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

工作总结：该项目是LMG和IRA之间的合作IRA LSB。它涉及到大肠杆菌DNA聚合酶III D的结构方面 负责复制大肠杆菌染色体的酶。我们的初选 重点放在polIII核心酶的结构上，它包括 三个紧密结合的亚基：α(135kD)，epsilon(27.5kD)和theta (8.6kD)。α亚基(dna E基因产物)是实际的 聚合酶，epsilon亚基(dna Q基因产物)是3‘ 核外溶解校对器，而theta亚基(HOLE基因产物) 功能未知，但可能是校对的修饰语 活动。这三种蛋白质以线性顺序结合在一起。 Alpha-epsilon-theta。亚基结构及亚基间质的研究 第三号刑警组织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 在大肠杆菌中合成DNA，并作为公认的模型系统发挥作用 用于一般的染色体复制酶的研究。在第一种方法中， 我们已经承担了对大量个体的提纯 Epsilon和theta亚基用于启动结构 用核磁共振技术进行分析。这两个相对较小的尺寸 亚基使核磁共振分析成为一种特别有吸引力的方法。而当 这些研究才刚刚开始，我们期望他们 最终将提供重要的见解，不仅是对 这两个重要的蛋白质，也进入催化机制 核外溶核校对，theta可能的功能作用 亚基，以及α-epsilon和epsilon-theta的性质 互动。

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.