Abstract
Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.
Highlights
Topoisomerase II is an enzyme that decatenates and disentangles DNA by passing one DNA helix through another [1,2,3,4,5]
Enzyme1⁄7drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide1⁄7 topoisomerase II interactions mediate cleavage complex formation
Topoisomerase II-targeted anticancer drugs greatly alter the DNA cleavage sites utilized by the enzyme (19, 40, 41, 50 –55)
Summary
Drosophila melanogaster topoisomerase II was purified from embryonic Kc cells as described previously by Shelton et al [43]. Cleavage reactions contained 1.4 nM (25 ng) labeled DNA, 3.5 nM (60 ng) Drosophila topoisomerase II, etoposide (at concentrations ranging from 1 to 100 M), or a dimethyl sulfoxide solvent control (final concentration, 1% (v/v)) in a total of 50 l of assay buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 2.5% glycerol (v/v)). Order of addition cleavage experiments were performed as described above, except that etoposide was preincubated in assay buffer with either topoisomerase II or DNA in the absence of the missing component for 2 min at 30 °C. During this period, the missing component was incubated at 30 °C. Filters were washed three times with 1 ml of ice-cold assay buffer, dried, and quantified by liquid scintillation counting
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