The basic mechanism(s) involved in the template directed substrate selection for the process of enzymatic synthesis of DNA have not been well understood. The enzymes catalyzing these reactions differ in their properties and structural makeup. The major objective of this proposal is to continue our investigations on the biochemical, enzymological and structural properties of the prototype enzyme, namely E. coli DNA polymerase I. The choice of this enzyme in the proposed studies is based on the facts that a) the three-dimensional anatomy of a large fragment (Klenow enzyme) of pol I has been resolved, b) significant information regarding the process of substrate and template-primer binding by pol I has been obtained through kinetic analyses, c) some of the sites (amino acid residues) participating in the binding of substrates and templates have been identified and, d) this enzyme continues to serve as a model for the mechanistic study of all DNA polymerases. In order to relate the important functional domains to a specific functional role(s) in the catalysis of DNA synthesis, we shall employ the site specific inhibitors, substrate analogues and UV mediated covalent bonding of substrate and template nucleotides to enzyme protein. The peptides containing the active sites will be isolated and sequenced. The overall structure-function analyses will be confirmed and complemented by similar analysis of enzymes modified by site specific mutagenesis of the pol I structural gene. The target residues for mutagenesis studies will be those revealed by chemical modification or affinity labeling studies as well as those implied by model of DNA polymerization reaction. The molecular mechanisms and functional anatomy of the DNA polymerase clarified in this manner will lead to a better understanding of the processes of DNA replication, DNA repair, and the mutagenic effects of chemicals and carcinogens. In addition, newer antimicrobial and antiviral agents can be synthesized which will target specific structural domains of the polymerase of the infectious agents.
