Direct observation of DNA overwinding by reverse gyrase

作者：Ogawa Taisaku; Yogo Katsunori; Furuike Shou; Sutoh Kazuo; Kikuchi Akihiko; Kinosita Kazuhiko Jr^*

来源：Proceedings of the National Academy of Sciences, 2015, 112(24): 7495-7500.

DOI：10.1073/pnas.1422203112

• reverse gyrase
• topoisomerase
• magnetic tweezers
• DNA overwinding
• torsion

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 degrees C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 degrees 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 degrees C. The unloaded reaction rate at 71 degrees 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 similar to 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.

• 出版日期2015-6-16