Single-molecule imaging of DNA gyrase activity in living Escherichia coli.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
10 01 2019
10 01 2019
Historique:
received:
12
07
2018
accepted:
07
11
2018
pubmed:
18
11
2018
medline:
29
8
2019
entrez:
17
11
2018
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30445553
pii: 5184721
doi: 10.1093/nar/gky1143
pmc: PMC6326794
doi:
Substances chimiques
DNA, Superhelical
0
DNA-Binding Proteins
0
DNA Gyrase
EC 5.99.1.3
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
210-220Subventions
Organisme : Wellcome Trust
ID : 200782/Z/16/Z
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BBS/E/J/00000201
Pays : United Kingdom
Références
Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9820-5
pubmed: 12902541
Elife. 2017 Aug 25;6:
pubmed: 28841133
Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11347-51
pubmed: 20498085
Proc Natl Acad Sci U S A. 2013 May 14;110(20):8063-8
pubmed: 23630273
Nucleic Acids Res. 2017 Sep 19;45(16):9611-9624
pubmed: 28934496
Nat Struct Mol Biol. 2007 Apr;14(4):264-71
pubmed: 17334374
Nat Commun. 2014 Dec 18;5:5820
pubmed: 25520215
Nat Methods. 2014 Feb;11(2):156-62
pubmed: 24390439
J Bacteriol. 1988 Oct;170(10):4983-5
pubmed: 2844734
Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):E4565-70
pubmed: 26240319
Biochemistry. 1993 Mar 16;32(10):2717-24
pubmed: 8383523
Genes Dev. 2004 Jul 15;18(14):1766-79
pubmed: 15256503
Integr Biol (Camb). 2009 Oct;1(10):602-12
pubmed: 20023777
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6879-83
pubmed: 6256766
Cell. 2018 Feb 8;172(4):771-783.e18
pubmed: 29358050
Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8654-9
pubmed: 12857958
Nat Rev Mol Cell Biol. 2011 Nov 23;12(12):827-41
pubmed: 22108601
Genes Dev. 2008 Sep 1;22(17):2426-33
pubmed: 18765793
PLoS Genet. 2012;8(8):e1002845
pubmed: 22916023
Nat Commun. 2016 Mar 30;7:11055
pubmed: 27025941
mBio. 2014 Feb 11;5(1):e01001-13
pubmed: 24520061
Science. 2010 Apr 23;328(5977):498-501
pubmed: 20413500
Bacteriol Rev. 1972 Dec;36(4):525-57
pubmed: 4568763
FEBS Lett. 2003 Jan 30;535(1-3):55-60
pubmed: 12560078
Genes Dev. 2000 Nov 15;14(22):2881-92
pubmed: 11090135
Cell. 1983 Aug;34(1):105-13
pubmed: 6309403
EcoSal Plus. 2015;6(2):
pubmed: 26435256
Biophys J. 2002 Nov;83(5):2681-92
pubmed: 12414700
Mol Microbiol. 2011 May;80(3):612-27
pubmed: 21414037
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
pubmed: 10829079
Cell. 2014 Jul 17;158(2):314-326
pubmed: 25036631
Phys Rev Lett. 1996 Aug 26;77(9):1897-1900
pubmed: 10063199
Biochimie. 2007 Apr;89(4):490-9
pubmed: 17397985
Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872-6
pubmed: 186775
Appl Microbiol Biotechnol. 2011 Nov;92(3):479-97
pubmed: 21904817
J Bacteriol. 2006 Apr;188(8):3002-11
pubmed: 16585761
FEBS Lett. 2014 Oct 1;588(19):3585-94
pubmed: 24859634
Cell. 2010 Aug 20;142(4):519-30
pubmed: 20723754
Nat Methods. 2008 Feb;5(2):155-7
pubmed: 18193054
Crit Rev Biochem Mol Biol. 1991;26(3-4):335-75
pubmed: 1657531
Open Biol. 2012 Jun;2(6):120090
pubmed: 22773951
Science. 2012 Oct 26;338(6106):528-31
pubmed: 23112333
Faraday Discuss. 2015;184:401-24
pubmed: 26419209
J Biol Chem. 2000 Mar 17;275(11):8103-13
pubmed: 10713132
Methods. 2015 Oct 15;88:81-8
pubmed: 25637032
Nature. 2006 Sep 21;443(7109):355-8
pubmed: 16971952
Genome Biol. 2004;5(11):R87
pubmed: 15535863
J Mol Biol. 1979 Jun 25;131(2):287-302
pubmed: 226717
Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15376-81
pubmed: 18832162
Cell Rep. 2015 Dec 22;13(11):2587-2596
pubmed: 26686641
Elife. 2017 Mar 31;6:
pubmed: 28362256
Cell. 2008 Apr 4;133(1):90-102
pubmed: 18394992
Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):E4390-9
pubmed: 26224838
Biophys Rev. 2016 Nov;8(Suppl 1):89-100
pubmed: 28510216
Nucleic Acids Res. 1981 Aug 11;9(15):3589-603
pubmed: 6269086
Microbiology. 1994 Sep;140 ( Pt 9):2371-82
pubmed: 7952188
Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024-7
pubmed: 2823250
PLoS Genet. 2013;9(8):e1003673
pubmed: 23990792
Proc Natl Acad Sci U S A. 1978 Apr;75(4):1773-7
pubmed: 347446
Front Immunol. 2018 May 22;9:1073
pubmed: 29872430
J Biol Chem. 1984 Dec 10;259(23):14472-80
pubmed: 6094559