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. We have been successful in obtaining a proteolytic fragment of the epsilon subunit, e186, which is fully active and which exhibits considerably better stability properties than the intact subunit. During the past year, we were able to obtain assign the backbone resonances of the epsilon catalytic domain (ECD ? residues 1-186). Using the chemical shift indexing method, we were then able to deduce the secondary structure of the enzyme.
