Abstract
DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. A critical step in this reaction is the formation of a chiral DNA wrap. Here we observe gyrase structural dynamics using a single-molecule assay in which gyrase drives the processive, stepwise rotation of a nanosphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and measurements of DNA contraction reveal multiple ATP-modulated structural transitions. DNA wrapping is coordinated with the ATPase cycle and proceeds by way of an unanticipated structural intermediate that dominates the kinetics of supercoiling. Our findings reveal a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction, a feature that may be common to the reaction cycles of other DNA and protein remodeling machines.
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Acknowledgements
We would like to thank J. Rubin and M. Yi for assistance with early assay development, and A. Spakowitz, L. Koslover, A. Vologodskii, A. Maxwell, A. Bates, J. Puglisi, D. Chowdhury, and members of the Bryant and Berger labs for helpful discussions and critical comments on the manuscript. This work was supported by a Pew Scholars Award and US National Institutes of Health (NIH) grant DP2 OD004690 to Z.B., by NIH grant CA077373 to J.M.B., and by a Stanford Bio-X Graduate Fellowship to A.B.
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A.B. and Z.B. designed the single-molecule experiments and developed the mechanochemical model. A.B. established the high-resolution assay, conducted the experiments and analyzed the data. A.J.S. and J.M.B. provided purified DNA gyrase subunits and gyrase expertise. A.B. and Z.B. wrote the paper in consultation with J.M.B. All authors discussed the results and commented on the manuscript.
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Basu, A., Schoeffler, A., Berger, J. et al. ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA. Nat Struct Mol Biol 19, 538–546 (2012). https://doi.org/10.1038/nsmb.2278
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DOI: https://doi.org/10.1038/nsmb.2278
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