This project deals with DNA polymerase III (Pol III), a replication- specific polymerase-exonuclease which is a member of a structurally unique bacterial DNA polymerase family. The ultimate objective of the research initiated by this project is to define, at the level of the enzyme's 3D structure, the molecular basis of: (a) dNTP binding and polymerization, (b) the unique reactivity of the dNTP binding domain for the HPUra class of inhibitory dNTP analogs, and (c) the enzyme's exonuclease function. The project exploits recombinant B.subtilis pol III as the model protein, and it applies experimental tools that include mutagenized forms of polC, the enzyme's structural gene, and a structurally diverse set of active site-directed dNTP analogs. The project has the following SPECIFIC AIMS. I TO LOCATE AND CHARACTERIZE THE dNTP AND ARYL INHIBITOR (HPUra) BINDING SITES OF THE ENZYME'S pol DOMAIN. Approaches include: (1) direct analysis of pol III-dNTP and pol III-inhibitor binding; (2) regional mutagenesis of polC; (3) transformation-mediated replacement of polC segments of wt B.subtilis with homologous mutagenized segments; (4) selection and analysis of specific classes of inhibitor-resistant transformants and their resident pol IIIs, and (5) the design and use of inhibitor and dNTP forms that can covalently link and identify site-specific aa residues. II TO LOCATE AND CHARACTERIZE THE ENZYME'S EXONUCLEASE (EXO) SITE. Approaches include: (1) random mutagenesis of the specific polC region encoding the putative exo domain; (2) transformation-mediated replacement of the exo-specific pol C segment of wt cells with homologous mutagenized segments; (3) isolation of exo-deficient transformants based on the expression of a mutator phenotype, and (4) analysis of the exo and pol activities of exo-deficient suspects. III TO PREPARE CRYSTALLINE FORMS OF POL III SUITABLE FOR X-RAY DIFFRACTION ANALYSIS. Approaches will draw on the results of Aims I and II and apply contemporary techniques for protein crystallization.
TO HEALTH ISSUES. This research should contribute significantly to the field of DNA replication and the pharmacology concerned with its control; specifically, it should: (1) provide new and useful insights into the structure and function of a structurally unique and relevant replication-specific DNA polymerase/exonuclease, and (2) facilitate the design of novel, therapeutically useful contravital agents selectively targeted for the replication-specific DNA polymerases of important human pathogens.
