis. Heteroduplexes containing wild type and mutant cI alleles will be constructed and scored for repair after transfection in E.coli. This will define which of the two possible mismatches (or both) leading to a base pair change is important. In addition to the wild type and mismatch repair deficient mutants we will also determine mutational specificity in mutD strains which are defective in polymerase III associated proof reading of errors. In such a strain frameshift mutations should not be prominent. The second approach involves constructing heteroduplexes using known base pair substitution mutations in all possible combinations, followed by transfection and scoring for repair. Additionally heteroduplexes derived from frameshift, deletion or insertion mutations will also be constructed and tested for repair. This information may increase our understanding of the mechanism of genetic diversity and the nature of spontaneous mutation which could potentially affect any process in living cells.