STRUCTURAL ANALYSIS OF EPSILON AND THETA SUBUNITS OF E COLI DNA POLYMERASE II

工作摘要：该项目是LMG和 LSB。 它涉及E.大肠杆菌DNA聚合酶Ⅲ D 负责复制E. coli染色体。 我们的首要 重点是pol III核心酶的结构，它由以下组成： 三个紧密结合的亚基：α（135 KD）、β（27.5 KD）和θ (8.6 KD）。 α亚基（dnaE基因产物）是 在聚合酶中，DNA亚基（dnaQ基因产物）是3'端的DNA聚合酶。 外切核酸酶校对器，而θ亚基（holE基因产物） 是未知的功能，但可能是一个修改器的校对 活动 这三种蛋白质以线性顺序结合在一起 α-θ-θ 结构和亚基间的研究 pol III核心的相互作用是高度相关的，因为它是一个 高效高保真染色体的主要决定因素 E.大肠杆菌，也作为一个公认的模型系统的功能 用于研究染色体复制酶。 在第一种方法中， 我们对大量的个体进行了净化， 为了启动一个结构性的 通过NMR技术分析。 这两个相对较小的尺寸 亚基使得NMR分析成为一种特别有吸引力的方法。 而 这些研究刚刚开始，我们希望它们 最终将提供重要的见解，不仅是结构， 这两个重要的蛋白质，而且还进入催化机制， 外切核酸校对，theta的可能功能作用 亚基，以及α-ε和ε-θ的性质 交互.

Summary of Work: This project is a Collaborative IRA between the LMG and LSB. It concerns structural aspects of E. coli DNA polymerase III D the enzyme responsible for replicating the E. coli chromosome. Our primary focus is on the structure of the pol III core enzyme, which consists of three tightly bound subunits: alpha (135 KD), epsilon (27.5 KD) and theta (8.6 KD). The alpha subunit (dnaE gene product) is the actual polymerase, the epsilon subunit (dnaQ gene product) is the 3' exonucleolytic proofreader, while the theta subunit (holE gene product) is of unknown function, but may be a modifier of the proofreading activity. The three proteins are bound together in the linear order alpha-epsilon-theta. A study of the structure as well as inter-subunit interactions of the pol III core is highly relevant, since it is a primary determinant for the high-efficiency and high-fidelity chromosomal DNA synthesis in E. coli and also functions as a recognized model system for the study of chromosomal replicases in general. In a first approach, we have undertaken the purification of large amounts of individual epsilon and theta subunits for the purpose of initiating a structural analysis by NMR techniques. The relatively small size of these two subunits makes analysis by NMR a particularly attractive approach. While these studies have just been started, it is our expectation that they will ultimately provide important insights into not just the structure of these two important proteins, but also into the catalytic mechanism of exonucleolytic proofreading, the possible functional role of the theta subunit, and the nature of the alpha-epsilon and epsilon-theta interactions.