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.

  • Letter
  • Published:

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Nature volume 404, pages 901–904 (2000)Cite this article

Abstract

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment1. This enables them to untangle DNA2,3,4,5,6 and relax the interwound supercoils (plectonemes) that arise in twisted DNA7. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death8,9. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled10,11,12,13. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

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: Extension versus supercoiling behaviour for stretched DNA.
Figure 2: Individual time courses for the topo II-mediated relaxation of positively supercoiled DNA stretched at F = 0.7 pN.
Figure 3: Reaction dependence on ATP concentration and force.
Figure 4: Evidence for topo II clamping a crossover between two DNA segments (no ATP).

Similar content being viewed by others

References

  1. Wang,J. C. Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q. Rev. Biophys. 31, 107–144 (1998).

    Article  CAS  Google Scholar 

  2. Liu,L. F., Liu,C. C. & Alberts,B. M. Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-stranded break. Cell 19, 697–707 (1980).

    Article  CAS  Google Scholar 

  3. Hsieh,T. Knotting of the circular duplex DNA by type II DNA topoisomerase from D. melanogaster . J. Biol. Chem. 258, 8413– 8420 (1983).

    CAS  PubMed  Google Scholar 

  4. Roca,J. & Wang,J. C. The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerase. Cell 71, 833– 840 (1992).

    Article  CAS  Google Scholar 

  5. Roca,J., Berger,J. M. & Harrison, S. C. & Wang,J. C. DNA transport by a type II topoisomerase: Direct evidence for a two-gate mechanism. Proc. Natl Acad. Sci. USA 93, 4057– 4062 (1996).

    Article  ADS  CAS  Google Scholar 

  6. Rybenkov,V. V., Ullsperger,C. Vologodskii, A. V. & Cozzarelli,N. R. Simplification of DNA topology below equilibrium values by type II topoisomerases. Science 277, 690–693 (1997).

    Article  CAS  Google Scholar 

  7. Osheroff,N., Shelton,E. R. & Brutlag, D. L. DNA topoisomerase II from D. melanogaster: relaxation of supercoiled DNA. J. Biol. Chem. 258, 9536–9543 (1983).

    CAS  PubMed  Google Scholar 

  8. Uemura,T. & Yanagida,M. Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis. EMBO J. 5, 1003– 1010 (1986).

    Article  CAS  Google Scholar 

  9. Ishida,R. et al. Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events. J. Cell Biol. 126, 1341–1351 (1994).

    Article  CAS  Google Scholar 

  10. Strick,T. R., Allemand,J. F., Bensimon, D., Bensimon,A. & Croquette,V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835– 1837 (1996).

    Article  ADS  CAS  Google Scholar 

  11. Strick,T. R., Allemand,J. F., Bensimon, D. & Croquette,V. The behavior of super-coiled DNA. Biophys. J. 74, 2016–2028 (1998).

    Article  ADS  CAS  Google Scholar 

  12. Strick,T. R., Croquette,V. & Bensimon, D. Homologous pairing in stretched supercoiled DNA. Proc. Natl Acad. Sci. USA 95, 10579– 10583 (1998).

    Article  ADS  CAS  Google Scholar 

  13. Allemand,J. F., Bensimon,D., Lavery,R. & Croquette,V. Stretched and overwound DNA forms a Pauling-like structure with exposed bases. Proc. Natl Acad. Sci. USA 95, 14152– 14157 (1998).

    Article  ADS  CAS  Google Scholar 

  14. Landau,L. & Lifchitz,E. Theory of Elasticity (Mir Editions, Moscow, 1967).

    Google Scholar 

  15. Moroz,J. D. & Nelson,P. Torsional directed walks, entropic elasticity and DNA twist stiffness. Proc. Natl Acad. Sci. USA 94, 14418–14422 (1998).

    Article  ADS  Google Scholar 

  16. Bouchiat,C. & Mézard,M. Elasticity model of a supercoiled DNA molecule. Phys. Rev. Lett. 80, 1556– 1559 (1998).

    Article  ADS  CAS  Google Scholar 

  17. Brown,P. O. & Cozzarelli,N. R. A sign inversion mechanism for enzymatic supercoiling of DNA. Science 206, 1081–1083 (1979).

    Article  ADS  CAS  Google Scholar 

  18. Hua,W., Young,E. C., Fleming,M. L. & Gelles,J. Coupling of kinesin steps to ATP hydrolysis. Nature 388, 390–393 (1997).

    Article  ADS  CAS  Google Scholar 

  19. Harkins,T. T. & Lindsley,J. E. Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis. Biochemistry 37, 7292–7298 (1998).

    Article  CAS  Google Scholar 

  20. Harkins,T. T., Lewis,T. J. & Lindsley, J. E. Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 2. Kinetic mechanism for the sequential hydrolysis of two ATP. Biochemistry 37, 7299–7312 (1998).

    Article  CAS  Google Scholar 

  21. Wang,M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).

    Article  ADS  CAS  Google Scholar 

  22. Visscher,K., Schnitzer,M. J. & Block, S. M. Single kinesin molecules studied with a molecular force clamp. Nature 400, 184– 189 (1999).

    Article  ADS  CAS  Google Scholar 

  23. Zechiedrich,E. L. & Osheroff,N. Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers. EMBO J. 9, 4555–4562 (1990).

    Article  CAS  Google Scholar 

  24. Roca,J., Berger,J. M. & Wang,J. C. On the simultaneous binding of eukaryotic DNA toposiomerase II to a pair of double-stranded DNA helices. J. Biol. Chem. 268, 14250–14255 (1993).

    CAS  PubMed  Google Scholar 

  25. Froelich-Ammon,S. J. & Osheroff,N. Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 270, 21429–21432 ( 1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Maier and J.-F. Allemand for helpful comments and O. Hyrien, J.-L. Sikorav, M. Duguet, V. Rybenkov, N. Crisona and N. Cozzarelli for stimulating conversations. We also thank N. Cozzarelli for a gift of cloned topo II. T.R.S. was supported by a CNRS BDI fellowship.

Author information

Authors and Affiliations

  1. Laboratoire de Physique Statistique de l'Ecole Normale Superieure, UMR 8550 CNRS, Universities of Paris VI and Paris VII, 24 rue Lhomond, Paris, 75231 Cedex 05, France

    Terence R. Strick, Vincent Croquette & David Bensimon

Authors
  1. Terence R. Strick
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vincent Croquette
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Bensimon
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Strick, T., Croquette, V. & Bensimon, D. Single-molecule analysis of DNA uncoiling by a type II topoisomerase . Nature 404, 901–904 (2000). https://doi.org/10.1038/35009144

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35009144

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