title: High-throughput microtitre plate-based assay for DNA topoisomerases.
abstract: We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of DNA topoisomerases. The assay utilizes intermolecular triplex formation between an immobilized triplex-forming oligo (TFO) and a triplex-forming region inserted into the plasmid substrate (pNO1), and capitalizes on the observation that supercoiled DNA forms triplexes more readily than relaxed DNA. Thus, supercoiled DNA is preferentially retained by the TFO under triplex-forming conditions while relaxed DNA can be washed away. Due to its high speed of sample analysis and reduced sample handling over conventional gel-based techniques, this assay can be used to screen chemical libraries for novel inhibitors of topoisomerases.
authors: Department of Biological Chemistry, John Innes Centre, Colney, Norwich NR47UH, UK.
authors: James A
authors: JA
authors: Taylor
authors: Nicolas P
authors: NP
authors: Burton
authors: Anthony
authors: A
authors: Maxwell
mesh_terms: D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D027081:DNA Gyrase / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification; D057075:Enzyme Assays / Q000379:methods*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification; D054852:Small Molecule Libraries; D059005:Topoisomerase II Inhibitors; D059003:Topoisomerase Inhibitors / Q000737:chemistry
chemical_list: D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D054852:Small Molecule Libraries; D059005:Topoisomerase II Inhibitors; D059003:Topoisomerase Inhibitors; C051802:triplex DNA; D004247:DNA; D027081:DNA Gyrase