Summary
DNA gyrase and DNA topoisomerase (topo) IV are the bacterial targets of coumarin and quinolone antimicrobial agents. Widespread resistance to clinically important antibiotics such as beta-lactams and macrolides has stimulated the development of novel gyrase and topo IV inhibitors especially against Streptococcus pneumoniae and other Gram-positive pathogens. Here, we describe how gyrase and topo IV activities are measured and how inhibitors of these enzymes may be assayed, focusing as a paradigm on DNA supercoiling by S. pneumoniae gyrase, DNA decatenation by S. pneumoniae topo IV, and DNA cleavage by both enzymes. These approaches provide mechanistic insight on inhibitor action and allow identification of dual gyrase/topo IV targeting agents that can minimize the emergence of bacterial resistance.
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References
Drlica, K., and Zhao, X. (1997) DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61,377–392.
Gellert, M., Mizuuchi, K., O’Dea, M. H., and Nash, H. A. (1976) DNA gyrase:An enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci.USA 73, 3872–3876.
Mizuuchi, K., Mizuuchi, M., O’Dea, M. H., and Gellert, M. (1984) Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins. J. Biol. Chem. 259, 9199–9201.
Pan, X.-S., and Fisher, L. M. (1999) Streptococcus pneumoniae DNA gyrase and topoisomerase IV: Overexpression, purification, and differential inhibition by fluoroquinolones. Antimicrob. Agents Chemother. 43, 1129–1136.
Aubry, A., Veziris, N., Cambau, E., Truffot-Pernot, C., Jarlier, V., and Fisher, L. M. (2006) Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis: Functional analysis of mutant enzymes. Antimicrob. Agents Chemother. 50, 104–112.
Mizuuchi, M., Fisher, L. M., O’Dea, M. H., and Gellert, M. (1980) DNA gyrase action involves the introduction of transient double strand breaks into DNA. Proc. Natl. Acad. Sci. USA. 77, 1847–1851.
Corbett, K. D., and Berger, J. M. (2004) Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Ann. Rev. Biophys. Biomol. Struct. 33, 95–118.
Corbett, K. D., Schoeffler A. J., Thomsen, N. D., and Berger, J. M. (2005) The structural basis for substrate specificity in DNA topoisomerase IV. J. Mol. Biol. 351, 545–561.
Kato, J., Nishimura, Y., Imamura, R., Niki, H., Higara, S., and Suzuki, S. (1990) New topoisomerase essential for chromosome segregation in E. coli. Cell 63, 393–404.
Maxwell, A., and Lawson, D. M. (2003) The ATP binding site of type II topoisomerases as a target of antibacterial drugs. Curr. Top. Med. Chem. 3, 283– 303.
Oram, M., Dosanjh, B., Gormley, N. A., Smith, C. V., Fisher, L. M., Maxwell, A., and Duncan, K. (1996) The mode of action of GR122222X, a novel inhibitor of DNA gyrase. Antimicrob. Agents Chemother. 40, 473–476.
Drlica, K., and Malik, M. (2003) Fluoroquinolones: Action and resistance. Curr. Top. Med. Chem. 3, 249–282.
Grossman, T. H., Bartels, D. J., Mullin, S., Gross, C. H., Parsons, J. D., Liao, Y., Grillot, A.L., Stamos, D., Olson, E. R., Charifson, P. S., and Mani, N. (2007) Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds. Antimicrob. Agents Chemother. 51, 657–666.
Pan, X.-S., and Fisher, L. M. (1997) Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: Selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrob. Agents Chemother. 41, 471–474.
Pan, X.-S., and Fisher, L. M. (1998) DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42, 2810–2816.
Zhanel, G. G., Fontaine, S., Adam, H., Schurek, K., Mayer, M., Noreddin, A. M., Gin, A. S., Rubinstein, E., and Hoban, D. J. (2006) A review of new fluoroquinolones: Focus on their use in respiratory tract infections. Treatment Respir. Med. 5, 437–465.
Staudenbauer, W. L., and Orr, E. (1982) DNA gyrase: Affinity chromatography on novobiocin-Sepharose and catalytic properties. Nucleic Acids Res. 9, 3589–3603.
Pan, X.-S., Yague, G., and Fisher, L. M. (2001) Quinolone resistance mutations in Streptococcus pneumoniae GyrA and ParC proteins: Mechanistic insights into quinolone action from enzymatic analysis, intracellular levels, and phenotypes of wild-type and mutant proteins. Antimicrob. Agents Chemother. 45, 3140–3147.
Sayer, P. J., Goble, M. L., Oram, M., and Fisher, L. M. (2001) Plasmid supercoiling by DNA gyrase. In Methods in Molecular Biology, Vol. 95: DNA Topoisomerase Protocols, Part II: Enzymology and Drugs (N. Osheroff and M. A. Bjornsti, eds.), Humana Press Inc, Totowa, NJ, pp. 25–33.
Acknowledgments
X.-S. Pan and this work were supported by Project Grant BBD01882X1 from the Biotechnology and Biological Sciences Council of the United Kingdom.
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Fisher, L.M., Pan, XS. (2008). Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV Activities. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_2
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DOI: https://doi.org/10.1007/978-1-59745-246-5_2
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