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.

  • Article
  • Published:

Visualizing single DNA-bound proteins using DNA as a scanning probe

Nature Methods volume 4, pages 1031–1036 (2007)Cite this article

Abstract

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Dual DNA manipulation assay.
Figure 2: DNA scanning scheme and reference scan.
Figure 3: Detection of individual DNA-bound restriction enzymes.
Figure 4: Nondestructive and destructive imaging.

Similar content being viewed by others

References

  1. West, S.C. Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4, 435–445 (2003).

    Article  CAS  Google Scholar 

  2. van den Bosch, M., Lohman, P.H. & Pastink, A. DNA double-strand break repair by homologous recombination. Biol. Chem. 383, 873–892 (2002).

    CAS  PubMed  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. van den Broek, B., Vanzi, F., Normanno, D., Pavone, F.S. & Wuite, G.J. Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI. Nucleic Acids Res. 34, 167–174 (2006).

    Article  CAS  Google Scholar 

  5. Luijsterburg, M.S., Noom, M.C., Wuite, G.J. & Dame, R.T. The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: A molecular perspective. J. Struct. Biol. 156, 262–272 (2006).

    Article  CAS  Google Scholar 

  6. Dame, R.T., Noom, M.C. & Wuite, G.J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444, 387–390 (2006).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. Gowers, D.M. & Halford, S.E. Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling. EMBO J. 22, 1410–1418 (2003).

    Article  CAS  Google Scholar 

  9. von Hippel, P.H. & Berg, O.G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  Google Scholar 

  10. Epshtein, V., Toulme, F., Rahmouni, A.R., Borukhov, S. & Nudler, E. Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727 (2003).

    Article  CAS  Google Scholar 

  11. Allersma, M.W., Gittes, F., deCastro, M.J., Stewart, R.J. & Schmidt, C.F. Two-dimensional tracking of ncd motility by back focal plane interferometry. Biophys. J. 74, 1074–1085 (1998).

    Article  CAS  Google Scholar 

  12. Gittes, F. & Schmidt, C.F. Interference model for back-focal-plane displacement detection in optical tweezers. Opt. Lett. 23, 7–9 (1998).

    Article  CAS  Google Scholar 

  13. Brewer, L.R., Corzett, M. & Balhorn, R. Protamine-induced condensation and decondensation of the same DNA molecule. Science 286, 120–123 (1999).

    Article  CAS  Google Scholar 

  14. Arai, Y. et al. Tying a molecular knot with optical tweezers. Nature 399, 446–448 (1999).

    Article  CAS  Google Scholar 

  15. Vermeulen, K.C. et al. Calibrating bead displacements in optical tweezers using acousto-optic deflectors. Rev. Sci. Instrum. 77, 013704 (2006).

    Article  Google Scholar 

  16. Gosule, L.C. & Schellman, J.A. Compact form of DNA induced by spermidine. Nature 259, 333–335 (1976).

    Article  CAS  Google Scholar 

  17. Shimamoto, N. One-dimensional diffusion of proteins along DNA - Its biological and chemical significance revealed by single-molecule measurements. J. Biol. Chem. 274, 15293–15296 (1999).

    Article  CAS  Google Scholar 

  18. Trabesinger, W., Schutz, G.J., Gruber, H.J., Schindler, H. & Schmidt, T. Detection of individual oligonucleotide pairing by single-molecule microscopy. Anal. Chem. 71, 279–283 (1999).

    Article  CAS  Google Scholar 

  19. Hiller, D.A. et al. Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence. Biochemistry 42, 14375–14385 (2003).

    Article  CAS  Google Scholar 

  20. Charvin, G., Vologodskii, A., Bensimon, D. & Croquette, V. Braiding DNA: experiments, simulations, and models. Biophys. J. 88, 4124–4136 (2005).

    Article  CAS  Google Scholar 

  21. Binnig, G., Quate, C.F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).

    Article  CAS  Google Scholar 

  22. Lindsay, S.M. et al. STM and AFM images of nucleosome DNA under water. J. Biomol. Struct. Dyn. 7, 279–287 (1989).

    Article  CAS  Google Scholar 

  23. Fritzsche, W., Takac, L. & Henderson, E. Application of atomic force microscopy to visualization of DNA, chromatin, and chromosomes. Crit. Rev. Eukaryot. Gene Expr. 7, 231–240 (1997).

    Article  CAS  Google Scholar 

  24. Kasas, S. et al. Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry 36, 461–468 (1997).

    Article  CAS  Google Scholar 

  25. van Noort, S.J. et al. Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope. Biophys. J. 74, 2840–2849 (1998).

    Article  CAS  Google Scholar 

  26. Huisstede, J.H.G., van der Werf, K.O., Bennink, M.L. & Subramaniam, V. Force detection in optical tweezers using backscattered light. Opt. Express 13, 1113–1123 (2005).

    Article  CAS  Google Scholar 

  27. Koch, S.J., Shundrovsky, A., Jantzen, B.C. & Wang, M.D. Probing protein-DNA interactions by unzipping a single DNA double helix. Biophys. J. 83, 1098–1105 (2002).

    Article  CAS  Google Scholar 

  28. 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 

  29. Dame, R.T., Wyman, C. & Goosen, N. Structural basis for preferential binding of H-NS to curved DNA. Biochimie 83, 231–234 (2001).

    Article  CAS  Google Scholar 

  30. Halford, S.E., Welsh, A.J. & Szczelkun, M.D. Enzyme-mediated DNA looping. Annu. Rev. Biophys. Biomol. Struct. 33, 1–24 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Svoboda, K. & Block, S.M. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994).

    Article  CAS  Google Scholar 

  33. Wuite, G.J.L., Davenport, R.J., Rappaport, A. & Bustamante, C. An integrated laser trap/flow control video microscope for the study of single biomolecules. Biophys. J. 79, 1155–1167 (2000).

    Article  CAS  Google Scholar 

  34. Gittes, F. & Schmidt, C.F. Signals and noise in micromechanical measurements. Methods Cell Biol. 55, 129–156 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank E.J.G. Peterman and R.T. Dame for useful discussions. We acknowledge D.A. Hiller (Yale) and J.J. Perona (University of California Santa Barbara) for the kind gift of EcoRV. This work is part of the research program of the 'Stichting voor Fundamenteel Onderzoek der Materie (FOM)', which is financially supported by the 'Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)' and was supported by a NWO-Vernieuwingsimpuls grant.

Author information

Author notes

  1. Maarten C Noom and Bram van den Broek: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics and Astronomy, Physics of Complex Systems, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, Amsterdam, 1081 HV, The Netherlands

    Maarten C Noom, Bram van den Broek, Joost van Mameren & Gijs J L Wuite

Authors
  1. Maarten C Noom
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bram van den Broek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joost van Mameren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gijs J L Wuite
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gijs J L Wuite.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Note (PDF 185 kb)

Supplementary Movie 1

Trapping beads, catching two DNA molecules and wrapping of the probing DNA around the scanned DNA. The movie (sped up 3×) shows how two optically trapped DNA molecules are wound around each other. First, beads are captured in the four optical traps. Next, two DNA molecules are caught between these beads. The displacements of the beads out of their traps (indicated by crosshairs) are a signal that DNA is tethered between them. By moving one of the traps in the direction perpendicular to the field of view, the DNA molecules are then wound around each other. Finally, the DNA molecules are brought into a crossed configuration, required for the DNA scanning experiments. (MOV 5015 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Noom, M., van den Broek, B., van Mameren, J. et al. Visualizing single DNA-bound proteins using DNA as a scanning probe. Nat Methods 4, 1031–1036 (2007). https://doi.org/10.1038/nmeth1126

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1126

This article is cited by

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