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: a (135 kD), e (27.5 kD) and q (8.6 kD). The a subunit (dnaE gene product) is the polymerase, the e subunit (dnaQ gene product) is the 3? exonucleolytic proofreader, while the q 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 a-e-q. 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 e subunit (e186) that is fully active and has improved stability and solubility compared to the intact protein. However, progress towards the solution structure has been slow due to still insufficient stability under NMR conditions. We have found two sets of conditions under which the e186 stability is enhanced, one of which is the formation of an e186-q complex. With regard to q, we have been able to express and purify both[15N] and [13C,15N]-labeled q in amounts sufficient for NMR studies and are currently working on the resonance assignments. We have also performed studies on isotopically labeled q, both by itself and in complex with e186. In general, q is a dynamically heterogeneous molecule which is characterized by ~50% helical secondary structure but which appears to have little tertiary structure in the absence of e. - E. Coli, DnaQ Gene, DNA Polymerase III, Structural Analysis, Replication Fidelity
