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

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JournalNucleic Acids Research
DateAccepted/In press - 7 Nov 2018
DateE-pub ahead of print - 16 Nov 2018
DatePublished (current) - 10 Jan 2019
Issue number1
Volume47
Number of pages11
Pages (from-to)210-220
Early online date16/11/18
Original languageEnglish

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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© The Author(s) 2018.

    Research areas

  • Catalysis, DNA Gyrase/chemistry, DNA, Superhelical/chemistry, DNA-Binding Proteins/chemistry, Escherichia coli/enzymology, Protein Binding, Single Molecule Imaging

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