This proposal describes an outstanding research program designed to continue electron microscopy structural studies on a number of important nucleo-protein complexes involved in the process of genetic recombination in both prokaryotic and eukaryotic cells. The work proposed here will be focused on the study of 1) filamentous complexes such as E. coli RecA-DNA complexes and its eukaryotic analog, Rad 51, which is likely to play a key role in recombination in yeast and also in humans; and 2) hexameric helicases, proteins that catalyze the opening of the DNA double strand to form single strand, utilizing the energy of ATP hydrolysis. Some of the proteins of this family have been characterized in Dr. Egelman's laboratory in the previous funding period. In both these cases, Dr. Egelman proposes to: 1) Improve the spatial resolution of the three-dimensional reconstruction of these nucleoprotein complexes, as derived from conventional, cryo and 3-D reconstruction electron microscopy methods. For the filaments, Dr. Egelman proposes to improve the resolution of the reconstructions by complexing the helical assembly with other proteins (such as UmuD, antibodies, Rad52, etc.) and also by examining other assemblies such as Dmc1. Improved resolution of the filament will be necessary to characterize the conformational change involved in the transition from inactive (for which X-ray data exist) to active RecA filaments (for which no crystal has been obtained). 2) To characterize the interactions between the helical filaments and the hexameric proteins with other proteins, in particular: complexes of RecA with UmuD, DnaB with DnaG, Rad51 with Rad52. 3) Scan for homologous proteins in other organisms to test the hypothesis, validated in part by the results obtained during the previous funding period, that both the helical filaments and the hexameric ring structure of the helicases are structures highly conserved throughout evolution.
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