MOLECULAR EFFECTORS OF ENZYMATIC DNA SYNTHESIS

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Our long-range goals are to understand and unravel some of the intricacies in the process of the enzymatic synthesis of DNA. Enzymes catalyzing DNA synthesis require muliple components and exhibit several common and a few unique properties. Yet the basis and/or components responsible for these properties have not been identified. In this proposal, our intention is to critically evaluate the process of substrate and template-primer binding through the identification and structural analysis of the involved functional domains using Klenow fragment of E. coli pol. I as a model enzyme. A large body of information on the catalytic properties and requirements for DNA synthesis, as well as the primary structure of this enzyme are now known. The results obtained will not only clarify the basic mechanisms of the DNA polymerase reaction but may provide insight into properties which may have important practical applications. We have developed procedures for the covalent linkage of reagents to specific enzyme sites and plan to develop new protocols and improve existing protocols for the efficient labeling of binding sites. Our goal is to isolate peptides bearing the substrate and template-primer binding sites from Klenow fragment of E. coli Pol. I and to determine their primary sequence. Thus, structural studies together with functional analyses of various structural domains within individual DNA polymerases will clarify the mechanisms of the complex multicomponent process of DNA synthesis.