STRUCTURE AND FUNCTION OF E. COLI POL A GENE

大肠杆菌的DNA聚合酶I(PolI)是一种重要的 在细胞中的作用既在复制过程中，在冈崎的加工中 片段，以及修复受损的DNA。它可以说是最好的型号 聚合酶作用的分子细节研究系统，以及 过程性酶的协调方式的研究 沿着大分子运动的合成。我们最近的工作是 为详细分析其作用机理提供了工具 POL I：我们已经确定了结构基因(Po1A)的序列， 过量生产功能性聚合酶片段的构建菌株 适合于结构研究，并建立了系统的快速 Po1A突变体的定位、克隆和测序。大部分工作 本提案中描述的目的是为了补充X光 通过定位DNA结合对我们的合作者进行了结晶学研究 并将功能分配给Pol I分子的区域。我们会 确定Pol I与其DNA底物之间的相互作用 亲和标记蛋白质，并通过化学和酶探针 DNA分子上的接触。我们将确定重要的功能 对po1a突变体进行测序和酶学研究。在……里面 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在细胞内的水平，以及Pol I是否起关联作用 与其他蛋白质结合。虽然这个项目与健康没有直接的关系 应用，显然，对这些过程的更好的理解 在DNA复制和修复中保持基因组的完整性 最终应该提供对诱变机制和 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.