STRUCTURE AND FUNCTION OF E. COLI POL A GENE

DNA聚合酶I（Pol I）。大肠杆菌是一种酶， 在细胞复制过程中的作用，在冈崎加工 修复受损的DNA。 它可以说是最好的模式 用于研究聚合酶作用的分子细节的系统， 对加工酶协调方式的研究 沿大分子沿着运动合成。 我们最近的工作 提供了详细分析药物作用机制的工具， Pol I：我们已经确定了结构基因（po1A）的序列， 过量产生功能性聚合酶片段的构建菌株 适合结构研究，并建立了一个系统， po1A突变体的定位、克隆和测序。 大部分工作 本提案中描述的是旨在补充X射线 我们合作者的晶体学研究，通过定位DNA结合 位点并将功能分配给Pol I分子的区域。 我们将 定义Pol I与其DNA底物之间的相互作用， 亲和标记的蛋白质，并通过化学和酶的探测 DNA分子上的接触点 我们将确定重要的功能 通过测序和po1A突变体的酶促研究， 在 此外，我们打算研究po1A的生物学作用的各个方面， 基因，决定基因是否是必需的，什么因素控制着 Pol I的细胞内水平，以及Pol I是否与 与其他蛋白质。 虽然这个项目没有直接的健康相关 应用程序，很明显，对这些过程的更好理解 在DNA复制和修复中维持基因组的完整性 最终将提供对诱变机制的深入了解， 因此是致癌的。

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.