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.

SSB protein diffusion on single-stranded DNA stimulates RecA filament formation

An Erratum to this article was published on 17 December 2009

Abstract

Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein–DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: FRET fluctuations arising from diffusional migration of SSB on ssDNA.
Figure 2: Analysis of SSB mobility on ssDNA.
Figure 3: SSB diffusion on ssDNA probed with three-colour FRET.
Figure 4: Mechanism of SSB displacement by an extending RecA filament.
Figure 5: SSB diffusion promotes RecA filament growth on DNA hairpin.

References

  1. Meyer, R. R. & Laine, P. S. The single-stranded DNA-binding protein of Escherichia coli . Microbiol. Rev. 54, 342–380 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M. & Keck, J. L. SSB as an organizer/mobilizer of genome maintenance complexes. Crit. Rev. Biochem. Mol. Biol. 43, 289–318 (2008)

    CAS  Article  Google Scholar 

  3. Kozlov, A. G. & Lohman, T. M. Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules. Biochemistry 41, 11611–11627 (2002)

    CAS  Article  Google Scholar 

  4. Kuznetsov, S. V., Kozlov, A. G., Lohman, T. M. & Ansari, A. Microsecond dynamics of protein–DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer. J. Mol. Biol. 359, 55–65 (2006)

    CAS  Article  Google Scholar 

  5. Roy, R., Kozlov, A. G., Lohman, T. M. & Ha, T. Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein. J. Mol. Biol. 369, 1244–1257 (2007)

    CAS  Article  Google Scholar 

  6. Lohman, T. M. & Ferrari, M. E. Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem. 63, 527–570 (1994)

    CAS  Article  Google Scholar 

  7. Raghunathan, S., Kozlov, A. G., Lohman, T. M. & Waksman, G. Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nature Struct. Biol. 7, 648–652 (2000)

    CAS  Article  Google Scholar 

  8. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996)

    CAS  Article  Google Scholar 

  9. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nature Methods 5, 507–516 (2008)

    CAS  Article  Google Scholar 

  10. Bujalowski, W. & Lohman, T. M. Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA. Biochemistry 25, 7799–7802 (1986)

    CAS  Article  Google Scholar 

  11. Lohman, T. M. & Overman, L. B. Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration. J. Biol. Chem. 260, 3594–3603 (1985)

    CAS  PubMed  Google Scholar 

  12. Griffith, J. D., Harris, L. D. & Register, J. Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments. Cold Spring Harb. Symp. Quant. Biol. 49, 553–559 (1984)

    CAS  Article  Google Scholar 

  13. McKinney, S. A., Joo, C. & Ha, T. Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951 (2006)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  15. Hohng, S., Joo, C. & Ha, T. Single-molecule three-color FRET. Biophys. J. 87, 1328–1337 (2004)

    CAS  Article  Google Scholar 

  16. Kowalczykowski, S. C. Initiation of genetic recombination and recombination-dependent replication. Trends Biochem. Sci. 25, 156–165 (2000)

    CAS  Article  Google Scholar 

  17. 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 

  18. Roca, A. I. & Cox, M. M. RecA protein: structure, function, and role in recombinational DNA repair. Prog. Nucleic Acid Res. Mol. Biol. 56, 129–223 (1997)

    CAS  Article  Google Scholar 

  19. Kuzminov, A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ. Microbiol. Mol. Biol. Rev. 63, 751–813 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Ennis, D. G., Amundsen, S. K. & Smith, G. R. Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage λ. Genetics 115, 11–24 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Glassberg, J., Meyer, R. R. & Kornberg, A. Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization. J. Bacteriol. 140, 14–19 (1979)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Golub, E. I. & Low, K. B. Indirect stimulation of genetic recombination. Proc. Natl Acad. Sci. USA 80, 1401–1405 (1983)

    CAS  Article  Google Scholar 

  23. Umezu, K., Chi, N. W. & Kolodner, R. D. Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein. Proc. Natl Acad. Sci. USA 90, 3875–3879 (1993)

    CAS  Article  Google Scholar 

  24. 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)

    CAS  Article  Google Scholar 

  25. Bork, J. M., Cox, M. M. & Inman, R. B. The RecOR proteins modulate RecA protein function at 5′ ends of single-stranded DNA. EMBO J. 20, 7313–7322 (2001)

    CAS  Article  Google Scholar 

  26. 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)

    CAS  Article  Google Scholar 

  27. Hobbs, M. D., Sakai, A. & Cox, M. M. SSB protein limits RecOR binding onto single-stranded DNA. J. Biol. Chem. 282, 11058–11067 (2007)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  29. Kowalczykowski, S. C., Clow, J., Somani, R. & Varghese, A. Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein–protein interaction. J. Mol. Biol. 193, 81–95 (1987)

    CAS  Article  Google Scholar 

  30. Kowalczykowski, S. C. & Krupp, R. A. Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. J. Mol. Biol. 193, 97–113 (1987)

    CAS  Article  Google Scholar 

  31. Muniyappa, K., Shaner, S. L., Tsang, S. S. & Radding, C. M. Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination. Proc. Natl Acad. Sci. USA 81, 2757–2761 (1984)

    CAS  Article  Google Scholar 

  32. Eggington, J. M., Kozlov, A. G., Cox, M. M. & Lohman, T. M. Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein. Biochemistry 45, 14490–14502 (2006)

    CAS  Article  Google Scholar 

  33. Bujalowski, W. & Lohman, T. M. Limited co-operativity in protein-nucleic acid interactions. A thermodynamic model for the interactions of Escherichia coli single strand binding protein with single-stranded nucleic acids in the “beaded”, (SSB)65 mode. J. Mol. Biol. 195, 897–907 (1987)

    CAS  Article  Google Scholar 

  34. Römer, R., Schomburg, U., Krauss, G. & Maass, G. Escherichia coli single-stranded DNA binding protein is mobile on DNA: proton NMR study of its interaction with oligo- and polynucleotides. Biochemistry 23, 6132–6137 (1984)

    Article  Google Scholar 

  35. Clendenning, J. B. & Schurr, J. M. A model for the binding of E. coli single-strand binding protein to supercoiled DNA. Biophys. Chem. 52, 227–249 (1994)

    CAS  Article  Google Scholar 

  36. Glikin, G. C., Gargiulo, G., Rena-Descalzi, L. & Worcel, A. Escherichia coli single-strand binding protein stabilizes specific denatured sites in superhelical DNA. Nature 303, 770–774 (1983)

    CAS  Article  Google Scholar 

  37. Sun, W. & Godson, G. N. Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis. J. Mol. Biol. 276, 689–703 (1998)

    CAS  Article  Google Scholar 

  38. Shereda, R. D., Bernstein, D. A. & Keck, J. L. A central role for SSB in Escherichia coli RecQ DNA helicase function. J. Biol. Chem. 282, 19247–19258 (2007)

    CAS  Article  Google Scholar 

  39. Lecointe, F. et al. Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks. EMBO J. 26, 4239–4251 (2007)

    CAS  Article  Google Scholar 

  40. Richard, D. J. et al. Single-stranded DNA-binding protein hSSB1 is critical for genomic stability. Nature 453, 677–681 (2008)

    CAS  Article  Google Scholar 

  41. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank C. Joo, S. A. McKinney, I. Rasnik, S. Hohng and S. Myong for experimental help and discussion; C. Murphy, M. Nahas and K. Raghunathan for discussion; T. Ho and A. Niedziela-Majka for help with DNA and protein preparation, respectively; and R. Porter for the SSB expression plasmid. T.H. is an employee of the Howard Hughes Medical Institute. These studies were supported by grants from the National Institutes of Health and the National Science Foundation.

Author Contributions R.R., A.G.K., T.M.L. and T.H. designed the experiments, A.G.K. prepared the wild-type SSB protein and the mutant SSB with fluorescent labels, R.R. performed the experiments and analysed the data; R.R., T.M.L. and T.H. wrote the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Taekjip Ha.

Supplementary information

Supplementary Information

This file contains Supplementary Material, Supplementary Methods including Table 1, Supplementary Figures 1-12 with legends, and Supplementary References. (PDF 2962 kb)

Supplementary Movie

SSB diffusion movie in three segments. In the first, SSB diffusion via the rolling mechanism is illustrated. In the second, RecA filament growth via monomer addition biases SSB diffusion in a directional manner. In the third, SSB can melt secondary structures transiently via diffusion and promotes RecA filament formation. (MOV 3985 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Roy, R., Kozlov, A., Lohman, T. et al. SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461, 1092–1097 (2009). https://doi.org/10.1038/nature08442

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08442

Further reading

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