Abstract
RAD51-filament formation on single-stranded DNA represents a undamental step in homologous recombination, an essential DNA-repair pathway. In vitro, nucleation, the initial step of filament formation, can take place on both single and double-stranded DNA. The fundamental mechanism of filament formation, selectivity for single-stranded DNA, and nucleus size are still under debate. We combined fluorescence microscopy, optical tweezers and micro-fluidics to quantify the assembly of RAD51 filaments with single-monomer resolution. Our assay allows the observation of nucleation and filament growth separately, permitting direct measurement of the cooperativity in RAD51-filament formation. We show that RAD51 nucleation process is intrinsically selective, strongly favouring single-stranded DNA and that the size of nuclei is heterogeneous. We propose that this heterogeneity arises from the energetic balance between RAD51 self-assembly in solution and the size-dependent nucleus-DNA interaction. Finally, we show that BRC4, one of the RAD51-binding domains of tumor suppressor BRCA2, acts as inhibitor of RAD51-filament formation.
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