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 substrat...

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Bibliographic Details
Published in:Analytical biochemistry 1993-04, Vol.210 (1), p.63-68
Main Authors: Onishi, Y., Azuma, Y., Kizaki, H.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate
Chromatography, High Pressure Liquid - methods
Chromatography, High Pressure Liquid - statistics & numerical data
DNA Topoisomerases, Type I - analysis
DNA Topoisomerases, Type I - isolation & purification
DNA Topoisomerases, Type II - analysis
DNA Topoisomerases, Type II - isolation & purification
DNA, Superhelical - chemistry
DNA, Superhelical - isolation & purification
Evaluation Studies as Topic
HeLa Cells
Humans
Nucleic Acid Conformation
Sensitivity and Specificity
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Description
Summary: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 DEAE-NPR 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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1993.1151