High-throughput assays for DNA gyrase and other topoisomerases.
We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.
Maxwell
Anthony
A
Department of Biological Chemistry, John Innes Centre Norwich Research Park, Colney, Norwich NR4 7UH, UK. tony.maxwell@bbsrc.ac.uk
Burton
Nicolas P
NP
O'Hagan
Natasha
N
D004796:Clinical Enzyme Tests / Q000379:methods*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D027101:DNA Topoisomerase IV / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D010957:Plasmids / Q000235:genetics
D004269:DNA, Bacterial; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase