Single-Molecule Measurements of Topoisomerase Activity with Magnetic Tweezers

  • Yeonee Seol
  • Keir C. NeumanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 778)


Magnetic tweezers provide a versatile tool enabling the precise application of force and torque on ­individual biomolecules. These properties make magnetic tweezers uniquely suited for the study of DNA topology and topoisomerases at the single-molecule level. Single-molecule approaches, which are complementary to ensemble biochemical and structural approaches, have provided remarkable insights into the mechanisms of topoisomerase activity and interactions with DNA. Here, we describe how to make single-molecule measurements of topoisomerase activity with a magnetic tweezers instrument. We provide detailed instructions for preparing and characterizing DNA substrates, flow cells, and supercoilable DNA tethers. We then describe magnetic tweezers measurements of supercoil relaxation by single topoisomerases.

Key words

Magnetic tweezers Single molecule DNA topology DNA topoisomerase 



The authors would like to thank Marie-Paule Strub, Vincent Croquette, Jean-Francois Allemand, Timothée Lionnet, Omar Saleh, and David Bensimon for assistance and enlightening discussions and Grace Liou for critical reading of the manuscript. This research was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health.


  1. 1.
    Bates, A. D., and Maxwell, A. (2005) DNA Topology, 2 ed., Oxford University Press, New York.Google Scholar
  2. 2.
    Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430–440.PubMedCrossRefGoogle Scholar
  3. 3.
    Travers, A., and Muskhelishvili, G. (2005) DNA supercoiling – a global transcriptional regulator for enterobacterial growth?, Nat. Rev. Microbiol. 3, 157–169.PubMedCrossRefGoogle Scholar
  4. 4.
    Espeli, O., and Marians, K. J. (2004) Untangling intracellular DNA topology. Mol. Microbiol. 52, 925–931.PubMedCrossRefGoogle Scholar
  5. 5.
    Schoeffler, A. J., and Berger, J. M. (2008) DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q Rev. Biophys. 41, 41–101.PubMedCrossRefGoogle Scholar
  6. 6.
    Corbett, K. D., and Berger, J. M. (2004) Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 33, 95–118.PubMedCrossRefGoogle Scholar
  7. 7.
    Champoux, J. J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413.PubMedCrossRefGoogle Scholar
  8. 8.
    Charvin, G., Strick, T. R., Bensimon, D., and Croquette, V. (2005) Tracking topoisomerase activity at the single-molecule level. Annu. Rev. Biophys. Biomol. Struct. 34, 201–219.PubMedCrossRefGoogle Scholar
  9. 9.
    Lipfert, J., Koster, D. A., Vilfan, I. D., Hage, S., and Dekker, N. H. (2009) Single-molecule magnetic tweezers studies of type IB topoisomerases. Methods Mol. Biol. 582, 71–89.PubMedCrossRefGoogle Scholar
  10. 10.
    Vilfan, I. D., Lipfert, J., Koster, D. A., Lemay, S. G., and Dekker, N. H. (2009) Magnetic Tweezers for Single-Molecule Experiments in Handbook of Single-Molecule Biophysics p 371, Springer.Google Scholar
  11. 11.
    Neuman, K. C., and Nagy, A. (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Meth. 5, 491–505.CrossRefGoogle Scholar
  12. 12.
    Neuman, K. C., Lionnet, T., and Allemand, J. F. (2007) Single-molecule micromanipulation techniques. Annual Review of Materials Research 37, 33–67.CrossRefGoogle Scholar
  13. 13.
    Lionnet, T., Allemand, J.-F., Revyakin, A., Strick, T. R., Saleh, O. A., Bensimon, D., and Croquette, V. (2008) Single-Molecule Studies Using Magnetic Traps in Single-Molecule Techniques: A Laboratory Manual. (Selvin, P. R., and Ha, T., Eds.), Cold Spring Harbor Laboratory Press.Google Scholar
  14. 14.
    Neuman, K. C. (2010) Single-molecule measurements of DNA topology and topoisomerases. J. Biol. Chem. 285, 18967–18971.PubMedCrossRefGoogle Scholar
  15. 15.
    Gosse, C., and Croquette, V. (2002) Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314–3329.PubMedCrossRefGoogle Scholar
  16. 16.
    Charvin, G., Allemand, J. F., Strick, T. R., Bensimon, D., and Croquette, V. (2004) Twisting DNA: single molecule studies. Contemporary Physics 45, 383–403.CrossRefGoogle Scholar
  17. 17.
    Nollmann, M., Stone, M. D., Bryant, Z., Gore, J., Crisona, N. J., Hong, S. C., Mitelheiser, S., Maxwell, A., Bustamante, C., and Cozzarelli, N. R. (2007) Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat. Struct. Mol. Biol. 14, 264–271.PubMedCrossRefGoogle Scholar
  18. 18.
    Revyakin, A., Ebright, R. H., and Strick, T. R. (2005) Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments. Nat. Methods 2, 127–138.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory of Molecular BiophysicsNational Heart, Lung, and Blood Institute, National Institutes of HealthBethesdaUSA

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