Abstract
We are interested in the mechanisms through which DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR). Understanding how cells maintain genome integrity when challenged with DSBs is of major importance, particularly since the discovery of multiple links between DSB metabolism and genome instability and cancer-predisposition disorders.
We analyzed HR in cells and in single molecule biochemical reactions. Using live cell video-microscopy we analyzed the accumulation of HR proteins in foci at sites of DNA damage. We will report on how the ATPase activity of RAD54 influences the core protein of HR, RAD51. RAD51 is the central catalyst in DSB repair through HR. It promotes DNA homology recognition and strand exchange between broken DNA and the repair template. At the biochemical level we analyzed the interaction of RAD51 with double-stranded DNA in detail. To promote repair, RAD51 polymerizes around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed. The molecular mechanism of RAD51 filament disassembly is poorly understood. We showed, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. Our integrative single-molecule approach allowed us to dissect the mechanism of this principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.
