Interactions of type II topoisomerases with divalent metal ions and therapeutic drugs

Abstract

All living organisms encode at least one type II topoisomerase. These enzymes help to regulate the superhelical density of the bacterial chromosome and remove knots and tangles from the double helix. Previous studies indicate that eukaryotic type II enzymes utilize a novel variant of the canonical two-metal-ion mechanism to catalyze DNA scission. However, the role of these metal ions in the reaction mediated by bacterial type II enzymes has been controversial. To resolve this issue, we characterized the cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities along with oligonucleotides that replaced bridging and non-bridging oxygen atoms at the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with sulfur-containing substrates. The metal ion dependence of DNA cleavage was sigmoidal in nature. Furthermore, rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3’-bridging atom of the scissile phosphate and facilitates DNA scission. Etoposide is a widely prescribed anticancer drug that stabilizes covalent topoisomerase II-cleaved DNA complexes. The drug contains a polycyclic ring system (rings A–D), a glycosidic moiety at C4, and a pendant ring (E–ring) at C1. Interactions between human topoisomerase IIα and etoposide in the enzyme-drug complex appear to be mediated by substituents on the A-, B-, and E-rings, but not the D-ring or C4 glycosidic moiety. To address the contributions of the D-ring to the activity of etoposide, drug derivatives were characterized. D-ring alterations diminished the ability of etoposide to enhance DNA cleavage mediated by human topoisomerase IIα in vitro and in cultured cells. They also decreased etoposide binding in the ternary enzyme-drug-DNA complex and altered sites of enzyme-mediated DNA cleavage. Based on these findings, we propose that the D-ring of etoposide has important interactions with DNA in the ternary topoisomerase II cleavage complex.