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

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          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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          Most cited references53

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          High-density mapping of single-molecule trajectories with photoactivated localization microscopy.

          We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
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            Supercoiling of the DNA template during transcription.

            L Liu, J. Wang (1987)
            Transcription of a right-handed double-helical DNA requires a relative rotation of the RNA polymerase and its nascent RNA around the DNA. We describe conditions under which the resistance to the rotational motion of the transcription ensemble around the DNA can be large. In such cases, the advancing polymerase generates positive supercoils in the DNA template ahead of it and negative supercoils behind it. Mutual annihilation of the positively and negatively supercoiled regions may be prevented by anchoring points on the DNA to a large structure, or, in the case of an unanchored plasmid, by the presence of two oppositely oriented transcription units. In prokaryotes, DNA topoisomerase I preferentially removes negative supercoils and DNA gyrase (topoisomerase II) removes positive ones. Our model thus provides an explanation for the experimentally observed high degree of negative or positive supercoiling of intracellular pBR322 DNA when DNA topoisomerase I or gyrase is respectively inhibited. We discuss the implications of our model in terms of supercoiling regulation, DNA conformational transitions, and gene regulation in both prokaryotes and eukaryotes.
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              High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.

              Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division. © 2011 Blackwell Publishing Ltd.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                10 January 2019
                16 November 2018
                16 November 2018
                : 47
                : 1
                : 210-220
                Affiliations
                [1 ]Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
                [2 ]Biological Physical Sciences Institute (BPSI), Departments of Physics and Biology, University of York, York YO10 5DD, UK
                [3 ]Molecular Biophysics Division, Faculty of Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
                [4 ]Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
                [5 ]NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
                Author notes
                To whom correspondence should be addressed. Email: zawadzki@ 123456amu.edu.pl
                Correspondence may also be addressed to Mark C. Leake. Email: mark.leake@ 123456york.ac.uk

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Author information
                http://orcid.org/0000-0002-5756-6430
                http://orcid.org/0000-0002-1715-1249
                http://orcid.org/0000-0002-9032-2315
                Article
                gky1143
                10.1093/nar/gky1143
                6326794
                30445553
                5f0d075e-031a-42de-9cc6-382b21e8d3bf
                © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 07 November 2018
                : 11 October 2018
                : 12 July 2018
                Page count
                Pages: 11
                Funding
                Funded by: National Science Centre Poland 10.13039/501100004281
                Award ID: 2015/19/P/NZ1/03859
                Funded by: Foundation for Polish Science 10.13039/501100001870
                Award ID: TEAM/2016-1/9
                Funded by: Medical Research Council 10.13039/501100000265
                Award ID: MwR/K01580X/1
                Funded by: Biotechnology and Biological Sciences Research Council 10.13039/501100000268
                Award ID: BB/N006453/1
                Award ID: BB/R001235/1
                Award ID: BB/J004561/1
                Award ID: BB/P012523/1
                Funded by: University of York 10.13039/100009001
                Award ID: 204829
                Funded by: Wellcome Trust 10.13039/100009001
                Award ID: 099204/Z/12Z
                Funded by: Sir Henry Wellcome
                Award ID: 204684/Z/16/Z
                Funded by: Leverhulme Trust 10.13039/501100000275
                Award ID: RP2013-K-017
                Categories
                Genome Integrity, Repair and Replication

                Genetics
                Genetics

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