The overall goal of this project is a complete description, at the molecular level, of the three reactions carried out by DNA polymerase I of E. coli: polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Together the polymerase and 3'-5' exonuclease (proofreading) functions ensure the accuracy of DNA synthesis, while the 5'-3' exonuclease coordinates with the polymerase activity in DNA repair and lagging strand replication. The enzymatic properties of polymerases are frequently exploited therapeutically in antiviral and chemotherapeutic strategies; moreover, replication errors made by DNA polymerases are the likely causes of a variety of human diseases. The 5'-3' exonuclease is a relative of the eukaryotic """"""""flap endonucleases"""""""" which play an essential role in various aspects of DNA replication, recombination and repair, and whose absence increases genome instability. Structural studies of a substantial number of polymerases, of several different types, show that all polymerases share a similar active site layout and reaction mechanism. Therefore, the comparatively simple E. coli DNA polymerase I (Pol I) serves as a valuable model for addressing issues relevant to all polymerases, many of which would be much less tractable as experimental systems. The structural data available for Pol I and its close relatives (including several cocrystal structures) provides a wealth of detail about the layout of the active sites, but poses many additional questions. Taking the structural information as a starting point, experiments are planned which will address the functional significance of the interactions seen in the cocrystal structures. Photo-crosslinking will be used to determine the path of the uncopied template strand of DNA, and a combination of mutagenesis and biochemical studies will be used to identify functionally important protein-DNA contacts. Moving beyond the static picture provided by the cocrystal complexes, a variety of fluorescence methods will be employed to probe several aspects of Pol I enzymology that involve movement: conformational transitions that take place within the polymerase reaction pathway, translocation during processive DNA synthesis, and shuttling of a DNA substrate between polymerase and 3'-5' exonuclease sites during proofreading. Many of the experiments in this proposal relate the biochemical properties of Pol I to its function in vivo: investigating how fidelity is achieved in the polymerase reaction, what distinguishes a DNA polymerase from an RNA polymerase, and how polymerase and 5'-3' exonuclease activities are coordinated so as to produce the correct biological end point.
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