title: Direct observation of DNA overwinding by reverse gyrase.
abstract: Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 °C. The unloaded reaction rate at 71 °C exceeds five turns per second, which is >10(2)-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s(-1), indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼ 5 pN ⋅ nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan;
authors.forename: Taisaku
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan;
authors.forename: Katsunori
authors.initials: K
authors.lastname: Yogo
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan;
authors.forename: Shou
authors.initials: S
authors.lastname: Furuike
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan;
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sutoh
authors.affiliation: Division of Molecular Mycology and Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan; kazuhiko@waseda.jp.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kinosita
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000378:metabolism; D055592:Biophysical Phenomena; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008280:Magnetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016944:Sulfolobus / Q000201:enzymology; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I
keywords: DNA overwinding; magnetic tweezers; reverse gyrase; topoisomerase; torsion