TOPOISOMERASE-II ETOPOSIDE INTERACTIONS DIRECT THE FORMATION OF DRUG-INDUCED ENZYME-DNA CLEAVAGE COMPLEXES

Topoisomerase II is the target for several highly active anticancer dr ugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cle avage in a site-specific fashion, little is understood regarding the m echanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carri ed out to determine the mechanistic basis by which the anticancer drug , etoposide, enhances cleavage complex formation at 22 specific nuclei c acid sequences. In general, maximal levels of DNA scission (i.e. C-m ax) varied over a considerably larger range than did the apparent affi nity of etoposide (i.e. K-m) for these sites, and there was no correla tion between these two kinetic parameters. Furthermore, enzyme drug bi nding and order of addition experiments indicated that etoposide and t opoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide topoisomerase II (rather t han etoposide DNA) interactions mediate cleavage complex formation. Fi nally, rates of religation at specific sites correlated inversely with C-max values, indicating that maximal levels of etoposide-induced sci ssion reflect the ability of the drug to inhibit religation at specifi c sequences rather than the affinity of the drug for site-specific enz yme-DNA complexes.