Toggle Main Menu Toggle Search

Open Access padlockePrints

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

Lookup NU author(s): Dr Adam WollmanORCiD

Downloads


Licence

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).


Abstract

© 2018 The Author(s). 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.


Publication metadata

Author(s): Stracy M, Wollman AJM, Kaja E, Gapinski J, Lee J-E, Leek VA, McKie SJ, Mitchenall LA, Maxwell A, Sherratt DJ, Leake MC, Zawadzki P

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2019

Volume: 47

Issue: 1

Pages: 210-220

Print publication date: 10/01/2019

Online publication date: 16/11/2018

Acceptance date: 07/11/2018

Date deposited: 10/02/2020

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: https://doi.org/10.1093/nar/gky1143

DOI: 10.1093/nar/gky1143

PubMed id: 30445553


Altmetrics

Altmetrics provided by Altmetric


Funding

Funder referenceFunder name
099204/Z/12Z
204684/Z/16/Z
2015/19/P/NZ1/03859
204829
BB/R001235/1
BB/J004561/1
BB/N006453/1
BB/P012523/1
First TEAM/2016-1/9
MwR/K01580X/1
RP2013-K-017

Share