Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for use in the biochemical and pharmacological characterization of topoisomerase II.
The ATP-dependent unknotting of phage P4 DNA is a highly specific assay for type II topoisomerases. Despite the unique specificity of the assay, however, its semiquantitative design has limited its use in studying the biochemical properties of these enzymes. To overcome this problem, we have modified the P4 DNA unknotting assay to provide a sensitive and reproducible method for quantifying topoisomerase II activity. Methods are described for accurate measurement of 10-100 ng of unknotted P4 DNA. Under the assay conditions employed, the initial rate of topoisomerase II activity was linear through 30 min. The quantitative assay has been used to determine biochemical and pharmacological parameters of purified topoisomerase II (p170). No topoisomerase II activity was observed in the absence of ATP; enzymatic activity was optimal between 0.5 and 1.0 mM ATP, but substrate inhibition occurred at concentrations above 1 mM. Eadie-Hofstee analysis with varying ATP concentrations gave an apparent Km for ATP of 0.24 mM and a maximal velocity under these conditions of 7.4 ng P4 DNA unknotted/min/ng topoisomerase II. IC50 values were determined for several topoisomerase inhibitors, including amsacrine, teniposide, and novobiocin. Inhibition by teniposide was found to be uncompetitive versus ATP, with a Ki of 3.7 microM. In contrast, inhibition by novobiocin was competitive versus ATP, indicating that teniposide and novobiocin inhibit topoisomerase II by different mechanisms.
Hofmann
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Research & Development, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939.
Mirabelli
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CK
Drake
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D000677:Amsacrine / Q000494:pharmacology; D001435:Bacteriophages / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000187:drug effects* / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D013713:Teniposide; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D004996:Ethidium