The overall goal of this research is to determine the molecular mechanism by which a DNA polymerase carries out accurate and processive template- directed DNA synthesis. The Klenow fragment of E. coli DNA polymerase I, as the only DNA polymerase for which high-resolution structural data are available, provides an excellent model system for understanding the reactions catalyzed by other more complex polymerases. Klenow fragment has two enzymatic activities, the polymerase and a 3'-5' exonuclease that serves a proofreading function. The two active sites are located on separate structural domains of the molecule. In our studies of Klenow fragment, we propose to use a combination of biochemical, structural and genetic approaches, continuing our close collaboration with the crystallographic group of T. Steitz. For the 3'-5' exonuclease, we propose (i) to test the reaction mechanism proposed from our previous studies, in particular the hypothesis that the attacking nucleophile is a metal-associated hydroxide ion; (ii) to improve the structural model of a catalytically competent enzyme-substrate complex; and (iii) to determine the location and extent of DNA binding sites relevant to exonucleolytic action. For the polymerase active site, we shall use site-directed mutagenesis and biochemical analysis of mutant proteins to extend the identification of residues important in substrate binding, catalysis and translocation. To address the mechanisms responsible for polymerase fidelity we shall screen for mutants with a mutator phenotype and study the biochemical properties of the resulting mutant enzymes. Applying our knowledge of polymerase-catalyzed base misincorporation reactions, we shall attempt to develop a localized mutagenesis procedure that should produce a wide spectrum of single amino acid changes at a high frequency. The procedure will be used to target specific regions of the Klenow fragment, complementing the site-directed mutagenesis strategy. The interaction of Klenow fragment with DNA will be carefully analyzed. Footprinting and phosphate ethylation interference will be used to determine the dimensions and geometry of the complex with duplex DNA. We shall investigate the role of contacts downstream of the primer terminus in determining the substrate preferences of both Klenow fragment and intact DNA polymerase I. We shall examine the extent of overlap between DNA binding modes used in polymerase and exonuclease reactions. Using """"""""high- resolution"""""""" footprinting we shall attempt to distinguish between the pre- and post-translocation positions of a primer terminus at the polymerase active site. To facilitate the crystallographic studies, particularly those of Klenow fragment complexes with duplex DNA, we shall construct appropriately engineered derivatives of Klenow fragment.
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