The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DNA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DNA. The question of polymerase accuracy has important health implications because the errors made by DNA polymerases can result in mutations leading to human disease. Moreover, DNA polymerases are frequently targeted in chemotherapeutic and antiviral strategies, as well as being important in a variety of diagnostic biotechnology applications, so an understanding of their reaction mechanisms is crucial. Experiments are proposed on two model DNA polymerases that have contrasting enzymatic properties: the highly accurate DNA polymerase I (Klenow fragment) o E. coli, and the much less accurate Dbh bypass polymerase from the archaeon S. solfataricus. Structural data are available for both these enzymes and several close homologues, and serve as the basis for many of the planned experiments. Moreover, because the important features of the polymerase active site and reaction mechanism are conserved throughout the polymerase family, the results obtained with these simple model systems will have much wider relevance. A major priority will be the investigation of noncovalent steps in the polymerase reaction pathway. These include the early steps involved in substrate recognition and rearrangement of the active site into a form poised for chemical catalysis, and the translocation step that must occur to vacate the active site for the next cycle of addition. A variety of fluorescence assays will be used, in combination with rapid single-turnover kinetics, with the goals of identifying the physical processes involved and understanding their roles in the specificity of the reaction. Fidelity mechanisms at the polymerase active site will be explored using mutants of Klenow fragment. Nucleotide analogues will be used to investigate the role of base pair shape and hydrogen-bonding interactions in the specificity of both Klenow fragment and Dbh polymerase. Mechanistic probes developed for Klenow fragment will be applied to studies of Dbh, in order to learn more about the reaction mechanism and the role of active site side chains in this recently discovered error-prone polymerase. This enzyme has a remarkable propensity for frameshifting, which may provide clues as to its in vivo function, perhaps indicating an ability to bypass certain types of bulky DNA lesions.
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