Stracy, Mathew, Wollman, Adam orcid.org/0000-0002-5501-8131, Kaja, Elzbieta et al. (9 more authors) (2019) Single-molecule imaging of DNA gyrase activity in living Escherichia coli. Nucleic Acids Research. pp. 210-220. ISSN 0305-1048
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2018. |
Keywords: | Catalysis,DNA Gyrase/chemistry,DNA, Superhelical/chemistry,DNA-Binding Proteins/chemistry,Escherichia coli/enzymology,Protein Binding,Single Molecule Imaging |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Physics (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 29 Oct 2018 12:40 |
Last Modified: | 22 Jan 2025 00:11 |
Published Version: | https://doi.org/10.1093/nar/gky1143 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gky1143 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:137893 |
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