ERROR PRONE DNA SYNTHESIS AND ONCOGENE MUTAGENESIS

在DNA中添加或删除核苷酸的突变 序列在病理学中起着重要作用。微卫星不稳定性 表明细胞对关键基因移码突变的易感性 在肿瘤发生中的重要性。三联体重复序列的扩展引发了 一组神经肌肉疾病的发病机制。天生的稳定性 DNA的稳定性是由其序列决定的，但序列的维持 是由一组蛋白质的监测活动确保的。我们 提议：a)确定结构因素的相对重要性 通过系统地测量DNA中的突变来导致滑动 重复不同长度和碱基组成的核苷酸序列。为 为此，我们计划建造一种衬底，在这种衬底中， 可以将序列放置在特定位置以设置报告基因 框架内或框架外；b)我们建议检验以下假设 复制型大肠杆菌DNA的dna E和dna N亚基 聚合酶通过将(重复的)DNA核苷酸锚定到 蛋白质中的特殊氨基酸；c)真核错配修复 蛋白质(MutS同源物)是专门用于碱基校正的 替换或滑移错误。我们将确定细菌是否 MutS蛋白可以通过以下方式区分两种类型的错误 选择错配修复突变体，主要纠正一个或 其他；d)校对活动需要与校对相关联 带有聚合酶亚单位的核酸外切酶。我们将确定是否 校对的结果来自核酸外切酶的修饰或 改变了与其他亚单位结构域的相互作用。我们的目标是学习 通过突变分析确定蛋白质中氨基酸的位置 它们识别DNA中的重复位置并与之相互作用，以防止或 控制滑移。我们认为，这种理解将导致 控制营养不良个体滑脱的干预策略 监视过程的特定阶段。

Mutations in which nucleotides are added or deleted from the DNA sequence play a major role in pathology. Microsatellite instability signals the susceptibility of cells to frameshift mutations of critical importance in tumorigenesis. Expansion of triplet repeats sets off the pathogenesis of a set of neuromuscular diseases. The innate stability of the DNA is determined by its sequence but the maintenance of sequence is ensured by the surveillance activities of a set of proteins. We propose: a) to determine the relative importance of structural factors in DNA that lead to slippage by systematically measuring mutations in repeat nucleotide runs of different length and base composition. For this purpose we plan to construct a substrate in which any target sequence can be placed in a particular position to set a reporter gene either in- or out-of-frame; b) We propose to test the hypothesis that the dnaE and dnaN subunits of the replicative Escherichia coli DNA polymerase reduce slippage by anchoring (repeated) DNA nucleotides to particular amino acids in protein; c) Eukaryotic mismatch repair proteins (mutS homologs) are specialized for the correction of base substitution or slippage errors. We will determine if the bacterial mutS protein can distinguish between the two types of errors by selecting for mismatch repair mutants correcting primarily one or the other; d) Proofreading activity requires association of the proofreading exonuclease with the polymerase subunit. We will determine whether proofreading results from modification of the exonuclease or from an altered interaction with other subunit domains. Our goal is to learn by mutational analysis the location of the amino acid sites in proteins which recognize and interact with repeated sites in DNA to prevent or control slippage. We suppose that this understanding will lead to intervention strategies to control slippage in individuals deficient in particular stages of the surveillance process.