Crystallographic Investigations of Replication Factor C

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DESCRIPTION (provided by applicant): An essential component of bacterial and eukaryotic DNA replication and repair is the so-called clamp loader, a five subunit complex that assembles dimeric or trimeric toroidal rings (clamps) around DNA using the energy of hydrolysis. The research plan presented here proposes 1) crystal structure determination of the eukaryotic clamp loader complex from S. cerevisiae, Replication Factor C (RFC), in the 'open' (ATP-bound) and "closed' (no nucleotide) conformations, and 2) crystal structure determination of a RFC:PCNA:DNA complex before and after a hydrolysis event has occurred. All five yeast RFC subunits can be coexpressed in E. coli and purified to homogeneity in quantities sufficient for crystallization. These structures will offer a tremendous amount of structural information for critical steps in the clamp loading cycle. Structures of the yeast RFC complex will be important for modeling the closely related human RFC complex, and comparison of prokaryotic and eukaryotic complexes should expand our understanding of fundamental properties governing clamp loaders.