ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings

Abstract

Extracts of Drosophila embryos contain an enzymatic activity that converts circular DNAs into huge networks of catenated rings in an ATP-dependent fashion. The catenated activity is resolved into two protein components during purification. One component is a novel DNA topoisomerase that requires the presence of ATP in order to relax supercoiled DNA. We have shown that the ATP-dependent DNA topoisomerase relaxes DNA by a mechanism distinct from that of nicking-closing enzymes. The Drosophila ATP-dependent topoisomerase allows one segment of a circular DNA to pass through transient breaks in both strands at another site on the DNA circle without any relative rotation between the ends at the transient break. This mechanism can convert negative supertwists to positive twists and vice versa until a relaxed equilibrium state is reached. The formation of catenated rings is mediated by an analogous bimolecular reaction which can occur between two nonhomologous DNA circles. The catenation reaction is fully reversible: in the presence of the second protein component, circular DNA is converted quantitatively into catenated forms; in its absence, the ATP-dependent topoisomerase resolves catenated networks back into monomer circles. The Drosophila ATP-dependent topoisomerase appears to be closely related to E. coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, both require ATP and both are inhibited by the antibiotic novobiocin. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but also may be involved in the folding and unfolding of eucaryotic chromosomes.

Nuclear envelope assembly around sperm chromatin in cell‐free preparations from Drosophila embryos

“…1E). In order to assess whether topoisomerase II activity is required for the nu- clear assembly, novobiocin was added to the extract at various stages of assembly [13]. When added at the onset of the reaction, the chromatin failed to decondense and remained in a slightly longer condensed form ( fig.3A).…”

“…The Drosophila ATP-dependent topoisomerase appears to be closely related to Escherichia coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, require ATP and are inhibited by novobiocin [13]. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but may also be involved in the folding and unfolding of eukaryotic chromosomes.…”

Catenation of DNA by eucaryotic topoisomerase II associated with simian virus 40 minichromosomes.

“…Catenation is only one of the possible reversible topoisomerase reactions. Catenation, as described here, or decatenation are activities of the same topoisomerase enzyme, the direction of the reaction being dependent on the influence of the surrounding salt concentration (Kreuzer and Cozzarelli, 1980;Hsieh and Brutlag, 1980;Tse and Wang, 1980). Both of the known types of topoisomerases (I and 1I) are able to catalyze catenation reactions (for review, see Cozzarelli, 1980b) using magnesium ions and polyamines as cofactors.…”

The probabilities of supercoil removal and decatenation by yeast DNA topoisomerase II

“…Elegant studies over a decade ago have established the mechanistic features that form the basis of their classification: a DNA bound enzyme makes a transient double-stranded cleavage in the DNA, and forms at the same time a pair of covalent DNA-enzyme links between the 5 0 ends of the severed DNA and a pair of active site residues in the homodimeric enzyme; a second DNA double helix is then passed through the transient break or gate in the first, after which the severed DNA is rejoined (Brown & Cozzarelli 1979;Liu et al 1980). The type II enzymes are ATP-dependent (Gellert et al 1976;Liu et al 1979;Hsieh & Brutlag 1980). Because DNA breakage and rejoining by all topoisomerases involve transesterification between enzyme tyrosyl and DNA phosphoryl groups (Tse et al 1980;Champoux 1981;Rowe et al 1984), these steps are not expected to require ATP, as illustrated by reactions catalysed by type I DNA topoisomerases that have no high-energy cofactor requirement (Wang 1971;Champoux & Dulbecco 1972).…”