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1
Kaufmann, S. H., and R. Hancock. "Topoisomerase II as a target for anticancer chemotherapy." Acta Biochimica Polonica 42, no. 4 (December 31, 1995): 381–93. http://dx.doi.org/10.18388/abp.1995_4892.
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Type II DNA topoisomerases are required for the segregation of genomic DNA at cell division in prokaryotic and eukaryotic cells, and inhibitors of these enzymes are potential cytotoxic agents in both prokaryotes and eukaryotes. The bacterial member of the topoisomerase II family, DNA gyrase, and the chemotherapeutic agents which target it are the subject of a recent review (Maxwell, A. et al., 1993, in Molecular Biology of DNA Topoisomerases, Andoh, T. et al., eds., pp. 21-30, CRC Press, Boca Raton). Here we present an overview of current knowledge of eukaryotic topoisomerase II and the anticancer agents which target this enzyme, focussing predominantly on new observations and recent reports and reviews.
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Takei, Masaya, Hideyuki Fukuda, Tokutaro Yasue, Masaki Hosaka, and Yasuo Oomori. "Inhibitory Activities of Gatifloxacin (AM-1155), a Newly Developed Fluoroquinolone, against Bacterial and Mammalian Type II Topoisomerases." Antimicrobial Agents and Chemotherapy 42, no. 10 (October 1, 1998): 2678–81. http://dx.doi.org/10.1128/aac.42.10.2678.
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ABSTRACT We determined the inhibitory activities of gatifloxacin againstStaphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 forS. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 μg/ml for S. aureustopoisomerase IV; IC50 = 0.109 μg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 μg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureustopoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.
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Austin, Caroline, Ka Lee, Rebecca Swan, Mushtaq Khazeem, Catriona Manville, Peter Cridland, Achim Treumann, Andrew Porter, Nick Morris, and Ian Cowell. "TOP2B: The First Thirty Years." International Journal of Molecular Sciences 19, no. 9 (September 14, 2018): 2765. http://dx.doi.org/10.3390/ijms19092765.
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Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and β. Topoisomerase IIβ was first reported in 1987. Here we review the research on DNA topoisomerase IIβ over the 30 years since its discovery.
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Gruger, Thomas, John L. Nitiss, Anthony Maxwell, E. Lynn Zechiedrich, Peter Heisig, Siegfried Seeber, Yves Pommier, and Dirk Strumberg. "A Mutation in Escherichia coli DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II." Antimicrobial Agents and Chemotherapy 48, no. 12 (December 2004): 4495–504. http://dx.doi.org/10.1128/aac.48.12.4495-4504.2004.
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ABSTRACT Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (GyrS83W) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of GyrS83W, and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for GyrS83W-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the GyrS83W mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.
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Blower, Tim R., Afif Bandak, Amy S. Y. Lee, Caroline A. Austin, John L. Nitiss, and James M. Berger. "A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents." Nucleic Acids Research 47, no. 15 (July 9, 2019): 8163–79. http://dx.doi.org/10.1093/nar/gkz579.
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AbstractType II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational ‘landscape’ of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2β) is additionally revealed to be significantly regulated by the enzymes’ adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.
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Strumberg, Dirk, John L. Nitiss, Jiaowang Dong, Jerrylaine Walker, Marc C. Nicklaus, Kurt W. Kohn, Jonathan G. Heddle, Anthony Maxwell, Siegfried Seeber, and Yves Pommier. "Importance of the Fourth Alpha-Helix within the CAP Homology Domain of Type II Topoisomerase for DNA Cleavage Site Recognition and Quinolone Action." Antimicrobial Agents and Chemotherapy 46, no. 9 (September 2002): 2735–46. http://dx.doi.org/10.1128/aac.46.9.2735-2746.2002.
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ABSTRACT We report that point mutations causing alteration of the fourth alpha-helix (α4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser740Trp, Gln743Pro, and Thr744Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr744Ala substitution had minimal effect on Ca2+- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the α4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser83Trp also changed the DNA cleavage pattern generated by Ca2+ or quinolones. Finally, Thr744Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the α4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the α4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.
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Yi, Lanhua, and Xin Lü. "New Strategy on Antimicrobial-resistance: Inhibitors of DNA Replication Enzymes." Current Medicinal Chemistry 26, no. 10 (June 20, 2019): 1761–87. http://dx.doi.org/10.2174/0929867324666171106160326.
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Background:Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms are effective weapons to fight against antibiotic-resistance.Objective:This review aims to find potential drugs which can be further developed into clinic practice and provide clues for developing more effective antimicrobials.Methods:DNA replication universally exists in all living organisms and is a complicated process in which multiple enzymes are involved in. Enzymes in bacterial DNA replication of initiation and elongation phases bring abundant targets for antimicrobial development as they are conserved and indispensable. In this review, enzyme inhibitors of DNA helicase, DNA primase, topoisomerases, DNA polymerase and DNA ligase were discussed. Special attentions were paid to structures, activities and action modes of these enzyme inhibitors.Results:Among these enzymes, type II topoisomerase is the most validated target with abundant inhibitors. For type II topoisomerase inhibitors (excluding quinolones), NBTIs and benzimidazole urea derivatives are the most promising inhibitors because of their good antimicrobial activity and physicochemical properties. Simultaneously, DNA gyrase targeted drugs are particularly attractive in the treatment of tuberculosis as DNA gyrase is the sole type II topoisomerase in Mycobacterium tuberculosis. Relatively, exploitation of antimicrobial inhibitors of the other DNA replication enzymes are primeval, in which inhibitors of topo III are even blank so far.Conclusion:This review demonstrates that inhibitors of DNA replication enzymes are abundant, diverse and promising, many of which can be developed into antimicrobials to deal with antibioticresistance.
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Garinther, W. I., and M. C. Schultz. "Topoisomerase function during replication-independent chromatin assembly in yeast." Molecular and Cellular Biology 17, no. 7 (July 1997): 3520–26. http://dx.doi.org/10.1128/mcb.17.7.3520.
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DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.
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Hirsch, Jana, and Dagmar Klostermeier. "What makes a type IIA topoisomerase a gyrase or a Topo IV?" Nucleic Acids Research 49, no. 11 (April 27, 2021): 6027–42. http://dx.doi.org/10.1093/nar/gkab270.
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Abstract Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.
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Kern, Gunther, Tiffany Palmer, David E. Ehmann, Adam B. Shapiro, Beth Andrews, Gregory S. Basarab, Peter Doig, et al. "Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914." Journal of Biological Chemistry 290, no. 34 (July 6, 2015): 20984–94. http://dx.doi.org/10.1074/jbc.m115.663534.
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We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467–474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β.
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Lin, Y. C. James, Jianhong Li, Chad R. Irwin, Heather Jenkins, Luke DeLange, and David H. Evans. "Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly." Journal of Virology 82, no. 12 (April 16, 2008): 5922–32. http://dx.doi.org/10.1128/jvi.02723-07.
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ABSTRACT Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIβ. One can prevent the interaction by introducing a C11-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIα/β in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIα and IIβ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIα/β in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.
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Blanche, F., B. Cameron, F. X. Bernard, L. Maton, B. Manse, L. Ferrero, N. Ratet, et al. "Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases." Antimicrobial Agents and Chemotherapy 40, no. 12 (December 1996): 2714–20. http://dx.doi.org/10.1128/aac.40.12.2714.
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Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.
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Forterre, Patrick, Christiane Eue, Mouldy Sioud, and Abdellah Hamal. "Studies on DNA polymerases and topoisomerases in archaebacteria." Canadian Journal of Microbiology 35, no. 1 (January 1, 1989): 228–33. http://dx.doi.org/10.1139/m89-035.
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We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.Key words: archaebacteria, DNA topoisomerases, DNA polymerases, DNA topology, gyrase.
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Mikulovich, Yu L., G. M. Sorokoumova, А. А. Selishcheva, and V. I. Shvets. "The antimicrobial activity of exogeno us anionic phospholipids against Mycobacterium tuberculosis and Escherichia coli." Fine Chemical Technologies 11, no. 3 (June 28, 2016): 64–73. http://dx.doi.org/10.32362/2410-6593-2016-11-3-64-73.
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The effect of anionic phospholipids, namely, cardiolipin, phosphatidylglycerol and phosphatidic acid, on the growth of gram-negative bacteria E. coli BL21(DE3), as well as gram-positive bacteria M. tuberculosis H37Rv was investigated in this study. The influence of all anionic phospholipids tested on the bacteria growth was shown to be dose-dependent. Lipids at concentrations below 335 μM didn’t affect, while at 335 μM and above they repressed bacteria growth and caused cellular death of both type of microorganisms. SOS response induction was observed by using strain E. coli CSH50 sfiA::lacZ during cultivation E. coli with cardiolipin, phosphatidylglycerol and phosphatidic acid. This indicates DNA damage through double-strand breaks. One reason of the DNA damage could be stabilization of transient complexes of DNA topoisomerase (types I and II) with DNA temporary broken by anionic phospholipids. However, neither phosphatidylglycerol nor phosphatidic acid affect the activity of types I and II DNA topoisomerases from E. coli in vitro. In contrast, cardiolipin inhibited DNA topoisomerase I and DNA gyrase (type II topoisomerase), but didn’t stabilize transient complexes of the enzyme with DNA. It indicates that DNA damage due to anionic phospholipids exposure didn’t result from inhibition of DNA topoisomerase activity through stabilization of the transient complex of the enzyme with DNA. The obtained results of cardiolipin, phosphatidylglycerol and phosphatidic acid bactericidal activity against grampositive M. tuberculosis and gram-negative E. coli make it possible to use anionic phospholipids as individual antimicrobial agents or as a matrix of effective and non-toxic liposomal drugs for tuberculosis treatment.
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Thakur, Devendra Singh. "Topoisomerase II Inhibitors in Cancer Treatment." International Journal of Pharmaceutical Sciences and Nanotechnology 3, no. 4 (February 28, 2011): 1173–81. http://dx.doi.org/10.37285/ijpsn.2010.3.4.2.
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Topoisomerase II constitutes a family of nuclear enzymes essential to all living cells. These enzymes are capable of transferring one DNA double helix through a transient break in another DNA double helix. Type II topoisomerases play important roles in DNA metabolic processes, in which they are involved in DNA replication, transcription, chromosome condensation and de-condensation. Topoisomerase II is also the cellular target for a number of widely used anticancer agents currently in clinical use, such as the anthracyclines (daunorubicin and doxorubicin), the epipodophyllotoxins (etoposide and teniposide), and the aminoacridines. These agents stimulate the topoisomerase II-cleavable complex, which is a transient configuration of topoisomerase II on DNA in which topoisomerase II is covalently attached to DNA. This causes the accumulation of cytotoxic nonreversible DNA double-strand breaks generated by the processing of such complexes by DNA metabolic processes. As of present, the clinical use of catalytic topoisomerase inhibitors as antineoplastic agents is limited to aclarubicin and MST-16. Both of these compounds are preferentially active toward hematological malignancies and show limited activity toward solid tumors. This review explains the role of topoisomerase inhibitors in cancer therapy.
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Duguet, M. "When helicase and topoisomerase meet!" Journal of Cell Science 110, no. 12 (June 15, 1997): 1345–50. http://dx.doi.org/10.1242/jcs.110.12.1345.
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Several examples of direct interactions between helicases and topoisomerases have recently been described. The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability. A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40. A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide. This enzyme may be required to maintain genomic stability at high temperature. A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast. This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells. A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences. Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom's and Werner's syndromes. The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation.
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Delgado, Justine L., Chao-Ming Hsieh, Nei-Li Chan, and Hiroshi Hiasa. "Topoisomerases as anticancer targets." Biochemical Journal 475, no. 2 (January 23, 2018): 373–98. http://dx.doi.org/10.1042/bcj20160583.
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Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However, traditional or nonspecific anticancer drugs are still important for the treatment of many cancer patients whose cancers either do not respond to or have developed resistance to cancer-specific anticancer agents. DNA topoisomerases, especially type IIA topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork arrest and double-strand break formation. Unfortunately, this unique mode of action is associated with the development of secondary cancers and cardiotoxicity. Structures of topoisomerase–drug–DNA ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. Recent advances in the field have suggested a possibility of designing isoform-specific human topoisomerase II poisons, which may be developed as safer anticancer drugs. It may also be possible to design catalytic inhibitors of topoisomerases by targeting certain inactive conformations of these enzymes. Furthermore, identification of various new bacterial topoisomerase inhibitors and regulatory proteins may inspire the discovery of novel human topoisomerase inhibitors. Thus, topoisomerases remain as important therapeutic targets of anticancer agents.
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Wang, Xilan. "Structural study of Topoisomerase IV-DNA-Levofloxacin complexes from Streptococcus pneumoniae." E3S Web of Conferences 131 (2019): 01021. http://dx.doi.org/10.1051/e3sconf/201913101021.
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S. pneumoniae is an important pathogen causing pulmonary infection, acute otitis media and purulent meningitis in infants and children. Type II topoisomerases are enzymes that play essential roles in DNA replication, chromosome segregation and recombination throughout all living organisms. Topoisomerases IV can make a transient break in DNA strands in one chromosome. These enzymes are very important antibacterial as well as anticancer targets and potential anti-trypanosomal targets. Levofloxacin has shown efficient inhibition of Type II topoisomerases in S. pneumoniae. Its mechanism of action is to inhibit the activity of DNA topoisomerase, prevent bacterial DNA synthesis and replication leading to bacterial death. We focused on solving the key structures of topoisomerase IV-DNA-levofloxacin complexes by negative staining electron microscopy and the resulting model was obtained at 32 Å by 3D autorefine in Relion 3.0. This study was to try and obtain the structure of the whole complex with DNA bound in the G-gate and the T-gate in order to study DNA capture and transport in type II topoisomerases.
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Bronstein, Joel C., Stacey L. Olson, Kristin LeVier, Mark Tomilo, and Peter C. Weber. "Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli." Antimicrobial Agents and Chemotherapy 48, no. 7 (July 2004): 2708–11. http://dx.doi.org/10.1128/aac.48.7.2708-2711.2004.
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ABSTRACT The subunits of DNA gyrase and topoisomerase IV from Staphylococcus haemolyticus were expressed in Escherichia coli, purified to homogeneity, and used to reconstitute active enzymes that were sensitive to known topoisomerase inhibitors. This represents the first description of a method for isolating type II topoisomerases of a coagulase-negative staphylococcal species.
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Heck, M. M., and W. C. Earnshaw. "Topoisomerase II: A specific marker for cell proliferation." Journal of Cell Biology 103, no. 6 (December 1, 1986): 2569–81. http://dx.doi.org/10.1083/jcb.103.6.2569.
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We have used an antibody probe to measure the levels of topoisomerase II in several transformed and developmentally regulated normal cell types. Transformed cells contain roughly 1 X 10(6) copies of the enzyme. During erythropoiesis in chicken embryos the enzyme level drops from 7.8 X 10(4) copies per erythroblast to less than 300 copies per erythrocyte concomitant with the cessation of mitosis in the blood. Cultured myoblasts also lose topoisomerase II upon fusion into nonproliferating myotubes. When peripheral blood lymphocytes (which lack detectable topoisomerase II) commence proliferation, they express topoisomerase II de novo. Appearance of the enzyme exactly parallels the onset of DNA replication. These results suggest that topoisomerase II is not required for transcription in higher eukaryotes, but that it may function during DNA replication. Furthermore, topoisomerase II is a sensitive and specific marker for proliferating cells.
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Morrissey, Ian, and John George. "Activities of Fluoroquinolones againstStreptococcus pneumoniae Type II Topoisomerases Purified as Recombinant Proteins." Antimicrobial Agents and Chemotherapy 43, no. 11 (November 1, 1999): 2579–85. http://dx.doi.org/10.1128/aac.43.11.2579.
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ABSTRACT Streptococcus pneumoniae topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptibleStreptococcus pneumoniae strain, from first-step mutants showing low-level resistance to ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin, and from two clinical isolates showing intermediate- and high-level fluoroquinolone resistance by a gene cloning method that produces recombinant proteins fromEscherichia coli. The concentrations of ciprofloxacin, sparfloxacin, levofloxacin, or ofloxacin required to inhibit wild-type topoisomerase IV were 8 to 16 times lower than those required to inhibit wild-type DNA gyrase. Furthermore, low-level resistance to these fluoroquinolones was entirely due to the reduced inhibitory activity of fluoroquinolones against topoisomerase IV. For all the laboratory strains, the 50% inhibitory concentration for topoisomerase IV directly correlated with the MIC. We therefore propose that withS. pneumoniae, ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin target topoisomerase IV in preference to DNA gyrase. Sitafloxacin, on the other hand, was found to be equipotent against either enzyme. This characteristic is unique for a fluoroquinolone. A reduction in the sensitivities of both topoisomerase IV and DNA gyrase are required, however, to achieve intermediate- or high-level fluoroquinolone resistance in S. pneumoniae.
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Lee, Joyce H., and James M. Berger. "Cell Cycle-Dependent Control and Roles of DNA Topoisomerase II." Genes 10, no. 11 (October 30, 2019): 859. http://dx.doi.org/10.3390/genes10110859.
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Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.
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Schoeffler, A. J., and J. M. Berger. "Recent advances in understanding structure–function relationships in the type II topoisomerase mechanism." Biochemical Society Transactions 33, no. 6 (October 26, 2005): 1465–70. http://dx.doi.org/10.1042/bst0331465.
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DNA topos (topoisomerases) are complex, multisubunit enzymes that remodel DNA topology. Members of the type II topo family function by passing one segment of duplex DNA through a transient break in another, a process that consumes two molecules of ATP and requires the co-ordinated action of multiple domains. Recent structural data on type II topo ATPase regions, which activate and enforce the directionality of DNA strand passage, have highlighted how ATP physically controls the catalytic cycle of the enzyme. Structural and biochemical studies of specialized DNA-binding domains in two paralogous bacterial type IIA topos (DNA gyrase and topo IV) show how these enzymes selectively negatively supercoil or decatenate DNA. Taken together, these findings expand our understanding of how disparate functional elements work together to co-ordinate the type II topo mechanism.
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Bellon, Steven, Jonathan D. Parsons, Yunyi Wei, Koto Hayakawa, Lora L. Swenson, Paul S. Charifson, Judith A. Lippke, Robert Aldape, and Christian H. Gross. "Crystal Structures of Escherichia coli Topoisomerase IV ParE Subunit (24 and 43 Kilodaltons): a Single Residue Dictates Differences in Novobiocin Potency against Topoisomerase IV and DNA Gyrase." Antimicrobial Agents and Chemotherapy 48, no. 5 (May 2004): 1856–64. http://dx.doi.org/10.1128/aac.48.5.1856-1864.2004.
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ABSTRACT Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 μM). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP Km increased ≈5-fold and the V max decreased ≈30%. In contrast, the topoisomerase IV ATP Km decreased by a factor of 6, and the V max increased ≈2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.
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Hong, George, and Kenneth N. Kreuzer. "An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo." Molecular and Cellular Biology 20, no. 2 (January 15, 2000): 594–603. http://dx.doi.org/10.1128/mcb.20.2.594-603.2000.
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ABSTRACT Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.
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Yague, Genoveva, Julia E. Morris, Xiao-Su Pan, Katherine A. Gould, and L. Mark Fisher. "Cleavable-Complex Formation by Wild-Type and Quinolone-Resistant Streptococcus pneumoniae Type II Topoisomerases Mediated by Gemifloxacin and Other Fluoroquinolones." Antimicrobial Agents and Chemotherapy 46, no. 2 (February 2002): 413–19. http://dx.doi.org/10.1128/aac.46.2.413-419.2002.
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ABSTRACT Gemifloxacin is a recently developed fluoroquinolone with potent activity against Streptococcus pneumoniae. We show that the drug is more active than moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin against S. pneumoniae strain 7785 (MICs, 0.03 to 0.06 μg/ml versus 0.25, 0.25, 1, and 1 to 2 μg/ml, respectively) and against isogenic quinolone-resistant gyrA-parC mutants (MICs, 0.5 to 1 μg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 μg/ml, respectively). Gemifloxacin was also the most potent agent against purified S. pneumoniae DNA gyrase and topoisomerase IV in both catalytic inhibition and DNA cleavage assays. The drug concentrations that inhibited DNA supercoiling or DNA decatenation by 50% (IC50s) were 5 to 10 and 2.5 to 5.0 μM, respectively. Ciprofloxacin and levofloxacin were some four- to eightfold less active against either enzyme; moxifloxacin and gatifloxacin showed intermediate activities. In assays of drug-mediated DNA cleavage by gyrase and topoisomerase IV, the same order of potency was seen: gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin ≈ ciprofloxacin. For gemifloxacin, the drug concentrations that caused 25% linearization of the input DNA by gyrase and topoisomerase IV were 2.5 and 0.1 to 0.3 μM, respectively; these values were 4-fold and 8- to 25-fold lower than those for moxifloxacin, respectively. Each drug induced DNA cleavage by gyrase at the same spectrum of sites but with different patterns of intensity. Finally, for enzymes reconstituted with quinolone-resistant GyrA S81F or ParC S79F subunits, although cleavable-complex formation was reduced by at least 8- to 16-fold for all the quinolones tested, gemifloxacin was the most effective; e.g., it was 4- to 16-fold more active than the other drugs against toposiomerase IV with the ParC S79F mutation. It appears that the greater potency of gemifloxacin against both wild-type and quinolone-resistant S. pneumoniae strains arises from enhanced stabilization of gyrase and topoisomerase IV complexes on DNA.
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Morimoto, Tsuda, Bunch, Sasanuma, Austin, and Takeda. "Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA." Genes 10, no. 11 (October 30, 2019): 868. http://dx.doi.org/10.3390/genes10110868.
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Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this ‘abortive catalysis’ can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.
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Coelho, João, and Alexandre Leitão. "The African Swine Fever Virus (ASFV) Topoisomerase II as a Target for Viral Prevention and Control." Vaccines 8, no. 2 (June 17, 2020): 312. http://dx.doi.org/10.3390/vaccines8020312.
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African swine fever (ASF) is, once more, spreading throughout the world. After its recent reintroduction in Georgia, it quickly reached many neighboring countries in Eastern Europe. It was also detected in Asia, infecting China, the world’s biggest pig producer, and spreading to many of the surrounding countries. Without any vaccine or effective treatment currently available, new strategies for the control of the disease are mandatory. Its etiological agent, the African swine fever virus (ASFV), has been shown to code for a type II DNA topoisomerase. These are enzymes capable of modulating the topology of DNA molecules, known to be essential in unicellular and multicellular organisms, and constitute targets in antibacterial and anti-cancer treatments. In this review, we summarize most of what is known about this viral enzyme, pP1192R, and discuss about its possible role(s) during infection. Given the essential role of type II topoisomerases in cells, the data so far suggest that pP1192R is likely to be equally essential for the virus and thus a promising target for the elaboration of a replication-defective virus, which could provide the basis for an effective vaccine. Furthermore, the use of inhibitors could be considered to control the spread of the infection during outbreaks and therefore limit the spreading of the disease.
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Aldred, Katie J., Erin J. Breland, Sylvia A. McPherson, Charles L. Turnbough, Robert J. Kerns, and Neil Osheroff. "Bacillus anthracis GrlAV96ATopoisomerase IV, a Quinolone Resistance Mutation That Does Not Affect the Water-Metal Ion Bridge." Antimicrobial Agents and Chemotherapy 58, no. 12 (September 22, 2014): 7182–87. http://dx.doi.org/10.1128/aac.03734-14.
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ABSTRACTThe rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit ofBacillus anthracistopoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.
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Caron, P. R., P. Watt, and J. C. Wang. "The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II." Molecular and Cellular Biology 14, no. 5 (May 1994): 3197–207. http://dx.doi.org/10.1128/mcb.14.5.3197.
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A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.
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Caron, P. R., P. Watt, and J. C. Wang. "The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II." Molecular and Cellular Biology 14, no. 5 (May 1994): 3197–207. http://dx.doi.org/10.1128/mcb.14.5.3197-3207.1994.
Full textAbstract:
A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.
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Eder, J. P., V. T. Chan, E. Niemierko, B. A. Teicher, and L. E. Schnipper. "Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells." Journal of Biological Chemistry 268, no. 19 (July 1993): 13844–49. http://dx.doi.org/10.1016/s0021-9258(19)85180-3.
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Stokes, Neil R., Helena B. Thomaides-Brears, Stephanie Barker, James M. Bennett, Joanne Berry, Ian Collins, Lloyd G. Czaplewski, et al. "Biological Evaluation of Benzothiazole Ethyl Urea Inhibitors of Bacterial Type II Topoisomerases." Antimicrobial Agents and Chemotherapy 57, no. 12 (September 16, 2013): 5977–86. http://dx.doi.org/10.1128/aac.00719-13.
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ABSTRACTThe type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 μg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci,Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 μg/ml forStreptococcus pneumoniaeto 0.25 μg/ml forStaphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance forS. aureuswere <2.3 × 10−10with compound A and <5.8 × 10−11with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.
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Freudenreich, C. H., and K. N. Kreuzer. "Mutational analysis of a type II topoisomerase cleavage site: distinct requirements for enzyme and inhibitors." EMBO Journal 12, no. 5 (May 1993): 2085–97. http://dx.doi.org/10.1002/j.1460-2075.1993.tb05857.x.
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Bakshi, RP, S. Galande, P. Bali, R. Dighe, and K. Muniyappa. "Developmental and hormonal regulation of type II DNA topoisomerase in rat testis." Journal of Molecular Endocrinology 26, no. 3 (June 1, 2001): 193–206. http://dx.doi.org/10.1677/jme.0.0260193.
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Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.
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Avemann, K., R. Knippers, T. Koller, and J. M. Sogo. "Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks." Molecular and Cellular Biology 8, no. 8 (August 1988): 3026–34. http://dx.doi.org/10.1128/mcb.8.8.3026.
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The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.
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Avemann, K., R. Knippers, T. Koller, and J. M. Sogo. "Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks." Molecular and Cellular Biology 8, no. 8 (August 1988): 3026–34. http://dx.doi.org/10.1128/mcb.8.8.3026-3034.1988.
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The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.
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Strahilevitz, Jacob, Ari Robicsek, and David C. Hooper. "Role of the Extended α4 Domain of Staphylococcus aureus Gyrase A Protein in Determining Low Sensitivity to Quinolones." Antimicrobial Agents and Chemotherapy 50, no. 2 (February 2006): 600–606. http://dx.doi.org/10.1128/aac.50.2.600-606.2006.
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ABSTRACT Fluoroquinolones target two bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. Acquired resistance to quinolones occurs stepwise, with the first mutation occurring in the more sensitive target enzyme. To limit the emergence of resistance, quinolones should ideally possess dual activities against the two enzymes. For reasons that are as yet unclear, Staphylococcus aureus gyrase is less sensitive to quinolones than topoisomerase IV, counter to its greater sensitivity in Escherichia coli, thereby limiting the use of quinolones for the treatment of staphylococcal infections. Mutations in the α4-helix domain of the GyrA subunit of gyrase are important in determining quinolone resistance. We replaced an extended region encompassing the α4 domain in the E. coli GyrA protein with its homolog in S. aureus and tested for its ability to complement a thermosensitive gyrase and its catalytic and noncatalytic properties. Purified gyrase reconstituted with chimeric GyrA was more resistant to ciprofloxacin than wild-type gyrase at both inhibition of catalytic activity and stimulation of cleavage complexes, and this difference was more apparent in the presence of K+-glutamate. The chimeric GyrA subunit was able to complement thermosensitive gyrase, similar to wild-type GyrA. Without supplemental K+-glutamate the MICs of ciprofloxacin for thermosensitive E. coli complemented with chimeric DNA gyrase were equal to those for E. coli complemented with wild-type gyrase but were twofold higher in the presence of K+-glutamate. Our findings suggest that the extended α4 domain of S. aureus GyrA is responsible, at least in part, for the increased resistance of S. aureus gyrase to quinolones and that this effect is modulated by K+-glutamate.
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Soteros, Chris, and Michael Szafron. "Crossing-sign discrimination and knot-reduction for a lattice model of strand passage." Biochemical Society Transactions 41, no. 2 (March 21, 2013): 576–81. http://dx.doi.org/10.1042/bst20120333.
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By performing strand-passages on DNA, type II topoisomerases are known to resolve topological constraints that impede normal cellular functions. The full details of this enzyme–DNA interaction mechanism are, however, not completely understood. To better understand this mechanism, researchers have proposed and studied a variety of random polygon models of enzyme-induced strand-passage. In the present article, we review results from one such model having the feature that it is amenable to combinatorial and asymptotic analysis (as polygon length goes to infinity). The polygons studied, called Θ-SAPs, are on the simple-cubic lattice and contain a specific strand-passage structure, called Θ, at a fixed site. Another feature of this model is the availability of Monte Carlo methods that facilitate the estimation of crossing-sign-dependent knot-transition probabilities. From such estimates, it has been possible to investigate how knot-reduction depends on the crossing-sign and the local juxtaposition geometry at the strand-passage site. A strong relationship between knot-reduction and a crossing-sign-dependent crossing-angle has been observed for this model. In the present article, we review these results and present heuristic geometrical arguments to explain this crossing-sign and angle-dependence. Finally, we discuss potential implications for other models of type II topoisomerase action on DNA.
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Su, Rina, Yunzhi Peng, Zhanli Wang, Hui Yu, and Qi Wu. "Identification of two novel type II topoisomerase mutations in Enterococcus spp. isolated from a hospital in China." Archives of Biological Sciences, no. 00 (2021): 34. http://dx.doi.org/10.2298/abs210628034s.
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Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacin-susceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the nonmutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation.
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Aubry, Alexandra, L. Mark Fisher, Vincent Jarlier, and Emmanuelle Cambau. "First functional characterization of a singly expressed bacterial type II topoisomerase: The enzyme from Mycobacterium tuberculosis." Biochemical and Biophysical Research Communications 348, no. 1 (September 2006): 158–65. http://dx.doi.org/10.1016/j.bbrc.2006.07.017.
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Akasaka, Takaaki, Mayumi Tanaka, Akihito Yamaguchi, and Kenichi Sato. "Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance." Antimicrobial Agents and Chemotherapy 45, no. 8 (August 1, 2001): 2263–68. http://dx.doi.org/10.1128/aac.45.8.2263-2268.2001.
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ABSTRACT The major mechanism of resistance to fluoroquinolones forPseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) ofgyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC orparE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations ingyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.
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Heaton, Victoria J., Jane E. Ambler, and L. Mark Fisher. "Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro." Antimicrobial Agents and Chemotherapy 44, no. 11 (November 1, 2000): 3112–17. http://dx.doi.org/10.1128/aac.44.11.3112-3117.2000.
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ABSTRACT We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniaeto gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC orgyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-stepgyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniaegyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.
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Defant, Andrea, Paolo Gatti, Alessandro Poli, Marta Serena, Alice Sosic, Barbara Gatto, and Ines Mancini. "DNA-Binding Properties of Cytotoxic Naphtindolizinedione-Carboxamides Acting as Type II Topoisomerase Inhibitors. A Combined In Silico and Experimental Study." Chemistry Proceedings 3, no. 1 (November 13, 2020): 96. http://dx.doi.org/10.3390/ecsoc-24-08103.
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Some clinically-used anticancer drugs are DNA intercalators acting as topoisomerase (Topo) IIα poisons in tumor cells highly expressing the enzyme. Synthetic naphtindolizinedione-carboxamides, previously designed as potential antitumor agents and showing relevant cytotoxic activities in vitro, have been now evaluated for their DNA-binding and inhibition of human Topo IIα, in comparison with the drug mitoxantrone. Docking calculation of each synthetic molecule as ligand with the CGCGAATTCGCG oligonucleotide model showed a stable intercalation in the DNA cut inside the enzyme. Moreover, molecular dynamics simulation indicated the stability of each DNA complex by evaluating the H-bonds involved as a function of time. These results are correlated to spectroscopic (binding constants and melting temperature by UV–VIS analysis, circular dichroism) and biological data (cytotoxicity and inhibition of human Topo IIα decatenation assay).
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Di Mauro, E., G. Camilloni, L. Verdone, and M. Caserta. "DNA topoisomerase I controls the kinetics of promoter activation and DNA topology in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 11 (November 1993): 6702–10. http://dx.doi.org/10.1128/mcb.13.11.6702.
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Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.
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Di Mauro, E., G. Camilloni, L. Verdone, and M. Caserta. "DNA topoisomerase I controls the kinetics of promoter activation and DNA topology in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, no. 11 (November 1993): 6702–10. http://dx.doi.org/10.1128/mcb.13.11.6702-6710.1993.
Full textAbstract:
Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.
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Black, Michael T., Thérèse Stachyra, Denis Platel, Anne-Marie Girard, Monique Claudon, Jean-Michel Bruneau, and Christine Miossec. "Mechanism of Action of the Antibiotic NXL101, a Novel Nonfluoroquinolone Inhibitor of Bacterial Type II Topoisomerases." Antimicrobial Agents and Chemotherapy 52, no. 9 (July 14, 2008): 3339–49. http://dx.doi.org/10.1128/aac.00496-08.
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ABSTRACT NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli, whereas the converse is true of enzymes from Staphylococcus aureus. This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.
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Schröder, Heinz C., Helmut Merz, Renate Steffen, Werner E. G. Müller, Prem S. Sarin, Susanne Trumm, Jutta Schulz, and Eckart Eich. "Differential in vitro Anti-HIV Activity of Natural Lignans." Zeitschrift für Naturforschung C 45, no. 11-12 (December 1, 1990): 1215–21. http://dx.doi.org/10.1515/znc-1990-11-1222.
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Abstract Two naturally occurring lignanolides, isolated from the tropical climbing shrub Ipomoea cairica, (-)-arctigen in and (-)-trachelogen in , were found to inhibit strongly replication of human immunodeficiency virus type 1 (HIV-1; strain HTLV-III B) in vitro. At a concentration of 0.5 (μм , (-)-arctigenin and (-)-trachelogenin inhibited the expression of HIV-1 proteins p 17 and p24 by 80 -90 % and 60 -70 % , respectively. The reverse transcriptase activity in the culture fluids was reduced by 80 -90 % when the cells (HTLV-III B/H 9) were cultivated in the presence of 0.5 μм (-)-arctigen in or 1 μм (-)-trachelogenin . At the same concentrations, the formation of syncytia in the HTLV-III B/H 9-Jurkat cell system was inhibited by the compounds by more than 80%. A series of other lignan type compounds displayed no anti-HIV activity. Studying the molecular mechanism of action of (-)-arctigenin and (-)-trachelogenin we found that both compounds are efficient inhibitors of the nuclear matrix-associated DNA topoisomerase II activity, particularly of the enzyme from HIV -1-infected cells. Our results suggest that both compounds prevent the increase of topoisomerase II activity, involved in virus replication, after infection of cells with HIV -1.
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Maxwell, A., L. Costenaro, S. Mitelheiser, and A. D. Bates. "Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases." Biochemical Society Transactions 33, no. 6 (October 26, 2005): 1460–64. http://dx.doi.org/10.1042/bst0331460.
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Type IIA topos (topoisomerases) catalyse topological conversions of DNA through the passage of one double strand through a transient break in another. In the case of the archetypal enzyme, DNA gyrase, it has always been apparent that the enzyme couples the free energy of ATP hydrolysis to the introduction of negative supercoiling, and the structural details of this process are now becoming clearer. The homologous type IIA enzymes such as topo IV and eukaryotic topo II also require ATP and it has more recently been shown that the energy of hydrolysis is coupled to a reduction of supercoiling or catenation (linking) beyond equilibrium. The mechanism behind this effect is less clear. We review the energy coupling process in both classes of enzyme and describe recent mechanistic and structural work on gyrase that addresses the mechanism of energy coupling.
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Aldred, Katie, Adeline Payne, and Olivia Voegerl. "A RADAR-Based Assay to Isolate Covalent DNA Complexes in Bacteria." Antibiotics 8, no. 1 (February 27, 2019): 17. http://dx.doi.org/10.3390/antibiotics8010017.
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Quinolone antibacterials target the type II topoisomerases gyrase and topoisomerase IV and kill bacterial cells by converting these essential enzymes into cellular poisons. Although much is known regarding the interactions between these drugs and enzymes in purified systems, much less is known regarding their interactions in the cellular context due to the lack of a widely accessible assay that does not require expensive, specialized equipment. Thus, we developed an assay, based on the “rapid approach to DNA adduct recovery,” or RADAR, assay that is used with cultured human cells, to measure cleavage complex levels induced by treating bacterial cultures with the quinolone ciprofloxacin. Many chemical and mechanical lysis conditions and DNA precipitation conditions were tested, and the method involving sonication in denaturing conditions followed by precipitation of DNA via addition of a half volume of ethanol provided the most consistent results. This assay can be used to complement results obtained with purified enzymes to expand our understanding of quinolone mechanism of action and to test the activity of newly developed topoisomerase-targeted compounds. In addition, the bacterial RADAR assay can be used in other contexts, as any proteins covalently complexed to DNA should be trapped on and isolated with the DNA, allowing them to then be quantified.