Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Counting RAD51 proteins disassembling from nucleoprotein filaments under tension


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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Assay for triggering of RAD51 disassembly.
Figure 2: RAD51 disassembly rate is reversibly reduced by DNA tension.
Figure 3: RAD51 disassembly occurs in bursts interspersed with pauses.
Figure 4: RAD51 disassembly pathway.


  1. 1

    Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998)

    CAS  Article  Google Scholar 

  2. 2

    Sung, P., Krejci, L., Van Komen, S. & Sehorn, M. G. Rad51 Recombinase and Recombination Mediators. J. Biol. Chem. 278, 42729–42732 (2003)

    CAS  Article  Google Scholar 

  3. 3

    Symington, L. S. & Heyer, W. D. Some disassembly required: role of DNA translocases in the disruption of recombination intermediates and dead-end complexes. Genes Dev. 20, 2479–2486 (2006)

    CAS  Article  Google Scholar 

  4. 4

    van Mameren, J. et al. Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers. Biophys. J. 91, L78–L80 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Brau, R. R. et al. Interlaced optical force-fluorescence measurements for single molecule biophysics. Biophys. J. 91, 1069–1077 (2006)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Kowalczykowski, S. C. & Eggleston, A. K. Homologous pairing and DNA strand-exchange proteins. Annu. Rev. Biochem. 63, 991–1043 (1994)

    CAS  Article  Google Scholar 

  7. 7

    Benson, F. E., Stasiak, A. & West, S. C. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 (1994)

    CAS  Article  Google Scholar 

  8. 8

    Chi, P. et al. Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst.) 5, 381–391 (2006)

    CAS  Article  Google Scholar 

  9. 9

    Hegner, M., Smith, S. B. & Bustamante, C. Polymerization and mechanical properties of single RecA–DNA filaments. Proc. Natl Acad. Sci. USA 96, 10109–10114 (1999)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Ristic, D. et al. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 33, 3292–3302 (2005)

    CAS  Article  Google Scholar 

  11. 11

    Galletto, R., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Noom, M. C., van den Broek, B., van Mameren, J. & Wuite, G. J. L. Visualizing single DNA-bound proteins using DNA as a scanning probe. Nat. Methods 4, 1031–1036 (2007)

    CAS  Article  Google Scholar 

  13. 13

    van den Broek, B., Noom, M. C. & Wuite, G. J. DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway. Nucleic Acids Res. 33, 2676–2684 (2005)

    CAS  Article  Google Scholar 

  14. 14

    Modesti, M. et al. Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule. Structure 15, 599–609 (2007)

    CAS  Article  Google Scholar 

  15. 15

    Bugreev, D. V. & Mazin, A. V. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl Acad. Sci. USA 101, 9988–9993 (2004)

    ADS  CAS  Article  Google Scholar 

  16. 16

    van der Heijden, T. et al. Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules. Nucleic Acids Res. 35, 5646–5657 (2007)

    CAS  Article  Google Scholar 

  17. 17

    Lindsley, J. E. & Cox, M. M. Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange. J. Biol. Chem. 265, 9043–9054 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Arenson, T. A., Tsodikov, O. V. & Cox, M. M. Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA. J. Mol. Biol. 288, 391–401 (1999)

    CAS  Article  Google Scholar 

  19. 19

    Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Wyman, C. Monomer networking activates recombinases. Structure 14, 949–950 (2006)

    CAS  Article  Google Scholar 

  21. 21

    Evans, E. Probing the relation between force—lifetime—and chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol. Struct. 30, 105–128 (2001)

    CAS  Article  Google Scholar 

  22. 22

    Kerssemakers, J. W. J. et al. Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712 (2006)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Brenner, S. L. et al. RecA protein-promoted ATP hydrolysis occurs throughout recA nucleoprotein filaments. J. Biol. Chem. 262, 4011–4016 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Tombline, G. & Fishel, R. Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis. J. Biol. Chem. 277, 14417–14425 (2002)

    CAS  Article  Google Scholar 

  25. 25

    Tombline, G., Shim, K. S. & Fishel, R. Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition. J. Biol. Chem. 277, 14426–14433 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Shim, K. S. et al. Magnesium influences the discrimination and release of ADP by human RAD51. DNA Repair (Amst.) 5, 704–717 (2006)

    CAS  Article  Google Scholar 

  27. 27

    Chen, Z., Yang, H. & Pavletich, N. P. Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures. Nature 453, 489–494 (2008)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Kiianitsa, K., Solinger, J. A. & Heyer, W. D. Terminal association of Rad54 protein with the Rad51–dsDNA filament. Proc. Natl Acad. Sci. USA 103, 9767–9772 (2006)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Zaitseva, E. M., Zaitsev, E. N. & Kowalczykowski, S. C. The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 274, 2907–2915 (1999)

    CAS  Article  Google Scholar 

  30. 30

    Li, X. et al. Rad51 and Rad54 ATPase activities are both required to modulate Rad51–dsDNA filament dynamics. Nucleic Acids Res. 35, 4124–4140 (2007)

    CAS  Article  Google Scholar 

Download references


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.

Author information



Corresponding authors

Correspondence to Erwin J. G. Peterman or Gijs J. L. Wuite.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures S1-S6 with Legends and Supplementary References (PDF 532 kb)

Supplementary Video 1

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

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)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

van Mameren, J., Modesti, M., Kanaar, R. et al. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature 457, 745–748 (2009).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links