MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

DESCRIPTION (adapted from applicant's abstract): The enzymatic synthesis of DNA is a multistep process that requires sequential binding of substrate, accompanied by several conformational changes within the enzyme protein. The molecular mechanisms involved in these steps are not well understood. The recent availability of a number of crystal structures of this class of enzymes, however, has made a significant advancement whereby the basic molecular mechanisms of DNA polymerization and their relationship to the structural makeup of enzyme may be clarified at the atomic level. The major objective of this proposal is to continue investigations on the biochemical, enzymological and structural properties of the prototype enzyme, namely E. coli polymerase 1. The choice of this enzyme in the proposed study is based on the fact that a) the three dimensional anatomy of large fragment (Klenow enzyme) of pol I family, complexed with substrates, has been resolved, b) significant information regarding the process of substrate and template-primer binding by pol I has been obtained from kinetic analysis, c) some of the sites (amino acid residues) participating in the substrates and template binding have been identified, d) a number of catalytic residues with some functional implication have been identified by site-directed mutagenesis, and e) this enzyme serves as the model system for mechanistic study of all DNA polymerases. In order to identify and relate important structural domains that carry out specific function in the catalysis of DNA synthesis, the following tripartite approach will be used: i) site directed mutagenesis of amino acid residues in conserved domains or implied by 3-D model structure examinations. An in depth analysis of the properties of mutant enzyme will clarify the role for the desired amino acid in specific domain structure, ii) photo-affinity labeling of enzyme proteins with template-primers and identification of sites of enzyme and template-primer contact and, iii) utilize all available structural information concerning DNA polymerases in the interpretation of mutagenesis results as well as to construct structural models which provide the detailed functional participation of various domain structures in atomic details and permit structural elucidation of the transition state, of the catalytic reaction. The molecular mechanism and functional anatomy of DNA polymerase clarified in this manner will lead to a better understanding of DNA replication, DNA repair and mutagenic effects of chemicals and carcinogenesis.