Mechanism of type II DNA topoisomerases: a tale of two gates

DNA topoisomerases are fascinating enzymes involved in many cellular processes including replication, transcription, and genetic recombination. They are capable of catalyzing topological rearrangements of DNA such as relaxation of supercoiled DNA and catenation and decatenation of DNA rings [1xCozzarelli, N.R. and Wang, J.C. (eds). See all References[1]. DNA topoisomerases accomplish this by breaking one or two DNA strands, allowing another single or double strand (the passing strand) to pass through the break before rejoining the broken DNA ends. DNA topoisomerases are classified according to their mechanism of action: type I enzymes cleave one DNA strand and pass single-stranded or double-stranded DNA through the break; type II enzymes cleave two DNA strands and pass a double strand through the break. In all cases, topoisomerases form transient phosphotyrosine bonds between the protein and DNA. Although some DNA topoisomerases require ATP for activity, it is not required for the breaking and religation steps. In the case where the passing strand or strands belong to the same molecule the result is relaxation, supercoiling, knotting or unknotting of DNA. If the passing strand or strands belong to a different DNA molecule the result is catenation or decatenation of the DNA molecules. Decatenation of DNA rings has proved to be one of the most illuminating reactions in deciphering the mechanism of DNA topoisomerases.A more unified view for the mechanism of many members of the DNA topoisomerase family is now emerging. To date, the best understood topoisomerase is Escherichia coli DNA topoisomerase I. Mechanistic studies of the decatenation reaction have shown that it forms an enzyme-bridged DNA opening or ‘gate’ in a nicked DNA molecule to allow passage of a double-stranded molecule through it (Figure 1aFigure 1a). This is clearly a one-gate mechanism, as it requires only one opening in the protein to allow strand passage. This mechanism has been lent support by the recent elucidation of the structure of a 67 kDa amino-terminal fragment of E. coli DNA topoisomerase I, where the toroidal shape of the protein and the location of the active site all favor a one-gate mechanism [2xThree- dimensional structure of the 67 K N-terminal fragment of E. coli DNA topoisomerase I. Lima, C.D., Wang, J.C., and Mondragon, A. Nature. 1994; 367: 138–146Crossref | PubMed | Scopus (203)See all References[2].Figure 1Schematic diagram of the mechanisms of action of topoisomerases. (a) E. coli DNA topoisomerase I (one-gate mechanism) and (b) yeast DNA topoisomerase II (two-gate mechanism). The proteins are depicted as toroids. The gated strand of the DNA is shown as a bar and the passing strand (P), which is viewed end-on in this figure, is shown as a circle. Thin bars represent single-stranded DNA; thick lines represent double-stranded DNA. The gated strand is labeled G and can be single-stranded (ssG) or double-stranded (dsG), depending on the reaction. Covalent attachment of the DNA to the protein is depicted as yellow circles. In both cases the reaction shown is the decatenation of DNA rings. See the text for details of the reactions.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe mechanism by which type II enzymes pass a double-stranded DNA molecule through a double-stranded break has been a subject of considerable interest. Now, Roca and Wang [3xDNA transport by a type II DNA topoisomerase: evidence in favor of a two-gate mechanism. Roca, J. and Wang, J.C. Cell. 1994; 77: 609–616Abstract | Full Text PDF | PubMed | Scopus (200)See all References[3] have elegantly and convincingly shown that for type II enzymes the reaction involves the opening of one gate to allow the entrance of a double-stranded DNA molecule into the protein, followed by concerted breaking of a double strand, strand passage, and opening of a second gate to release the passing molecule.Wang and co-workers had previously shown that yeast DNA topoisomerase II, and probably all other type II enzymes, form a clamp around DNA in an ATP-dependent manner. Upon ATP binding the molecule clamps DNA, releasing it after ATP hydrolysis. Non-hydrolyzable ATP analogs lock the clamp in the closed position, trapping the DNA within the protein. Roca and Wang have now gone one step further and shown that the passing strand can leave the clamp independently of ATP hydroysis. To do this, they constructed catenated molecules consisting of one supercoiled double-stranded DNA ring and a nicked or relaxed double-stranded molecule and studied the decatenation reaction. Their results strongly suggest the existence of a second gate that opens independently of the first one which serves to release the passing strand after it has gone through the double-stranded break. The mechanism for type II DNA topoisomerases necessarily involves two large conformational changes in the protein to open two different gates (Figure 1bFigure 1b). In the first step, the clamp is opened to allow both the strand to be cleaved and the strand to be passed to enter the protein; ATP binding closes the clamp, after which there is concerted cleavage and gating of the DNA to form the open second gate. Both strands of the broken DNA molecule remain attached to the protein during the reaction. Clearly the second opening is at a different position, probably near the active site tyrosines of the enzyme. Once the second gate is opened, the passing strand can exit the protein and the gate can close, with concomitant rejoining of the broken DNA strands. ATP hydrolysis then serves to open the first gate again and the enzyme is now ready for another catalytic cycle. The experiments of Roca and Wang give clear support for this two-gate mechanism for type II DNA topoisomerases.There are some parallels and differences in the proposed mechanisms for E. coli DNA topoisomerase I and yeast DNA topoisomerase II. In both cases large conformational changes are needed to open a gate or gates to allow strand passage. The proteins necessarily have to clamp or enclose DNA during part of the reaction forming, at least in the case of E. coli DNA topoisomerase I, a transient intermediate. The gating mechanism is probably not the only one used by all topoisomerases. Experimental data seem to indicate that most eukaryotic type I DNA topoisomerases do not use an enzyme-bridging mechanism.To date, there is limited structural information on type II enzymes. The X-ray structure of a 42 kDa amino-terminal fragment of the GyrB subunit of E. coli DNA topoisomerase II (DNA gyrase) shows that it forms a dimer in the presence of a non- hydrolyzable ATP analog [4xCrystal structure of an N-terminal fragment of the DNA gyrase B protein. Wigley, D.B., Davies, G.J., Dodson, E.J., Maxwell, A., and Dodson, G. Nature. 1991; 351: 624–629Crossref | PubMedSee all References[4]. Furthermore, the dimer forms a toroidal structure which could correspond to the clamp of the Roca and Wang model. Sequence similarity between the amino termini of yeast DNA topoisomerase II and GyrB further supports the idea that this region of the protein is involved in forming the gate. The ATP-binding site in the GyrB fragment is in a location that could certainly influence the opening and closing of the gate. Electron microscopy studies also indicate a conformational change in gyrase upon ATP hydrolysis. The sequence similarity between yeast DNA topoisomerase II and gyrase clearly suggests a structural similarity, and locates one of the gates at the amino terminus of the yeast protein. Mechanistically there are some differences between the two enzymes but the main features of the Roca and Wang mechanism probably apply to all type II enzymes.Mechanistic studies of E. coli DNA topoisomerase I predicted a toroidal shape for the enzyme, and this was strikingly confirmed by the X-ray structure. Now the studies of Roca and Wang make a similar prediction for yeast DNA topoisomerase II and probably all other type II enzymes. We look forward to the structure of yeast DNA topoisomerase II in anticipation that it will confirm such an elegant and attractive mechanism.