MUTAGENESIS AND ITS CONTROL IN E COLI

这项资助的长期目标是从分子水平上了解两个 E.突变率及其控制的相关问题。coli：（1）机制 提供正常DNA复制的保真度;（2） SOS反应诱导的突变率和复制修复增强 DNA损伤。在这段资助期内，主要的具体目标是 了解：（1）基地选择的相对贡献和机制 和DNA聚合酶III全酶的保真度的编辑;（2） 多蛋白SOS诱导点突变途径的生物化学 （UmuCD途径）;（3）点突变途径之间的关系 以及其他涉及非诱变复制修复的SOS途径， 重复突变这三个领域的目标是定义 每种蛋白质对整个途径的贡献， 用纯蛋白质做的生化实验与结果相关联 进行基因和生理分析。对突变的理解 率和DNA损伤的致突变作用与 致癌作用癌基因显然可以通过突变激活， 并且在致突变和致癌作用之间存在相关性， 破坏DNA的物质 碱基选择的实验将涉及：（i）更全面的研究 核苷酸插入的特异性;（ii）更精确的动力学 正确和不正确掺入的反应途径分析 个核苷酸编辑工作将解决：（i）内在核酸外切酶 聚合酶III的编辑亚基的特异性;（ii） 聚合酶亚基对编辑特异性的影响，特别是通过动力学 从错配的碱基延伸的链。对于SOS的工作， 将寻求用纯化的蛋白质重建诱变途径， 探索非致突变途径的机制， 复制-重启。实验将分为四组：（i）使用 简单的复制系统与目前可用的蛋白质;（ii）研究 可能的额外蛋白质组分;（iii）蛋白质-蛋白质分析 （iv）研究更复杂的双链复制 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.