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• W2143162207 startingPage "e133" @default.
• W2143162207 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." @default.
• W2143162207 created "2016-06-24" @default.
• W2143162207 creator A5002873716 @default.
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• W2143162207 creator A5039868655 @default.
• W2143162207 date "2013-05-16" @default.
• W2143162207 modified "2023-10-12" @default.
• W2143162207 title "Real-time detection of DNA topological changes with a fluorescently labeled cruciform" @default.
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• W2143162207 doi "https://doi.org/10.1093/nar/gkt413" @default.
• W2143162207 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3711437" @default.
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• W2143162207 hasPublicationYear "2013" @default.
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