MUTAGENESIS AND ITS CONTROL IN E COLI

这笔赠款的长期目标是对两个分子的理解 大肠杆菌中突变率及其控制的相关问题：(1)机制 提供正常DNA复制的保真度；(2) SOS反应诱导的高突变率和复制修复 造成DNA损伤。这一授权期的主要具体目标是 理解：(1)基地选择的相对贡献和作用机制 和编辑DNA聚合酶III全酶的保真度；(2) 多蛋白SOS诱导的点突变途径的生物化学 (3)点突变途径之间的关系。 和其他SOS途径涉及非突变复制修复和 重复突变。这三个领域的目标都是定义 每个单独的蛋白质对整个途径的贡献和对 将使用纯蛋白质的生化实验与结果相关联 在活体内进行遗传和生理分析。对突变的理解 DNA损伤的诱变率和诱变效应与 人类的致癌作用。癌基因显然可以通过突变来激活， 而且在诱变和致癌作用之间存在相关性 破坏DNA的特工。 关于基地选择的实验将涉及：(I)更全面的研究 核苷酸插入的特异性；(Ii)更精细的动力学 正确与错误并入的反应途径分析 核苷酸。编辑工作将涉及：(I)内在核酸外切酶 聚合酶III编辑亚基的特异性；(Ii) 聚合酶亚基对编辑的特异性，特别是通过动力学 从错配的碱基开始链延长。对于SOS工作，实验 将寻求用纯化的蛋白质重建诱变途径 探讨非诱变途径的作用机制 复制-重新启动。实验将分为四组：(I)使用 利用现有蛋白质的简单复制系统；(Ii)研究 可能增加的蛋白质成分；(3)蛋白质-蛋白质分析 相互作用；(Iv)更复杂的双链复制的研究 系统。

The long-term objectives of this grant are a molecular understanding of two related aspects of mutation rate and its control in E. coli: (1) mechanisms that provide for the fidelity of normal DNA replication; (2) basis for the enhanced mutation rate and replicational repair induced by the SOS response to DNA damage. The major specific aims for this grant period are to understand: (1) the relative contribution and mechanism of base selection and editing for the fidelity of DNA polymerase III holoenzyme; (2) the biochemistry of the multi-protein SOS-induced pathway for point mutations (UmuCD pathway); (3) the relationship between the point-mutation pathway and other SOS pathways involving nonmutagenic replicational repair and duplication mutations. The goal in all three areas is to define the contribution of each individual protein to the overall pathway and to correlate the biochemical experiments using pure proteins with the results of genetic and physiological analysis in vivo. An understanding of mutation rate and the mutagenic effects of DNA lesions has relevance to carcinogenesis in humans. Oncogenes can clearly be activated by mutation, and there is a correlation between the mutagenic and carcinogenic effect of agents that damage DNA. Experiments on base selection will involve: (i) a more complete study of the specificity of nucleotide insertion; (ii) a more refined kinetic analysis of the reaction pathway for incorporation of correct and incorrect nucleotides. Work on editing will address: (i) the intrinsic exonuclease specificity of the editing subunit of polymerase III; (ii) contribution of the polymerase subunit to editing specificity, especially by kinetics of chain elongation from a mispaired base. For the SOS work, the experiments will seek to reconstitute the mutagenic pathway with purified proteins and to explore the mechanism of the nonmutagenic pathway termed replication-restart. Experiments will fall into four groups: (i) use of a simple replication system with currently available proteins; (ii) study of possible additional protein components; (iii) analysis of protein-protein interactions; (iv) investigation of more complex double strand replication systems.