title: Real-time detection of DNA topological changes with a fluorescently labeled cruciform.
abstract: Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.
authors.affiliation: Department of Molecular and Cellular Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720-3220, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Jude
authors.forename: Abbey
authors.initials: A
authors.lastname: Hartland
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D027081:DNA Gyrase / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D045566:DNA, Cruciform / Q000737:chemistry* / Q000378:metabolism; D004278:DNA, Superhelical / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D055029:Inverted Repeat Sequences; D010957:Plasmids / Q000235:genetics; D059003:Topoisomerase Inhibitors / Q000494:pharmacology*
chemical_list: D000951:Antigens, Neoplasm; D045566:DNA, Cruciform; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D059003:Topoisomerase Inhibitors; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II