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 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 and inter- subunit interactions of the core is highly relevant, since it is a primary determinant for the high-efficiency and high-fidelity chromosomal DNA synthesis in E. coli. It also functions as a model system for chromosomal replicases in general. We have succeeded in obtaining a proteolytic fragment of the epsilon subunit which is fully active and which exhibits considerably better stability properties than the intact subunit. Nevertheless, the fragment undergoes slow precipitation under the conditions of the NMR study, so that additional work is required to determine conditions under which it will be fully stable. With regard to theta, we have been able to express and purify both[15N] and [13C,15N]-labeled theta. Its behavior is however not ideal, and it shows substantial dynamic broadening of the resonances due to conformational exchange or reversible aggregation. This leads to significant degeneracy of the resonances, limiting the assignments which can be made. Thus far, attempts to define solution conditions which more strongly favor a specific conformation of the theta subunit have been unsuccessful; however, these studies are continuing.
