Proceedings of the National Academy of Sciences of the United States of America
Vol. 112, No. 24, Pages 7495-7500, June 16, 2015   doi:10.1073/pnas.1422203112
Originally published at http://www.pnas.org/ on May 28 2015

Direct observation of DNA overwinding by reverse gyrase

Taisaku Ogawa^a, Katsunori Yogo^a,1, Shou Furuike^a,2, Kazuo Sutoh^a, Akihiko Kikuchi^b, and Kazuhiko Kinosita, Jr.<sup>a,3

a</sup>Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan;
^bDivision of Molecular Mycology and Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
¹Present address: Graduate School of Medical Sciences, Kitasato University, Sagamihara, Kanagawa 252-0373, Japan.
²Present address: Department of Physics, Osaka Medical College, Takatsuki 569-8686, Japan.
³To whom correspondence should be addressed.

Edited by James M. Berger, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 27, 2015 (received for review November 20, 2014)

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 >10²-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.

Significance
Reverse gyrase resides in bacteria that live in hot conditions. The enzyme further intertwines the double helix of DNA to make it tighter. In biochemical assays, reverse gyrase appears quite inefficient, requiring many enzyme molecules per DNA and yet taking many minutes to wind it up. Here, we show that one reverse gyrase molecule rapidly overwinds relaxed DNA but begins to idle as torsion accumulates. Excess torsion would hamper replication/transcription activities, whereas quick restoration of modest torsion prevents thermal denaturation of DNA. We discuss how reverse gyrase lets one strand of DNA pass the other in a preferred direction to achieve overwinding.