An assay method for DNA topoisomerase activity based on separation of relaxed DNA from supercoiled DNA using high-performance liquid chromatography.
A method for assaying the activities of DNA topoisomerases I and II has been developed. The assay for type I and II enzymes is based on monitoring relaxation of supercoiled plasmid DNA in the absence or presence of ATP, respectively. The reaction product, relaxed DNA, was separated from the substrate, supercoiled pBR329 plasmid, by a linear gradient of NaCl using high-performance ion-exchange chromatography with a DEAENPR column. In this method, nanogram amounts of relaxed DNA are detectable within 30 min, indicating that a subtle change in the activity in the cells can be assayed precisely.
Department of Biochemistry, Tokyo Dental College, Mihama-ku, Japan.
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Onishi
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Y
Azuma
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H
Kizaki
D000255:Adenosine Triphosphate; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000302:isolation & purification; D004250:DNA Topoisomerases, Type II / Q000032:analysis* / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D012680:Sensitivity and Specificity
D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II