Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV Activities
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 WordsDNA gyrase DNA topoisomerase IV DNA supercoiling DNA decatenation cleavage complex ciprofloxacin agarose gel electrophoresis Streptococcus pneumoniae
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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