This project is concerned with the structural and functional aspects of the core assembly of DNA polymerase III, the enzyme responsible for replicating the bacterial chromosome. Pol III core is composed of three tightly-bound subunits: alpha, epsilon, theta (in this linear order). Alpha (dnaE gene product) is the 135 kD DNA polymerase, epsilon (28 kD, dnaQ gene product) is the 3' exonucleolytic proofreader, and theta (8kD, holE gene product) has an as yet unknown function. In addition to its catalytic proofreading function, epsilon also has an important structural function within pol III core, as evidenced by the conditional lethality of dnaQ deletion mutants. Our experiments have entailed: (i) a structure-function analysis of epsilon by detailed analysis of a series of dnaQ mutator mutants isolated previously in our laboratory. This has provided new insight in the functional organization of epsilon, including the function of the three conserved Exo motifs and of the C-terminal domain, which we showed responsible for epsilon-alpha interaction; (ii) analysis of the cellular function of theta, using a strain deleted for the holE gene. These studies have revealed a role for theta in stabilizing epsilon; and (iii) NMR structural studies of epsilon and theta, individually and in complex. These studies have lead to a structural model for epsilon, consistent with the reported structure of other proofreading exonucleases, and to definition of the theta interaction site on epsilon. Based on this work we have also identified a possible function of the theta subunit, namely stabilization of the epsilon proofreading subunit. This stabilizing function can also be assumed by the theta homolog residing in bacteriophage P1 (hot gene product). The NMR structures of both theta and Hot, as well as the crystal structure of the epsilon-Hot complex have been obtained, providing insight into the stabilizing function of Hot (and by implication theta) on the epsilon proofreading function.
