Nature 439, 100-104 (5 January 2006) | doi:10.1038/nature04319; Received 13 June 2005; Accepted 11 October 2005

Mechanochemical analysis of DNA gyrase using rotor bead tracking

Jeff Gore1,6, Zev Bryant2,3,6,5, Michael D. Stone2,6,5, Marcelo Nöllmann2, Nicholas R. Cozzarelli2 and Carlos Bustamante1,2,3,4

DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA1, 2, 3. The directionality of supercoiling is ensured by chiral wrapping of the DNA4, 5 around a specialized domain6, 7, 8, 9 of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule10. In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two11. Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.

  1. Department of Physics, University of California, Berkeley, California 94720, USA
  2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
  3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
  4. Howard Hughes Medical Institute, USA
  5. †Present addresses: Department of Biochemistry, Stanford University, Stanford, California 94305, USA (Z.B.); Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA (M.D.S.)
  6. *These authors contributed equally to this work

Correspondence to: Nicholas R. Cozzarelli2Carlos Bustamante1,2,3,4 Correspondence and requests for materials should be addressed to C.B. (Email: carlos@alice.berkeley.edu) or N.R.C. (Email: ncozzare@berkeley.edu).

Received 13 June 2005 | Accepted 11 October 2005


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