Counting RAD51 proteins disassembling from nucleoprotein filaments under tension

Abstract

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 it1,2. After strand exchange, the nucleoprotein filament should disassemble in order for the recombination process to complete3. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here, we have combined optical tweezers with single-molecule fluorescence microscopy and microfluidics4,5 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.

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…Assuming that, as for dsDNA (van Mameren et al , 2009b), disassembly occurs strictly from filament ends, the observed rates depend on the number of NPFs bound. Correcting for differences in initial DNA coverage yields comparable disassembly rates of (9 ± 2) 10 −4  s −1 for ADP‐bound filaments and (11 ± 2) 10 −4  s −1 for ATP‐bound filaments (Fig EV1A–D).…”

“…Subsequently, this ssDNA molecule was exposed to a buffer containing a fluorescent variant of hRAD51 (hRAD51‐K313‐C319S‐Alexa Fluor 555, referred to as hRAD51 in this study, Appendix Fig S1A–C). Because hRAD51 disassembly is triggered by ATP hydrolysis (van Mameren et al , 2009b), we measured the disassembly rate of hRAD51 in the presence of Mg 2+ . After incubating (for 10 s to 5 min) an ssDNA molecule in the protein channel (containing 0.18 μM hRAD51), the construct was brought back to the protein‐free buffer channel where the fluorescence signal was used to confirm a high coverage of the ssDNA with hRAD51 (Fig 1C, top panel).…”

“…It is known that hRAD51 disassembly from dsDNA slows down at increased tension on the dsDNA template, resulting in complete stalling of disassembly at forces exceeding 50 pN (van Mameren et al , 2009b). We therefore tested whether similar force dependence occurs in hRAD51 disassembly from ssDNA by measuring the average disassembly times at three different forces (Fig 2A–C).…”

“…Our previous single‐molecule work on the interaction of hRAD51 with ssDNA focused mainly on the assembly of the NPF (Candelli et al , 2014) and on the disassembly of hRAD51 from double‐stranded DNA (dsDNA; van Mameren et al , 2009b). The latter study showed that hRAD51 disassembles through a pause–burst mechanism from NPF ends, dominated by ATP hydrolysis of the RAD51 monomers at filament ends.…”

“…This process is highly dependent on the tension in the dsDNA template: disassembly stalls completely at forces above 50 pN. In addition, although the NPF can readily form in the presence of both ATP (van Mameren et al , 2009b) and ADP (Hilario et al , 2009), hRAD51 disassembly from dsDNA is critically dependent on ATP hydrolysis (van Mameren et al , 2009b). X‐ray crystallography has identified the location of the ATP‐binding pocket between adjacent monomers in the NPF (Conway et al , 2004).…”