This project is concerned with the structural and functional aspects of the DNA polymerase III epsilon subunit, the product of the dnaQ gene. Epsilon is a multifunctional protein that plays an essential in the pol III holoenzyme complex. It is a component of the tightly-bound pol III core enzyme that, besides epsilon, is comprised of the alpha (= polymerase) subunit and theta subunit (of unknown function). Epsilon contains the 3' exonuclease function that serves as the polymerase proofreader. In addition, it tightly binds both the alpha and theta subunits and, on that basis, likely fulfills a structural function as well. The importance of the catalytic and structural functions is evidenced by the conditional lethality of dnaQ mutator mutants that are specifically defective in proofreading activity or dnaQ mutator mutants resulting from a dnaQ deletion. We have begun the structure-function analysis of epsilon by a detailed analysis of a series of dnaQ mutator mutants previously isolated in our laboratory. Specifically, we will (i) sequence the mutant genes to reveal to location and nature of the responsible mutations, (ii) determine the magnitude and specificity of the mutator effect, (iii) determine whether these mutations are either recessive or dominant in a genetic complementation assay, and (iv) attempt to assay the interaction of the mutant epsilon subunits with the alpha and theta subunits using the yeast two-hybrid system.
