Skip to main content

Thank you for visiting nature.com. 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.

  • Letter
  • Published:

Direct observation of individual RecA filaments assembling on single DNA molecules

Abstract

Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination1. Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth2,3,4,5,6,7. However, many aspects of this process remain unclear, including the rates of nucleation and growth and the involvement of ATP hydrolysis, largely because visualization at the single-filament level is lacking. Here we report the direct observation of filament assembly on individual double-stranded DNA molecules using fluorescently modified RecA. The nucleoprotein filaments saturate the DNA and extend it 1.6-fold. At early time points, discrete RecA clusters are seen, permitting analysis of single-filament growth from individual nuclei. Formation of nascent RecA filaments is independent of ATP hydrolysis but is dependent on the type of nucleotide cofactor and the RecA concentration, suggesting that nucleation involves binding of 4–5 ATP–RecA monomers to DNA. Individual RecA filaments grow at rates of 3–10 nm s-1. Growth is bidirectional and, in contrast to nucleation, independent of nucleotide cofactor, suggesting addition of 2–7 monomers s-1. These results are in accord with extensive genetic and biochemical studies, and indicate that assembly in vivo is controlled at the nucleation step. We anticipate that our approach and conclusions can be extended to the related eukaryotic counterpart, Rad51 (see ref.8), and to regulation by assembly mediators9,10,11.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nucleation and growth of RecA filaments visualized on individual dsDNA molecules.
Figure 2: Nucleation of RecA filaments is independent of ATP hydrolysis but dependent on the type of nucleoside triphosphate and the RecA concentration.
Figure 3: Growth of individual RecA filaments is independent of nucleoside triphosphate.
Figure 4: RecA filaments can grow bidirectionally in the presence of ATP.

Similar content being viewed by others

References

  1. Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D. & Rehrauer, W. M. Biochemistry of homologous recombination in Escherichia coli.. Microbiol. Rev. 58, 401–465 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kowalczykowski, S. C., Clow, J. & Krupp, R. A. Properties of the duplex DNA-dependent ATPase activity of Escherichia coli recA protein and its role in branch migration. Proc. Natl Acad. Sci. USA 84, 3127–3131 (1987)

    Article  ADS  CAS  Google Scholar 

  3. Pugh, B. F. & Cox, M. M. Stable binding of recA protein to duplex DNA. Unraveling a paradox. J. Biol. Chem. 262, 1326–1336 (1987)

    CAS  PubMed  Google Scholar 

  4. Shivashankar, G. V., Feingold, M., Krichevsky, O. & Libchaber, A. RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis. Proc. Natl Acad. Sci. USA 96, 7916–7921 (1999)

    Article  ADS  CAS  Google Scholar 

  5. 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)

    Article  ADS  CAS  Google Scholar 

  6. Kowalczykowski, S. C. Biochemistry of genetic recombination: energetics and mechanism of DNA strand exchange. Annu. Rev. Biophys. Biophys. Chem. 20, 539–575 (1991)

    Article  CAS  Google Scholar 

  7. Cazenave, C., Toulme, J. J. & Helene, C. Binding of RecA protein to single-stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides. EMBO J. 2, 2247–2251 (1983)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Kowalczykowski, S. C. Cancer: catalyst of a catalyst. Nature 433, 591–592 (2005)

    Article  ADS  CAS  Google Scholar 

  10. Sung, P., Krejci, L., Van Komen, S. & Sehorn, M. G. Rad51 recombinase and recombination mediators. J. Biol. Chem. 278, 42729–42732 (2003)

    Article  CAS  Google Scholar 

  11. Yang, H., Li, Q., Fan, J., Holloman, W. K. & Pavletich, N. P. The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA–ssDNA junction. Nature 433, 653–657 (2005)

    Article  ADS  CAS  Google Scholar 

  12. Zaitsev, E. N. & Kowalczykowski, S. C. A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange. Genes Dev. 14, 740–749 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kim, J. I. & Cox, M. M. The RecA proteins of Deinococcus radiodurans and Escherichia coli promote DNA strand exchange via inverse pathways. Proc. Natl Acad. Sci. USA 99, 7917–7921 (2002)

    Article  ADS  CAS  Google Scholar 

  14. Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001)

    Article  ADS  CAS  Google Scholar 

  15. Egelman, E. H. & Stasiak, A. Structure of helical RecA–DNA complexes. Complexes formed in the presence of ATP-γS or ATP. J. Mol. Biol. 191, 677–697 (1986)

    Article  CAS  Google Scholar 

  16. Sattin, B. D. & Goh, M. C. Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy. Biophys. J. 87, 3430–3436 (2004)

    Article  ADS  CAS  Google Scholar 

  17. van der Heijden, T. et al. Torque-limited RecA polymerization on dsDNA. Nucleic Acids Res. 33, 2099–2105 (2005)

    Article  CAS  Google Scholar 

  18. Menetski, J. P., Varghese, A. & Kowalczykowski, S. C. Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein. Biochemistry 27, 1205–1212 (1988)

    Article  CAS  Google Scholar 

  19. Zaitsev, E. N. & Kowalczykowski, S. C. Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay. Nucleic Acids Res. 26, 650–654 (1998)

    Article  CAS  Google Scholar 

  20. Kowalczykowski, S. & Steinhardt, J. Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity. J. Mol. Biol. 115, 201–213 (1977)

    Article  CAS  Google Scholar 

  21. Fulconis, R. et al. Twisting and untwisting a single DNA molecule covered by RecA protein. Biophys. J. 87, 2552–2563 (2004)

    Article  ADS  CAS  Google Scholar 

  22. Register, J. C. & Griffith, J. The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange. J. Biol. Chem. 260, 12308–12312 (1985)

    CAS  PubMed  Google Scholar 

  23. Shaner, S. L., Flory, J. & Radding, C. M. The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends. J. Biol. Chem. 262, 9220–9230 (1987)

    CAS  PubMed  Google Scholar 

  24. Shaner, S. L. & Radding, C. M. Translocation of Escherichia coli recA protein from a single-stranded tail to contiguous duplex DNA. J. Biol. Chem. 262, 9211–9219 (1987)

    CAS  PubMed  Google Scholar 

  25. Menetski, J. P., Bear, D. G. & Kowalczykowski, S. C. Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc. Natl Acad. Sci. USA 87, 21–25 (1990)

    Article  ADS  CAS  Google Scholar 

  26. Chow, S. A., Honigberg, S. M. & Radding, C. M. DNase protection by recA protein during strand exchange. Asymmetric protection of the Holliday structure. J. Biol. Chem. 263, 3335–3347 (1988)

    CAS  PubMed  Google Scholar 

  27. Kowalczykowski, S. C. Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins. Biochimie 73, 289–304 (1991)

    Article  CAS  Google Scholar 

  28. Anderson, D. G. & Kowalczykowski, S. C. The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a χ-regulated manner. Cell 90, 77–86 (1997)

    Article  CAS  Google Scholar 

  29. Morimatsu, K. & Kowalczykowski, S. C. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Mol. Cell 11, 1337–1347 (2003)

    Article  CAS  Google Scholar 

  30. Spies, M. & Kowalczykowski, S. C. The RecA binding locus of RecBCD is a general domain for recruitment of DNA strand exchange proteins. Mol. Cell 21, 573–580 (2006)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Spies, J. Siino and A. Forget for suggestions and discussions, and members of the Kowalczykowski laboratory for comments. This work was supported by NIH grants to S.C.K. and R.J.B. R.G. was supported partially by a Fellowship from the Jeane B. Kempner Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stephen C. Kowalczykowski.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Figures 1–13, Supplementary Methods and related text. Supplementary Figure 1 is a simple schematic that summarizes the main finding of the paper. The Supplementary Methods describe data analysis and preparation of fluorescent RecA; Supplementary Data provide biochemical characterization the fluorescent RecA. The data included show RecA–ATPγS filaments are highly stable; nucleation becomes rate-limiting for RecA assembly at high NaCl concentrations; RecA preferentially nucleates at AT-rich DNA; the relationship between DNA elongation and RecA filament formation; and stabilization of ATP–RecA clusters by Ca2+ ions. (PDF 471 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Galletto, R., Amitani, I., Baskin, R. et al. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006). https://doi.org/10.1038/nature05197

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05197

This article is cited by

Comments

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing