The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment1,2. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed3. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here we show, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics4,5, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. By 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. After relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results indicate 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 principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.
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We thank B. van den Broek and R.T. Dame for discussions and a critical reading of the manuscript and J. Kerssemakers for kindly providing his step fitting algorithm. This work was supported by the Biomolecular Physics program of the Dutch organization for Fundamental Research of Matter (FOM) (to R.K., C.W., E.J.G.P. and G.J.L.W.), and grants from the Dutch Cancer Society (KWF), the Netherlands Organization for Scientific Research (NWO), the Netherlands Genomics Initiative/NWO, the Association for International Cancer Research and the European Commission Integrated Projects Molecular Imaging and DNA Repair and a National Cancer Institute–National Institutes of Health USA program project (to C.W. and R.K.). E.J.G.P. and G.J.L.W. are recipients of NWO Vidi grants; C.W. of an NWO Vici grant.
This file contains Supplementary Methods, Supplementary Figures S1-S6 with Legends and Supplementary References (PDF 532 kb)
Supplementary Video 1 on which the data in Figure 1b is based, shows an individual RAD51–DNA complex, suspended in a buffer flow from a single optically trapped bead. At about one third of the video, the complex is moved to a Mg2+-containing buffer channel, triggering disassembly as evidenced by the concurrent intensity decrease and shrinkage of the entire complex. The video is sped up 200 ×. (MOV 262 kb)
Supplementary Video 2 on which the data in Figure 2a is based, shows that RAD51 filament disassembly is slowed down by tension applied to the DNA. The video displays a graph of the decreasing fluorescence intensity (white) and the increasing tension (red) signals. The video is sped up 300 ×. (MOV 646 kb)
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van Mameren, J., Modesti, M., Kanaar, R. et al. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature 457, 745–748 (2009). https://doi.org/10.1038/nature07581
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