Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate

Abstract

The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.

Acidic pH induces topoisomerase II-mediated DNA damage

“…Above pH 7.5 or below pH 4.0, little DNA cleavage was detectable. The strongest cleavage was observed at pH 5.0, consistent with the results obtained with both calf thymus and Drosophila TOP2s (19,20). Similar results were obtained with human TOP2␤ (data not shown).…”

“…Previous studies have demonstrated that both calf thymus and Drosophila TOP2s induce DNA cleavage at pH below 7.0, with an optimum about 5 (19,20). In the current study, we show that TOP2 is involved in acidic pH-induced DNA damage, mutagenesis, and cytotoxicity.…”

“…These results thus suggest that acidic pH stabilizes reversible TOP2 cleavage complexes in vitro. Previous studies have demonstrated that acidic pH-induced DNA breaks are primarily single-strand breaks (20). As shown in Fig.…”

DNA topoisomerase II, genotoxicity, and cancer

“…Thus, the enzyme generates cleaved DNA molecules that contain 4-base single-stranded ends at their 5′-termini. [71,72] During its cleavage event, topoisomerase II covalently attaches to these newly generated 5′-termini [48,[71][72][73]. This covalent enzyme-cleaved DNA complex is known as the "cleavage complex.…”

Bioflavonoids as Poisons of Human Topoisomerase IIα and IIβ

“…To maintain genomic integrity during this cleavage event, the enzyme forms covalent bonds between active site tyrosyl residues and the 5′-DNA termini created by scission of the double helix (54)(55)(56). These covalent topoisomerase IIcleaved DNA intermediates are known as cleaVage complexes.…”