STRUCTURE AND FUNCTION OF E. COLI POL A GENE

大肠杆菌 POL A 基因的结构和功能

• 批准号: 3275805
• 负责人: NIGEL David GRINDLEY
• 金额: $ 16.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-05-01 至 1986-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275805
• 关键词: DNA directed RNA polymerase; DNA repair; Escherichia coli; alleles; bacterial DNA; bacterial genetics; bacteriophage lambda; computer data analysis; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic transcription; molecular cloning; mutant; nucleic acid sequence; radiotracer; structural genes

DNA polymerase I (Pol I) of E. coli is an enzyme that plays an important role in the cell both during replication, in the processing of Okazaki fragments, and in the repair of damaged DNA. It is arguably the best model system for the study of the molecular details of polymerase action, and for an investigation of the way in which processive enzymes can co-ordinate synthesis with movement along a macromolecule. Our recent work has provided the tools for a detailed analysis of the mechanism of action of Pol I: we have determined the sequence of the structural gene (po1A), constructed strains which overproduce a functional polymerase fragment suitable for structural studies, and established a system for the rapid mapping, cloning and sequencing of po1A mutants. Most of the work described in this proposal is designed to complement the X-ray crystallographic studies of our collaborators, by locating the DNA binding site and assigning functions to regions of the Pol I molecule. We shall define the interaction between Pol I and its DNA substrate both nby affinity labeling the protein, and by chemical and enzymatic probing of contacts on the DNA molecule. We shall identify functionally important regions of Pol I by sequencing and enzymatic studies of po1A mutants. In addition, we intend to examine aspects of the biological role of the po1A gene, determining whether the gene is essential, what factors control the intracellular level of Pol I, and whether Pol I functions in association with other proteins. While this project has no direct health-related application, it is clear that an improved understanding of those processes in DNA replication and repair that maintain the integrity of the genome should eventually provide insights into the mechanisms of mutagenesis and thus of carcinogenesis.

大肠杆菌的DNA聚合酶I（pol I）是一种重要的酶 在复制过程中，在冈崎的处理过程中的角色 碎片，并修复受损的DNA。 可以说是最好的模型 研究聚合酶作用的分子细节的系统，以及用于 对加工酶可以协调的方式的调查 沿大分子运动的合成。 我们最近的工作有 提供了用于详细分析作用机理的工具 pol I：我们确定了结构基因（PO1A）的顺序， 构造的菌株过量产生功能性聚合酶片段 适合结构研究，并建立了一个快速的系统 PO1A突变体的映射，克隆和测序。 大多数工作 该提案中描述的旨在补充X射线 通过定位DNA结合，我们的合作者的晶体学研究 位点并将功能分配给Pol I分子的区域。 我们将 定义pol I和其DNA底物之间的相互作用均可 亲和力标记蛋白质，并通过化学和酶促探测 DNA分子的接触。 我们将确定功能很重要 PO1A突变体的测序和酶促研究通过POL I的区域。 在 此外，我们打算检查PO1A生物学作用的各个方面 基因，确定基因是否必不可少，哪些因素控制 pol I的细胞内水平，以及Pol I是否合并 与其他蛋白质。 虽然该项目与健康无关 应用，很明显，对这些过程有了改进的理解 在维持基因组完整性的DNA复制和修复中 最终应该提供有关诱变机制和 因此是致癌作用的。