SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

DNA中错配碱基的修复可能是生命中普遍存在的 然而，人们对这种修复的特异性知之甚少。 在 除了在重组中的作用外，合成后错配修复在 大肠杆菌被认为可以纠正复制错误。 我们提出 这种校正系统是消除潜在的 移码突变 本提案旨在确定 使用大肠杆菌和λ噬菌体修复特异性。 两 将使用的方法。 首先，将产生λ中的cI突变， 来自错配修复缺陷的菌株。 这些菌株显示高 突变率，因为碱基对错配产生的复制错误 没有得到纠正。cI突变将通过遗传作图来定义， DNA序列分析 含有野生型和突变型cI的异源双链体 将构建等位基因并在转染后对修复进行评分， E.coli. 这将定义两个可能的不匹配中的哪一个（或两者） 导致碱基对改变是重要的。 除了野生型 和错配修复缺陷突变体，我们也将确定突变 在聚合酶III缺陷的mutD菌株中的特异性 相关的错误校对阅读。 在这样的菌株中， 不应该突出。 第二种方法涉及使用已知的碱基构建异源双链体， 在所有可能的组合中配对取代突变，然后是 转染和评分用于修复。 另外，异源双链体衍生 从移码，缺失或插入突变也将被构建 并进行了修复测试 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性质 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.