The overall goal of this project is a complete understanding of the way in which DNA polymerase I of E. coli carries out accurate and processive template-directed DNA synthesis. Replication errors made by DNA polymerases are implicated in the causes of some human diseases; moreover the enzymatic properties of the polymerases themselves are frequently exploited in antiviral and chemotherapeutic strategies. Since evidence is accumulating that all polymerases share a similar active site layout and reaction mechanism, it is reasonable to assume that studies carried out on a relatively simple model system will have much wider relevance. DNA polymerase I provides a good model system since the high-resolution structure of the Klenow fragment portion of the molecule, containing the polymerase and 3'-5' (proofreading) exonuclease activities, provides an important foundation for relating polymerase function to the molecular structure using a combination of biochemistry and molecular genetics. The polymerase and 3'-5' exonuclease active sites of Klenow fragment catalyze analogous phosphoryl transfer reactions, and are thought to do so using a pair of divalent metal ions appropriately positioned by coordination to a cluster of carboxylate side chains. For both active sites, a variety of substrate analogs will be used to test she proposed reaction mechanism and to investigate interactions between the substrate molecules and active site side chains. Experiments are proposed to characterize the polymerase-DNA interactions specifically addressing the location and orientation of the DNA, the extent to which interactions change between polymerase and 3'-5' exonuclease modes, and the identification of specific contacts between protein and DNA. Experiments will also be carried out to analyze the way in which the protein maintains accuracy in the polymerase reaction, discriminating against both base substitution errors and DNA misalignments that can give rise to frameshifts. DNA polymerase I also contains a third activity, the 5'-3' exonuclease which plays an important role in """"""""nick translation' activities in vivo. Site-directed mutagenesis will be used to locate active-site residues, complementing ongoing structural studies. A variety of biochemical approaches will be used to determine the way in which the 5'-3' exonuclease activity is coordinated with the polymerase to accomplish the in vivo functions of DNA polymerase I in replication and repair.
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