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.

Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

Abstract

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA1. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA–ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange2,3, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA–ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed ‘intersegmental contact sampling’, in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: DNA pairing by RecA, imaged using single-molecule TIRFM, indicates that the three-dimensional conformation of target dsDNA is important in the homology search.
Figure 2: Visualization of RecA-promoted DNA pairing with an individual optically trapped DNA dumbbell, imaged by epifluorescence.
Figure 3: DNA three-dimensional conformation and nucleoprotein filament length contribute to the homology search.
Figure 4: RecA nucleoprotein filaments exhibit transient non-homologous interactions and loop-release events.

References

  1. 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. 2

    Menetski, J. P. & Kowalczykowski, S. C. Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J. Mol. Biol. 181, 281–295 (1985)

    CAS  Article  Google Scholar 

  3. 3

    Kowalczykowski, S. C. & Krupp, R. A. DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions. Proc. Natl Acad. Sci. USA 92, 3478–3482 (1995)

    ADS  CAS  Article  Google Scholar 

  4. 4

    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)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Adzuma, K. No sliding during homology search by RecA protein. J. Biol. Chem. 273, 31565–31573 (1998)

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Fulconis, R., Miné, J., Bancaud, A., Dutreix, M. & Viovy, J. L. Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation. EMBO J. 25, 4293–4304 (2006)

    CAS  Article  Google Scholar 

  8. 8

    van der Heijden, T. et al. Homologous recombination in real time: DNA strand exchange by RecA. Mol. Cell 30, 530–538 (2008)

    CAS  Article  Google Scholar 

  9. 9

    Forget, A. L. & Kowalczykowski, S. C. Single-molecule imaging brings Rad51 nucleoprotein filaments into focus. Trends Cell Biol. 20, 269–276 (2010)

    CAS  Article  Google Scholar 

  10. 10

    McEntee, K., Weinstock, G. M. & Lehman, I. R. Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J. Biol. Chem. 256, 8835–8844 (1981)

    CAS  PubMed  Google Scholar 

  11. 11

    Honigberg, S. M., Gonda, D. K., Flory, J. & Radding, C. M. The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5′-(gamma-thio)triphosphate. J. Biol. Chem. 260, 11845–11851 (1985)

    CAS  PubMed  Google Scholar 

  12. 12

    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 

  13. 13

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

    ADS  CAS  Article  Google Scholar 

  14. 14

    Julin, D. A., Riddles, P. W. & Lehman, I. R. On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli. J. Biol. Chem. 261, 1025–1030 (1986)

    CAS  PubMed  Google Scholar 

  15. 15

    Gonda, D. K. & Radding, C. M. By searching processively recA protein pairs DNA molecules that share a limited stretch of homology. Cell 34, 647–654 (1983)

    CAS  Article  Google Scholar 

  16. 16

    Tsang, S. S., Chow, S. A. & Radding, C. M. Networks of DNA and recA protein are intermediates in homologous pairing. Biochemistry 24, 3226–3232 (1985)

    CAS  Article  Google Scholar 

  17. 17

    Berg, O. G., Winter, R. B. & von Hippel, P. H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20, 6929–6948 (1981)

    CAS  Article  Google Scholar 

  18. 18

    Berg, O. G. in The Biology of Nonspecific DNA Protein Interactions (ed. Revzin, A.) 71–85 (CRC, 1990)

    Google Scholar 

  19. 19

    Mirshad, J. K. & Kowalczykowski, S. C. Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. Biochemistry 42, 5945–5954 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Harmon, F. G. & Kowalczykowski, S. C. RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev. 12, 1134–1144 (1998)

    CAS  Article  Google Scholar 

  21. 21

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

    ADS  CAS  Article  Google Scholar 

  22. 22

    Perkins, T. T., Quake, S. R., Smith, D. E. & Chu, S. Relaxation of a single DNA molecule observed by optical microscopy. Science 264, 822–826 (1994)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to members of the laboratory for their comments on this work. A.L.F. was funded by an American Cancer Society Postdoctoral Fellowship (PF-08–046–01-GMC) and S.C.K. was supported by the National Institutes of Health (GM-62653 and GM-64745).

Author information

Affiliations

Authors

Contributions

A.L.F. and S.C.K. conceived the general ideas, designed the experiments and interpreted the data. A.L.F. performed experiments. A.L.F. and S.C.K. wrote the manuscript.

Corresponding author

Correspondence to Stephen C. Kowalczykowski.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4 with legends and legends for Supplementary Movies 1-3. (PDF 531 kb)

Supplementary Movie 1

Composite movie depicting the experimental procedure used to visualize DNA pairing on single DNA-dumbbell molecules by optical trapping (see Supplementary Information file for full legend). (MP4 21540 kb)

Supplementary Movie 2

Movie showing RecA nucleoprotein filaments, both heterologously- and homologously-bound (left and right red spots, respectively) during the extension step (Fig. 2b, step 6) of a pairing assay performed using the 1,762 nt ssDNA (see Supplementary Information file for full legend). (MOV 553 kb)

Supplementary Movie 3

Movie showing RecA nucleoprotein filaments, both heterologously- and homologously-bound (left and right red spots, respectively) during the extension step (Fig. 2b, step 6) of a pairing assay performed using the 430 nt ssDNA (see Supplementary Information file for full legend). (MOV 631 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Forget, A., Kowalczykowski, S. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482, 423–427 (2012). https://doi.org/10.1038/nature10782

Download citation

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