title: The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction.
abstract: Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. Strand passage requires an opening of the so-called DNA-gate by approximately 2 nm. A single-molecule FRET study reported equal populations of open and closed DNA-gate in topoisomerase II. We present here single-molecule FRET experiments that monitor the conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted. DNA distortion requires cleavage, but neither ATP nor the presence of a tDNA. At the same time, the DNA-gate of gyrase is predominantly in the closed conformation. In agreement with the single molecule data and with the danger of dsDNA breaks for genome integrity, <5% of cleavage complexes are detected in equilibrium. Quinolone inhibitors favor DNA cleavage by B. subtilis gyrase, but disfavor DNA distortion, and the DNA-gate remains in the closed conformation. Our results demonstrate that DNA binding, distortion and cleavage, and gate-opening are mechanistically distinct events. During the relaxation and supercoiling reactions, gyrase with an open DNA-gate is not significantly populated, consistent with gate-opening as a very rare event that only occurs briefly to allow for strand passage.
authors: Biozentrum, Department of Biophysical Chemistry, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
authors: Airat
authors: A
authors: Gubaev
authors: Manuel
authors: M
authors: Hilbert
authors: Dagmar
authors: D
authors: Klostermeier
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D027081:DNA Gyrase / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D059005:Topoisomerase II Inhibitors
chemical_list: D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D010710:Phosphates; D059005:Topoisomerase II Inhibitors; D027081:DNA Gyrase