Publications by authors named "Lesley A Mitchenall"

25 Publications

  • Page 1 of 1

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

Nat Commun 2021 02 16;12(1):1053. Epub 2021 Feb 16.

School of Physics and Astronomy, University of Leeds, Leeds, UK.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.
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http://dx.doi.org/10.1038/s41467-021-21243-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887228PMC
February 2021

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

J Antimicrob Chemother 2020 10;75(10):2835-2842

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Objectives: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches.

Methods: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863.

Results: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive.

Conclusions: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.
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http://dx.doi.org/10.1093/jac/dkaa286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556816PMC
October 2020

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

Nucleic Acids Res 2019 01;47(1):210-220

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

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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http://dx.doi.org/10.1093/nar/gky1143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326794PMC
January 2019

The EU approved antimalarial pyronaridine shows antitubercular activity and synergy with rifampicin, targeting RNA polymerase.

Tuberculosis (Edinb) 2018 09 11;112:98-109. Epub 2018 Aug 11.

Collaborations Pharmaceuticals, Inc., 840 Main Campus Drive, Lab 3510, Raleigh, NC 27606, USA; Collaborative Drug Discovery, 1633 Bayshore Highway, Suite 342, Burlingame, CA 94403, USA. Electronic address:

The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5 μg/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of C-uracil and C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5 μg/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009 μg/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
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http://dx.doi.org/10.1016/j.tube.2018.08.004DOI Listing
September 2018

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress.

Mol Microbiol 2018 03 5;107(6):734-746. Epub 2018 Feb 5.

Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland.

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F F ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (Nov ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.
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http://dx.doi.org/10.1111/mmi.13911DOI Listing
March 2018

A rapid high-resolution method for resolving DNA topoisomers.

BMC Res Notes 2018 Jan 16;11(1):37. Epub 2018 Jan 16.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Objective: Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis.

Results: We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.
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http://dx.doi.org/10.1186/s13104-018-3147-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771066PMC
January 2018

Quinolone-resistant gyrase mutants demonstrate decreased susceptibility to triclosan.

J Antimicrob Chemother 2017 10;72(10):2755-2763

Institute of Microbiology & Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B152TT, UK.

Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance.

Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined.

Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance.

Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials.
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http://dx.doi.org/10.1093/jac/dkx201DOI Listing
October 2017

DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana.

J Biol Chem 2016 Feb 9;291(7):3136-44. Epub 2015 Dec 9.

From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom,

The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.
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http://dx.doi.org/10.1074/jbc.M115.689554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751362PMC
February 2016

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases.

Sci Rep 2014 Aug 21;4:6158. Epub 2014 Aug 21.

Department of Biological Chemistry, John Innes Centre Norwich Research Park, Norwich NR4 7UH, United Kingdom.

DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the 'exit gate' may be an important determinant of this process.
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http://dx.doi.org/10.1038/srep06158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139952PMC
August 2014

Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI.

PLoS One 2013 26;8(2):e58010. Epub 2013 Feb 26.

Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, United Kingdom.

DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058010PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3582512PMC
August 2013

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

J Biol Chem 2013 Feb 28;288(7):5149-56. Epub 2012 Dec 28.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.
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http://dx.doi.org/10.1074/jbc.M112.419069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576119PMC
February 2013

A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

PLoS One 2012 28;7(9):e46499. Epub 2012 Sep 28.

Departament of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, United Kingdom.

Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046499PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896PMC
February 2013

The role of Ca²⁺ in the activity of Mycobacterium tuberculosis DNA gyrase.

Nucleic Acids Res 2012 Oct 27;40(19):9774-87. Epub 2012 Jul 27.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.
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http://dx.doi.org/10.1093/nar/gks704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479174PMC
October 2012

The dimer state of GyrB is an active form: implications for the initial complex assembly and processive strand passage.

Nucleic Acids Res 2011 Oct 10;39(19):8488-502. Epub 2011 Jul 10.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

In a previous study, we presented the dimer structure of DNA gyrase B' domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a 'sluice-like' model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B' protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA-GyrB-DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.
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http://dx.doi.org/10.1093/nar/gkr553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201873PMC
October 2011

Inhibition of DNA gyrase and DNA topoisomerase IV of Staphylococcus aureus and Escherichia coli by aminocoumarin antibiotics.

J Antimicrob Chemother 2011 Sep 21;66(9):2061-9. Epub 2011 Jun 21.

Pharmaceutical Biology, Pharmaceutical Institute, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.

Objectives: Aminocoumarin antibiotics are potent inhibitors of bacterial DNA gyrase. We investigated the inhibitory and antibacterial activity of naturally occurring aminocoumarin antibiotics and six structural analogues (novclobiocins) against DNA gyrase and DNA topoisomerase IV from Escherichia coli and Staphylococcus aureus as well as the effect of potassium and sodium glutamate on the activity of these enzymes.

Methods: The inhibitory concentrations of the aminocoumarins were determined in gyrase supercoiling assays and topoisomerase IV decatenation assays. Both subunits of S. aureus topoisomerase IV were purified as His-Tag proteins in E. coli. The MIC was tested in vivo for the control organisms E. coli ATCC 25922 and S. aureus ATCC 29213.

Results: DNA gyrase is the primary target in vitro of all investigated aminocoumarins. With the exception of simocyclinone D8, all other aminocoumarins inhibited S. aureus gyrase on average 6-fold more effectively than E. coli gyrase. Potassium glutamate is essential for the activity of S. aureus gyrase and increases the sensitivity of E. coli gyrase to aminocoumarins ≥ 10-fold. The antibacterial activity of the tested compounds mirrored their relative activities against topoisomerases.

Conclusions: The study provides insights about the substituents that are important for the inhibitory activity of aminocoumarins against the target enzymes, which will facilitate the rational design of improved antibiotics.
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http://dx.doi.org/10.1093/jac/dkr247DOI Listing
September 2011

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV.

Nucleic Acids Res 2011 Jun 7;39(11):4808-17. Epub 2011 Feb 7.

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.
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http://dx.doi.org/10.1093/nar/gkr018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113566PMC
June 2011

Vibrio cholerae ParE2 poisons DNA gyrase via a mechanism distinct from other gyrase inhibitors.

J Biol Chem 2010 Dec 15;285(51):40397-408. Epub 2010 Oct 15.

Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Tufts University School of Medicine, Boston, Massachusetts 02115, USA.

DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.
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http://dx.doi.org/10.1074/jbc.M110.138776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001019PMC
December 2010

Structural and biochemical analysis of the pentapeptide repeat protein EfsQnr, a potent DNA gyrase inhibitor.

Antimicrob Agents Chemother 2011 Jan 11;55(1):110-7. Epub 2010 Oct 11.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC(50)) of 1.2 μM, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.
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http://dx.doi.org/10.1128/AAC.01158-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019687PMC
January 2011

A crystal structure of the bifunctional antibiotic simocyclinone D8, bound to DNA gyrase.

Science 2009 Dec;326(5958):1415-8

Department of Biological Chemistry, John Innes Centre, Colney, Norwich NR4 7UH, UK.

Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets.
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http://dx.doi.org/10.1126/science.1179123DOI Listing
December 2009

Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage-reunion domain of Escherichia coli DNA gyrase.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2009 Aug 30;65(Pt 8):846-8. Epub 2009 Jul 30.

Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, England.

Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage-reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 A from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development.
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http://dx.doi.org/10.1107/S1744309109028097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720350PMC
August 2009

How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisomerases.

J Mol Biol 2009 Feb 7;385(5):1397-408. Epub 2008 Dec 7.

Department of Biological Chemistry, John Innes Centre, Colney, Norwich, UK.

DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range approximately 2-9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.
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http://dx.doi.org/10.1016/j.jmb.2008.11.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343537PMC
February 2009

DNA gyrase requires DNA for effective two-site coordination of divalent metal ions: further insight into the mechanism of enzyme action.

Biochemistry 2008 Aug 22;47(33):8538-45. Epub 2008 Jul 22.

Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy.

The catalytic properties of DNA gyrase, an A 2B 2 complex, are modulated by the presence of divalent metal ions. Using circular dichroism, protein melting experiments and enzyme activity assays, we investigated the correlation between the A 2B 2 conformation, the nature of the metal ion cofactor and the enzyme activity in the presence and absence of DNA substrate. At room temperature, DNA gyrase structure is not appreciably affected by Ca (2+) or Mg (2+) but is modified by Mn (2+). In addition, metal ions strongly affect the enzyme's thermal transitions, rendering the A 2B 2 structure more flexible. Using the B subunit, we were able to identify two distinct complexes with manganese ions. The first one exhibits a 1:1 stoichiometry and is not affected by the presence of DNA. The second complex is associated with a large protein structural modification that can be remarkably modulated by addition of the DNA substrate. This behavior is conserved in the reconstituted protein. Studies with two GyrB mutants indicate that Mn (2+) interference with the TOPRIM region modulates gyrase supercoiling activity. In particular, considering the need for two divalent metal ions for an efficient catalytic cleavage of the phosphodiester bond, our data suggest that residue D500 participates in the first complexation event (DNA-independent), whereas residue D498 is involved mainly in the second process. In conclusion, a combination of the ion features (ionic size, electronegativity, coordination sphere) operating at the level of the catalytic region and of the ion-driven modifications in overall enzyme structure and flexibility contribute to the mechanism of gyrase activity. An effectual role for DNA recruiting the second catalytic metal ion is envisaged.
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http://dx.doi.org/10.1021/bi800480jDOI Listing
August 2008

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases.

Nucleic Acids Res 2007 30;35(18):6075-85. Epub 2007 Aug 30.

Department of Pharmaceutical Sciences, University of Padova, 35131 Padova, Italy.

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase-quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions -2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme-drug complex.
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http://dx.doi.org/10.1093/nar/gkm653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094056PMC
November 2007

A fluoroquinolone resistance protein from Mycobacterium tuberculosis that mimics DNA.

Science 2005 Jun;308(5727):1480-3

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Fluoroquinolones are gaining increasing importance in the treatment of tuberculosis. The expression of MfpA, a member of the pentapeptide repeat family of proteins from Mycobacterium tuberculosis, causes resistance to ciprofloxacin and sparfloxacin. This protein binds to DNA gyrase and inhibits its activity. Its three-dimensional structure reveals a fold, which we have named the right-handed quadrilateral beta helix, that exhibits size, shape, and electrostatic similarity to B-form DNA. This represents a form of DNA mimicry and explains both its inhibitory effect on DNA gyrase and fluoroquinolone resistance resulting from the protein's expression in vivo.
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http://dx.doi.org/10.1126/science.1110699DOI Listing
June 2005

Arabidopsis thaliana DNA gyrase is targeted to chloroplasts and mitochondria.

Proc Natl Acad Sci U S A 2004 May 10;101(20):7821-6. Epub 2004 May 10.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

DNA gyrase is the bacterial DNA topoisomerase (topo) that supercoils DNA by using the free energy of ATP hydrolysis. The enzyme, an A(2)B(2) tetramer encoded by the gyrA and gyrB genes, catalyses topological changes in DNA during replication and transcription, and is the only topo that is able to introduce negative supercoils. Gyrase is essential in bacteria and apparently absent from eukaryotes and is, consequently, an important target for antibacterial agents (e.g., quinolones and coumarins). We have identified four putative gyrase genes in the model plant Arabidopsis thaliana; one gyrA and three gyrB homologues. DNA gyrase protein A (GyrA) has a dual translational initiation site targeting the mature protein to both chloroplasts and mitochondria, and there are individual targeting sequences for two of the DNA gyrase protein B (GyrB) homologues. N-terminal fusions of the organellar targeting sequences to GFPs support the hypothesis that one enzyme is targeted to the chloroplast and another to the mitochondrion, which correlates with supercoiling activity in isolated organelles. Treatment of seedlings and cultured cells with gyrase-specific drugs leads to growth inhibition. Knockout of A. thaliana gyrA is embryo-lethal whereas knockouts in the gyrB genes lead to seedling-lethal phenotypes or severely stunted growth and development. The A. thaliana genes have been cloned in Escherichia coli and found to complement gyrase temperature-sensitive strains. This report confirms the existence of DNA gyrase in eukaryotes and has important implications for drug targeting, organelle replication, and the evolution of topos in plants.
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http://dx.doi.org/10.1073/pnas.0400836101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419690PMC
May 2004