ATP Binding is Prerequisite to the Helical Structure of Human Rad51 Presynaptic Filament

Abstract

Human Rad51 is a key protein component of homologous recombination, the error free repair process of double strand DNA breaks. It is a 37 kDa protein of two domains. The full structure of the protein is not known in atomic details, X-ray crystallography has been successful only in the case of the larger C-domain in complex with a regulatory protein, BRCA2. The structure of the N-domain has been determined by NMR. It is known that in the repair process, the recombinases form a helical filament around ssDNA overhangs - the presynaptic filament (PSF) - at the double strand breaks, the structure of which is of vital significance in the proceeding of the repair. It is also known that the recombinases possess ATPase activity, and ATP is needed for the successful repair. While in the case of the bacterial-, archeal,- and yeast - homologues of HsRad51, data were presented concerning the parameters of their ATP-dependent PSF structures in crystalline conditions, such results are non-existent for HsRad. In this study our goal was to provide evidence for the purely structural role of ATP in forming the PSF structure by HsRad51. Therefore, the hydrolysis of ATP has been excluded by adding Ca to the solutions instead of Mg. The topology of HsRad structures without and with added ATP/Ca and 75mer ssDNA has been determined by transmission electron microscopy. The formations were labeled by the fluorescent dye ANS, and pressure perturbation fluorescence spectroscopy has been applied to characterize the strength of interaction at protomeric interfaces in the Rad51 filaments. We found that the formation of the ordered filamentous structure clearly requires the presence of ATP/Ca, and that the interface binding strength is the highest in the presynaptic filament of helical structure.