Structural Studies of Nucleoprotein Complexes

University Of Virginia, Charlottesville VA

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Abstract

DESCRIPTION (provided by applicant): Homologous genetic recombination is responsible, in part, for the introduction of genetic diversity within a population. This diversity is essential to the long-term viability of that population under many different stresses and challenges. Homologous genetic recombination is now also widely accepted as one of the mechanisms of DNA repair within individuals, and defects in the recombination/repair pathways have been tied to genetic instability and cancers. The bacterial RecA protein has been one of the main model systems for understanding homologous genetic recombination, and we know that the human RAD51 and Dmc1 proteins are structural homologs of RecA that play key roles in DNA repair and meiosis, respectively. However, our detailed knowledge of the manner in which these proteins actually function in catalyzing the recognition of homology between different molecules and in strand exchange is quite limited, in part because we only have high resolution structures for components, and low resolution structures for the functional assemblies. We will greatly extend our existing electron microscopic studies of RecA and RAD51 filaments through applications of the unique image processing method that we have developed for studying helical polymers. We have the ability to look at the complexes formed by these proteins on DNA, while such complexes cannot be directly studied by crystallography or NMR. We will also use electron microscopy and image analysis to study ring complexes, such as helicases and branch migration pumps, that act in many areas of DNA replication, recombination and repair. Specifically, we will work on the archaeal and eukaryotic MCM proteins, an essential set of eukaryotic RuvB-like proteins (Rvb1p/Rvb2p), and a RecA-like circadian clock protein (KaiC)