Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV Activities

  • L. Mark Fisher
  • Xiao-Su Pan
Part of the Methods In Molecular Medicine™ book series (MIMM, volume 142)

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.

Key Words

DNA gyrase DNA topoisomerase IV DNA supercoiling DNA decatenation cleavage complex ciprofloxacin agarose gel electrophoresis Streptococcus pneumoniae 

References

  1. 1.
    Drlica, K., and Zhao, X. (1997) DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61,377–392.PubMedGoogle Scholar
  2. 2.
    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.CrossRefPubMedGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    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.PubMedGoogle Scholar
  5. 5.
    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.CrossRefPubMedGoogle Scholar
  6. 6.
    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.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    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.CrossRefPubMedGoogle Scholar
  9. 9.
    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.CrossRefPubMedGoogle Scholar
  10. 10.
    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.CrossRefPubMedGoogle Scholar
  11. 11.
    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.PubMedGoogle Scholar
  12. 12.
    Drlica, K., and Malik, M. (2003) Fluoroquinolones: Action and resistance. Curr. Top. Med. Chem. 3, 249–282.CrossRefPubMedGoogle Scholar
  13. 13.
    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.CrossRefPubMedGoogle Scholar
  14. 14.
    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.PubMedGoogle Scholar
  15. 15.
    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.PubMedGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    Staudenbauer, W. L., and Orr, E. (1982) DNA gyrase: Affinity chromatography on novobiocin-Sepharose and catalytic properties. Nucleic Acids Res. 9, 3589–3603.CrossRefGoogle Scholar
  18. 18.
    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.CrossRefPubMedGoogle Scholar
  19. 19.
    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.Google Scholar

Copyright information

© Humana Press Inc. 2008

Authors and Affiliations

  • L. Mark Fisher
    • 1
  • Xiao-Su Pan
    • 2
  1. 1.Molecular and Metabolic Signalling Centre, Division of Basic Medical SciencesSt George’s, University of LondonUK
  2. 2.Department of Basic Medical SciencesSt George’s, University of LondonUK

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