No abstract

The ability of type II DNA topoisomerases to perturb the equilibrium distributions of DNA topoisomers is a consequence of their ability to hydrolyse ATP. A sliding mechanism of topoisomerase action has been proposed to account for this phenomenon.

The mechanism of type II DNA topoisomerases involves the formation of an enzyme-operated gate in one double-stranded DNA segment and the passage of another segment through this gate. DNA gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a feature that requires the enzyme to dictate the directionality of strand passage. Although it is known that this is a consequence of the characteristic wrapping of DNA by gyrase, the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate is largely unknown. We have addressed this mechanism by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. We propose a model in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA substrate, setting up an equilibrium of the transported segment across the DNA gate. The overall efficiency of strand passage is determined by the position of this equilibrium, which depends on the superhelical density of the DNA substrate. This mechanism is concerted, in that capture of the transported segment by the ATP-operated clamp induces opening of the DNA gate, which in turn stimulates ATP hydrolysis.The topological state of DNA plays an important role in its biological activity and is maintained by a group of enzymes called DNA topoisomerases (1, 2). These enzymes perform the formidable task of passing one DNA segment through another. This feat is achieved by the cleavage of DNA and the formation of a transient DNA gate through which a segment of the same or another DNA molecule is passed. Mechanistic differences in this reaction distinguish topoisomerases into two types. Type I enzymes introduce a single-stranded break in DNA and facilitate the passage of a second strand through this gate. Type II topoisomerases create a double-stranded break and allow the passage of a double-stranded DNA segment.Type II topoisomerases are able to catalyze a number of reactions, including the negative supercoiling of DNA, the relaxation of negative and positive supercoils, the catenation and decatenation of DNA circles, and the knotting and unknotting of DNA (3). These reactions generally require the hydrolysis of ATP. Most type II enzymes [e.g., prokaryotic topoisomerase (topo) IV and eukaryotic topo II] favor DNA relaxation and decatenation reactions. DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA in an ATP-dependent manner (3, 4). All type II enzymes share a degree of sequence similarity but tend to differ at their C termini (5). DNA gyrase is composed of two subunits, DNA gyrase A protein (GyrA) (97 kDa in Escherichia coli) and DNA gyrase B protein (GyrB) (90 kDa in E. coli), the active form being an A 2 B 2 heterotetramer. Eukaryotic topo IIs are homodimers where the monomer is equivalent to a fusion of the A and B subunits of gyrase, such that the N terminus is homologous to the B subunit, and the C terminus corresponds approximately ...