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

TY - JOUR

T1 - Single-molecule imaging of DNA gyrase activity in living Escherichia coli

AU - Stracy, Mathew

AU - Wollman, Adam

AU - Kaja, Elzbieta

AU - Gapinski, Jacek

AU - Lee, Ji-Eun

AU - Leek, Victoria A.

AU - McKie, Shannon J.

AU - Mitchenall, L. A.

AU - Maxwell, Anthony

AU - Sherratt, David J

AU - Leake, Mark Christian

AU - Zawadzki, Pawel

N1 - © The Author(s) 2018.

PY - 2019/1/10

Y1 - 2019/1/10

N2 - 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.

AB - 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.

KW - Catalysis

KW - DNA Gyrase/chemistry

KW - DNA, Superhelical/chemistry

KW - DNA-Binding Proteins/chemistry

KW - Escherichia coli/enzymology

KW - Protein Binding

KW - Single Molecule Imaging

UR - http://www.scopus.com/inward/record.url?scp=85059796420&partnerID=8YFLogxK

U2 - 10.1093/nar/gky1143

DO - 10.1093/nar/gky1143

M3 - Article

C2 - 30445553

SN - 0305-1048

VL - 47

SP - 210

EP - 220

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 1

ER -