Topoisomerase II·Etoposide Interactions Direct the Formation of Drug-induced Enzyme-DNA Cleavage Complexes

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. 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...

Molecular Analysis of Yeast and Human Type II Topoisomerases

“…Recent results from Osheroff and colleagues (19,53) have stressed the importance of the enzyme in determining cleavage site specificity with non-intercalating top2 poisons. The results described here indicate that the enzyme plays a very important role in the cleavage specificity of intercalating top2 poisons as well.…”

DNA topoisomerase II, genotoxicity, and cancer

“…All available evidence indicates that non-covalent topoisomerase II poisons act within the active site of the enzyme at the interface between the protein and DNA substrate [15,16,[133][134][135][136][137]. Furthermore, mechanistic studies suggest that it is actually the interactions between topoisomerase II and these compounds that serve as the point of entry into the enzyme-DNA complex [135][136][137][138]. Although the non-intercalative/intercalative nature of compounds appears to have little effect on the action of drugs within the ternary topoisomerase II-DNA-drug complex, it has the potential to modulate the efficacy of these agents in a physiological setting.…”

“…Although topoisomerase II-targeted anticancer drugs act at the enzyme-DNA interface [15,16,104,[133][134][135][136][137], the accumulation of drugs in the double helix has the potential to inhibit enzyme binding or activity. Because the generation of positive superhelical twists by DNA intercalation induces torsional stress in the double helix [130,189], the ability of these molecules to absorb intercalative compounds is limited.…”

EndoG is dispensable in embryogenesis and apoptosis

“…24 Etoposide induces DNA breaks by inhibiting the ability of topoisomerase II to ligate cleaved nucleic acid molecules. 25 Menadione is a quinone that generates quinone radicals and free radicals in mitochondria resulting in reactive oxygen species stress. 26 Thapsigargin and tunicamycin induce endoplasmic reticulum-stress.…”