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. C max ) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. K m ) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme⅐drug 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 etoposide⅐ topoisomerase II (rather than etoposide⅐DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with C max 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.Topoisomerase II is an enzyme that decatenates and disentangles DNA by passing one DNA helix through another (1-5). Due to the requirement for such a DNA strand passage activity in a number of critical nuclear processes, including replication, recombination, and chromosome segregation (1, 3, 4, 6 -10), topoisomerase II is essential for the survival of proliferating eukaryotic cells (11)(12)(13)(14).As a prerequisite for its DNA passage reaction, topoisomerase II generates transient double-stranded breaks in the nucleic acid backbone (3-5). In order to maintain the integrity of the cleaved genetic material during this process, the enzyme forms a proteinaceous bridge that spans the nucleic acid break. This bridge is anchored by covalent phosphotyrosyl bonds established between the active site residues of the homodimeric enzyme and the newly created 5Ј-DNA termini (15-18). Because the covalent topoisomerase II-cleaved DNA complex (referred to as the cleavage complex) is normally a short-lived intermediate in the catalytic cycle of the enzyme, it is tolerated by the cell. However, when present in high concentrations, cleavage complexes become potentially toxic, promoting frameshift mutations, permanent double-stranded DNA breaks, illegitimate recombination, and apoptosis (6, 8, 19 -23).The cytotoxic potential of topoisomerase II has been exploited clinically by the development of anticancer drugs that generate high levels of covalent enzyme-DNA cleavage complexes (19,(22)(23)(24)(25)(26). Because rapidly proliferating cells contain high concentrations of topoisomerase II (27-31), aggressive malignancies are most susceptibl...

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

Given the avid and selective metal binding properties of naturally-occurring metalloproteins, it is possible to exploit these systems in the development of novel sensors, i.e., "biosensors", for the detection of trace quantities of metal ions. Here, we exploit the high affinity of human carbonic anhydrase II (CAII) for zinc in the detection of nanomolar concentrations of this metal ion by fluorescence anisotropy using a fluorescein-derivatized arylsulfonamide probe, 4-aminosulfonyl[1-(4-N-(5-fluoresceinylthioureido)butyl)]benzamide (3). This probe was designed through an iterative, structure-based approach and was demonstrated to bind tightly only to the zinc-bound holoenzyme (K d ) 2.3 nM) and not the metal-free apoenzyme. Furthermore, the probe exhibits anisotropy that is proportional to the concentration of bound zinc, and this behavior can be exploited in the detection of zinc in the 10-1000 nM range. Strategies for the structure-based design of improved CAII-based metal ion biosensors are considered in view of these results.