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Dynamic coupling between conformations and nucleotide states in DNA gyrase

Abstract

Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.

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Fig. 1: Gyrase mechanochemical cycle and single-molecule rotor bead tracking assay.
Fig. 2: Conformations of the DNA:gyrase complex probed using RBT.
Fig. 3: Structural dynamics of DNA gyrase associated with ADP/ATP exchange.
Fig. 4: Structural dynamics of DNA gyrase associated with ATP hydrolysis.
Fig. 5: Structural dynamics of DNA gyrase affected by tension, and [ATP]-dependent analysis of the ν state.

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Acknowledgements

This work was supported by National Institutes of Health R01 grants GM106159 to Z.B. and CA077373 to J.M.B., a Stanford Bio-X Graduate Fellowship to A.B. and a Stanford Interdisciplinary Graduate Fellowship to P.M.L. NIH training grant 5T32GM008403-24 supported M.H.

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A.B. designed and performed single-molecule experiments, analyzed single-molecule data and carried out theoretical modeling. M.H. designed and performed bulk biochemical experiments. A.B., P.L. and L.E.F. developed and characterized single-molecule tools and methods. M.H. and E.M.T. provided purified proteins. A.B., Z.B. and M.H. wrote the paper. All authors commented on the paper and discussed the results. Z.B. and J.M.B. supervised research.

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Correspondence to Zev Bryant.

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Basu, A., Hobson, M., Lebel, P. et al. Dynamic coupling between conformations and nucleotide states in DNA gyrase. Nat Chem Biol 14, 565–574 (2018). https://doi.org/10.1038/s41589-018-0037-0

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