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Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases

Arnoldi, E; Pan, XS; Fisher, LM (2013) Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases. NUCLEIC ACIDS RESEARCH, 41 (20). 9411 - 9423 (13). ISSN 0305-1048 https://doi.org/10.1093/nar/gkt696
SGUL Authors: Fisher, Larry Mark Pan, Xiao Su

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Abstract

Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca2+-induced cleavage distinguished ‘intrinsic recognition’ of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for −4G, −2A and −1T bases preceding the breakage site (between −1 and +1) and enzyme-unique or degenerate determinants at −3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advanc

Item Type: Article
Additional Information: Copyright © The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Anti-Bacterial Agents, DNA Cleavage, DNA Gyrase, DNA Topoisomerase IV, DNA, Bacterial, Fluoroquinolones, Naphthyridines, Plasmids, Streptococcus pneumoniae, Topoisomerase II Inhibitors, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, BIOCHEMISTRY & MOLECULAR BIOLOGY, DOUBLE-STRAND BREAKS, DIVALENT METAL-IONS, STREPTOCOCCUS-PNEUMONIAE, ESCHERICHIA-COLI, STRUCTURAL BASIS, WILD-TYPE, GYRASE, IV, SITE, MECHANISM
SGUL Research Institute / Research Centre: Academic Structure > Molecular and Clinical Sciences Research Institute (MCS)
Academic Structure > Molecular and Clinical Sciences Research Institute (MCS) > Cell Sciences (INCCCS)
Journal or Publication Title: NUCLEIC ACIDS RESEARCH
ISSN: 0305-1048
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Dates:
DateEvent
1 November 2013Published
PubMed ID: 23939623
Web of Science ID: 23939623
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URI: https://openaccess.sgul.ac.uk/id/eprint/103578
Publisher's version: https://doi.org/10.1093/nar/gkt696

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