of Work: This project is a Collaborative IRA between the LMG and LSB. It concerns structural aspects of E. coli DNA polymerase III - 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.
