Covalent attachment of ethidium to DNA results in enhanced topoisomerase II-mediated DNA cleavage

Academic Article

Abstract

  • The classic DNA intercalator, ethidium, was used to probe the effects of (i) intercalation and (ii) covalent modification of the DNA on the catalytic activity of topoisomerase II. Ethidium bromide, which binds reversibly to DNA via intercalation, does not stimulate topoisomerase II-mediated DNA cleavage at concentrations up to 100 μM, indicating that the intercalative binding of this molecule to DNA is not sufficient to alter the activity of the enzyme. In contrast, covalent attachment of the photoreactive ethidium analog to DNA resulted in marked enhancement of topoisomerase II-mediated single- and double-stranded DNA cleavage. This increase in DNA cleavage was observed at very low drug binding densities (< 1 drug per 10-80 base pairs) which correspond to nanomolar concentrations, as compared with other topoisomerase II poisons such as etoposide or m-AMSA which require micromolar concentrations to elicit comparable DNA cleavage levels. Over the past decade, topoisomerase II has been an important target for a variety of clinically relevant anticancer agents due to the abilities of these agents to convert this enzyme to a cellular toxin resulting in an increase in the levels of enzyme-mediated DNA breaks. Modification of DNA by covalently attaching a DNA-targeting intercalating agent (i.e., ethidium bromide) resulted in a marked shift of the cleavage/religation equilibrium of the enzyme toward the cleaved state 'poison' topoisomerase II as observed by the enhancement in single- and double-stranded cleavage; thus, key insight was gained into the mechanism(s) through which DNA binding agents may influence the catalytic properties of topoisomerase II. These data demonstrate that conversion of a reversible ethidium-DNA complex to an irreversible adduct results in the transformation of an ineffective intercalating drug into a potent topoisomerase II-targeted agent. Finally, they provide support for the recently proposed 'positional poisoning model' for the actions of DNA lesions and anticancer drugs on the type II enzyme.
  • Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Author List

  • Marx G; Zhou H; Graves DE; Osheroff N
  • Start Page

  • 15884
  • End Page

  • 15891
  • Volume

  • 36
  • Issue

  • 50