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Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque

Nature Structural & Molecular Biology volume 14, pages 264–271 (2007)Cite this article

Abstract

E. coli DNA gyrase uses the energy of ATP hydrolysis to introduce essential negative supercoils into the genome, thereby working against the mechanical stresses that accumulate in supercoiled DNA. Using a magnetic-tweezers assay, we demonstrate that small changes in force and torque can switch gyrase among three distinct modes of activity. Under low mechanical stress, gyrase introduces negative supercoils by a mechanism that depends on DNA wrapping. Elevated tension or positive torque suppresses DNA wrapping, revealing a second mode of activity that resembles the activity of topoisomerase IV. This 'distal T-segment capture' mode results in active relaxation of left-handed braids and positive supercoils. A third mode is responsible for the ATP-independent relaxation of negative supercoils. We present a branched kinetic model that quantitatively accounts for all of our single-molecule results and agrees with existing biochemical data.

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Figure 1: Gyrase subunit composition and mechanism of action.
Figure 2: Magnetic tweezers experimental setup.
Figure 3: Activities of gyrase at low forces.
Figure 4: Activity of gyrase at high DNA tensions.
Figure 5: Passive mode of relaxation by gyrase.
Figure 6: Gyrase unbraids DNA.
Figure 7: Force-velocity curves and proposed mechanochemical model.

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Acknowledgements

The authors would like to dedicate this work to our friend and colleague Nicholas Cozzarelli, who passed away during completion of this research. We thank A. Schoeffler and J. Berger (University of California, Berkeley) for the gift of enzyme and P. Higgins (University of Alabama at Birmingham) for plasmids. This work was supported by US National Institutes of Health Grants GM31655 (to N.R.C.) and GM32543 (to C.B.), the Human Frontiers Science Organization through a long-term fellowship (to M.N.), the Program in Mathematics and Molecular Biology from the Burroughs Wellcome Foundation (M.N.), US Department of Energy Grant KP1102-DE-AC0376SF00098 (to C.B.), and the Fannie and John Hertz Foundation (J.G.).

Author information

Author notes

  1. Michael D Stone, Zev Bryant, Jeff Gore & Seok-Cheol Hong

    Present address: Present addresses: Department of Bioengineering, Stanford University, Stanford, California 94305, USA (Z.B.), Department of Physics, Korea University, Seoul, South Korea (S.C.H.), Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA (M.D.S.), and Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (J.G.).,

  2. Nicholas R Cozzarelli: Deceased.

  3. Marcelo Nöllmann, Michael D Stone, Zev Bryant and Jeff Gore: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Marcelo Nöllmann, Michael D Stone, Zev Bryant, Nancy J Crisona, Seok-Cheol Hong, Carlos Bustamante & Nicholas R Cozzarelli

  2. Deparment of Physics, University of California, Berkeley, 94720, California, USA

    Jeff Gore, Seok-Cheol Hong & Carlos Bustamante

  3. John Innes Centre, Norwich, NR4 7UH, UK

    Sylvain Mitelheiser & Anthony Maxwell

  4. Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Zev Bryant & Carlos Bustamante

  5. Howard Hughes Medical Institute, USA

    Carlos Bustamante

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Contributions

M.N., M.D.S, Z.B. and J.G collected and interpreted single-molecule data, supervised by C.B. and N.R.C. N.J.C. collected and analyzed ensemble data. S.-C.H. helped with the construction of single-molecule instruments. S.M. and A.M. contributed ideas and reagents. M.N., M.D.S., Z.B., J.G., N.J.C., C.B. and N.R.C. wrote the paper.

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Correspondence to Carlos Bustamante.

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Supplementary information

Supplementary Fig. 1

Gyrase activity on (+) and (−) supercoiled substrates in bulk. (PDF 319 kb)

Supplementary Fig. 2

The rate of (+) supercoil relaxation at high forces is force-independent. (PDF 44 kb)

Supplementary Fig. 3

The passive mode requires the presence of (−) supercoils. (PDF 31 kb)

Supplementary Data

Derivation of kinetic equations. (PDF 78 kb)

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Nöllmann, M., Stone, M., Bryant, Z. et al. Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat Struct Mol Biol 14, 264–271 (2007). https://doi.org/10.1038/nsmb1213

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