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Mechanochemical analysis of DNA gyrase using rotor bead tracking


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.

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Figure 1: Experimental design and single-molecule observations of gyrase activity.
Figure 2: Modulation of gyrase activity by DNA tension.
Figure 3: Proposed mechanochemical model.
Figure 4: Gyrase activity observed at high resolution.


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We thank N. Crisona, P. Arimondo, A. Vologodskii, A. Edelstein, S. Mitelheiser, A. Schoeffler, and F. Mueller-Planitz for discussions; A. Maxwell and J. Berger for enzymes; P. Higgins for plasmids; and C. Hodges, M. Le and D. Jennings for technical assistance. J.G. acknowledges funding from the Hertz Foundation. This work was supported by the NIH and the DOE.

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Correspondence to Nicholas R. Cozzarelli or Carlos Bustamante.

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Supplementary information

Supplementary Notes

A description of the function used to fit processivity and initiation rate data and a discussion of the applicability of the simple mechanochemical model from which this function is derived. (DOC 18 kb)

Supplementary Figure

A histogram of pause durations in high resolution bursts. The data support a model in which the rate-limiting step lies at the end of the reaction cycle. (DOC 88 kb)

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Gore, J., Bryant, Z., Stone, M. et al. Mechanochemical analysis of DNA gyrase using rotor bead tracking. Nature 439, 100–104 (2006).

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