Single-molecule imaging of DNA gyrase activity in living Escherichia coli

Nucleic Acids Research
Mathew StracyPawel Zawadzki

Abstract

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

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Citations

Jul 6, 2019·Nucleic Acids Research·Jorge B SchvartzmanAndrzej Stasiak
Jan 14, 2020·Nucleic Acids Research·Jack W ShepherdMark C Leake
May 30, 2020·PLoS Computational Biology·Martín Carballo-PachecoBartlomiej Waclaw
Sep 20, 2020·Nature Communications·Rohit Agarwal, Karl E Duderstadt
Jul 24, 2020·Nature Communications·Jason CosgroveMark C Coles
Nov 11, 2019·The Journal of Chemical Physics·Haw YangTao Liu
Jul 20, 2019·BMC Molecular and Cell Biology·Charles J Dorman
Jul 1, 2020·International Journal of Molecular Sciences·Syed Moiz Ahmed, Peter Dröge
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Jan 23, 2021·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Shannon J McKieAnthony Maxwell
Mar 7, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Dagmar Klostermeier
Apr 28, 2021·Nucleic Acids Research·Jana Hirsch, Dagmar Klostermeier
Jun 26, 2021·Methods : a Companion to Methods in Enzymology·Mark C Leake

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Methods Mentioned

BETA
electrophoresis
electron microscopy
fluorescence microscopy

Software Mentioned

MicrobeTracker
spotFinder
MATLAB

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