The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes a homologous duplex DNA segment and invades it,. After strand exchange, the nucleoprotein filament should disassemble in order for the recombination process to complete. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here, we have combined optical tweezers with single-molecule fluorescence microscopy and microfluidics, to reveal that disassembly results from the interplay between ATP hydrolysis and release of the tension stored in the nucleoprotein filament. Applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. Upon relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results imply that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this key homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.
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