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Fluoroquinolone-Gyrase-DNA Cleaved Complexes

  • Gan Luan
  • Karl Drlica
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.

Key words

DNA topoisomerase Quinolone DNA gyrase Cleaved complexes Fluoroquinolone Plasmid DNA isolation 

Notes

Acknowledgments

We thank the following for critical comments on the manuscript: Arkady Mustaev and Marila Gennaro.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Microbiology, Biochemistry & Molecular Genetics, Public Health Research Institute, New Jersey Medical School, Rutgers Biomedical and Health SciencesRutgers UniversityNewarkUSA

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