This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.DNA replication is a fundamental biological process required for cellular reproduction. The central feature of DNA replication is the template-induced nucleotidyl transfer reaction mediated by DNA polymerases. Recently, several structural studies have been done in elucidating the mechanism of enzymatic polymerization, including the determination of the structures of several DNA polymerases and their complexes with substrate and substrate analogues. These structures suggest a two-metal ion mechanism of nucleotide incorporation, a feature thought to be shared by all families of DNA polymerases. One metal is associated with dNTP binding and the other involved in metal ion catalysis in the chemical step. In addition, a conformational change step was suggested from crystal structures of Pol beta and Pol I upon binding of dNTP substrate. There have been major efforts recently to investigate metal ions interactions and proposed conformational change of DNA polymerases upon the binding of the incoming dNTP. These efforts have exploited a myriad of techniques including computational, functional, and crystallographic studies, even though the conformational flexibility of the enzyme, substrates, and complexes makes it difficult to unambiguously predict the dynamic behavior of a polymerase during catalytic cycling. Alternatively, small-angle x-ray scattering (SAXS) has been shown to be sensitive to the conformational changes of protein and nucleic acids. Thus, our goal in this proposal is to provide the structural insights into the interactions among divalent metal ions, DNA and dNTP substrates, with three polymerases, Pol beta, Pol I and ASFV Pol X, and further correlated these studies to our biochemical and functional studies. By monitoring conformational changes induced by incorporation of DNA and dNTP substrate and by titration of divalent metal ions, we will experimentally determine forms and energetics of enzyme-substrate interactions and enzyme-metal ion interactions.
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