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.

Repetitive shuttling of a motor protein on DNA

Nature volume 437, pages 1321–1325 (2005)Cite this article

Abstract

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3′ to 5′ direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Blockade-induced repetitive shuttling.
Figure 2: Physical mechanism of repetitive shuttling.
Figure 3: Potential roles of repetitive shuttling.

References

  1. Lohman, T. M. & Bjornson, K. P. Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214 (1996)

    Article  CAS  Google Scholar 

  2. Byrd, A. K. & Raney, K. D. Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nature Struct. Mol. Biol. 11, 531–538 (2004)

    Article  CAS  Google Scholar 

  3. Fairman, M. E. et al. Protein displacement by DExH/D “RNA helicases” without duplex unwinding. Science 304, 730–734 (2004)

    Article  ADS  CAS  Google Scholar 

  4. Kaplan, D. L. & O'Donnell, M. DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands. Mol. Cell 10, 647–657 (2002)

    Article  CAS  Google Scholar 

  5. Lee, M. S. & Marians, K. J. Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein. J. Biol. Chem. 265, 17078–17083 (1990)

    CAS  PubMed  Google Scholar 

  6. Kawaoka, J., Jankowsky, E. & Pyle, A. M. Backbone tracking by the SF2 helicase NPH-II. Nature Struct. Mol. Biol. 11, 526–530 (2004)

    Article  CAS  Google Scholar 

  7. von Hippel, P. H. Helicases become mechanistically simpler and functionally more complex. Nature Struct. Mol. Biol. 11, 494–496 (2004)

    Article  CAS  Google Scholar 

  8. Krejci, L. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305–309 (2003)

    Article  ADS  CAS  Google Scholar 

  9. Veaute, X. et al. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423, 309–312 (2003)

    Article  ADS  CAS  Google Scholar 

  10. Veaute, X. et al. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli. EMBO J. 24, 180–189 (2005)

    Article  CAS  Google Scholar 

  11. Sandler, S. J. Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12. Genetics 155, 487–497 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Marians, K. J. Mechanisms of replication fork restart in Escherichia coli. Phil. Trans. R. Soc. Lond. B 359, 71–77 (2004)

    Article  CAS  Google Scholar 

  13. Scott, J. F., Eisenberg, S., Bertsch, L. L. & Kornberg, A. A mechanism of duplex DNA replication revealed by enzymatic studies of phage φX174: catalytic strand separation in advance of replication. Proc. Natl Acad. Sci. USA 74, 193–197 (1977)

    Article  ADS  CAS  Google Scholar 

  14. Rasnik, I., Myong, S., Cheng, W., Lohman, T. M. & Ha, T. DNA-binding orientation and domain conformation of the E. coli Rep helicase monomer bound to a partial duplex junction: Single-molecule studies of fluorescently labelled enzymes. J. Mol. Biol. 336, 395–408 (2004)

    Article  CAS  Google Scholar 

  15. Cheng, W., Hsieh, J., Brendza, K. M. & Lohman, T. M. E. coli Rep oligomers are required to initiate DNA unwinding in vitro. J. Mol. Biol. 310, 327–350 (2001)

    Article  CAS  Google Scholar 

  16. Ha, T. et al. Initiation and reinitiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002)

    Article  ADS  CAS  Google Scholar 

  17. Brendza, K. M. et al. Auto-inhibition of E. coli Rep monomer helicase activity by its 2B sub-domain. Proc. Natl Acad. Sci. USA 102, 10081 (2005)

    Article  Google Scholar 

  18. 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)

    Article  ADS  CAS  Google Scholar 

  19. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999)

    Article  ADS  CAS  Google Scholar 

  20. Ha, T. Single molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001)

    Article  CAS  Google Scholar 

  21. Mukhopadhyay, J. et al. Translocation of σ70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 106, 453–463 (2001)

    Article  CAS  Google Scholar 

  22. Zhuang, X. W. et al. A single-molecule study of RNA catalysis and folding. Science 288, 2048–2051 (2000)

    Article  ADS  CAS  Google Scholar 

  23. Blanchard, S. C., Kim, H. D., Gonzalez, R. L. Jr, Puglisi, J. D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl Acad. Sci. USA 101, 12893–12898 (2004)

    Article  ADS  CAS  Google Scholar 

  24. Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84 (1999)

    Article  CAS  Google Scholar 

  25. Perkins, T. T., Li, H. W., Dalal, R. V., Gelles, J. & Block, S. M. Forward and reverse motion of single RecBCD molecules on DNA. Biophys. J. 86, 1640–1648 (2004)

    Article  ADS  CAS  Google Scholar 

  26. Dessinges, M. N., Lionnet, T., Xi, X. G., Bensimon, D. & Croquette, V. Single-molecule assay reveals strand switching and enhanced processivity of UvrD. Proc. Natl Acad. Sci. USA 101, 6439–6444 (2004)

    Article  ADS  CAS  Google Scholar 

  27. Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ATP. Cell 90, 635–647 (1997)

    Article  CAS  Google Scholar 

  28. Murphy, M. C., Rasnik, I., Cheng, W., Lohman, T. M. & Ha, T. Probing single stranded DNA conformational flexibility using fluorescence spectroscopy. Biophys. J. 86, 2530–2537 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Cheng, W. et al. The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity. Proc. Natl Acad. Sci. USA 99, 16006–16011 (2002)

    Article  ADS  CAS  Google Scholar 

  30. Margittai, M. et al. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc. Natl Acad. Sci. USA 100, 15516–15521 (2003)

    Article  ADS  CAS  Google Scholar 

  31. Denhardt, D. T., Dressler, D. H. & Hathaway, A. The abortive replication of φX174 DNA in a recombination deficient mutant of Escherichia coli. Proc. Natl Acad. Sci. USA 57, 813–820 (1967)

    Article  ADS  CAS  Google Scholar 

  32. Scott, J. F. & Kornberg, A. Purification of the rep protein of Escherichia coli. An ATPase which separates duplex DNA strands in advance of replication. J. Biol. Chem. 253, 3292–3297 (1978)

    CAS  PubMed  Google Scholar 

  33. Petit, M. A. & Ehrlich, D. Essential bacterial helicases that counteract the toxicity of recombination proteins. EMBO J. 21, 3137–3147 (2002)

    Article  CAS  Google Scholar 

  34. 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 

Download references

Acknowledgements

We thank S. A. McKinney for writing the data acquisition program and the National Institute of Health for grants (to T.H. and T.M.L.).

Author information

Authors and Affiliations

  1. Physics Department, University of Illinois, Urbana-Champaign,

    Sua Myong, Ivan Rasnik, Chirlmin Joo & Taekjip Ha

  2. Howard Hughes Medical Institute, Urbana, Illinois, 61801, USA

    Taekjip Ha

  3. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Missouri, 63110, St Louis, USA

    Timothy M. Lohman

Authors
  1. Sua Myong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivan Rasnik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chirlmin Joo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Timothy M. Lohman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Taekjip Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Taekjip Ha.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Notes

This file contains Supplementary Methods and Supplementary Figures 1-7. (DOC 113 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Myong, S., Rasnik, I., Joo, C. et al. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005). https://doi.org/10.1038/nature04049

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04049

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

Advanced 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