Direct observation of DNA overwinding by reverse gyrase
Direct observation of DNA overwinding by reverse gyrase
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DOI：
10.1073/pnas.1422203112
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发表时间：
2015-06-16
影响因子：
11.1
通讯作者：
Kinosita, Kazuhiko, Jr.
中科院分区：
文献类型：
--
作者：
Ogawa, Taisaku;Yogo, Katsunori;Kinosita, Kazuhiko, Jr.
在超嗜热菌中发现的反向旋转酶是已知的唯一一种过度缠绕（将正超螺旋引入）DNA的酶。在>70 ℃下检测到的ATP依赖性活性迄今为止仅通过凝胶电泳进行研究;因此，反应动力学仍然不清楚。在这里，我们在显微镜下在71摄氏度下对过度缠绕反应进行成像，使用含有连续30个错配碱基对的DNA作为明确的底物位点。单个反向旋转酶分子可使DNA顺时针旋转超过100圈。结合的酶显示出适度的温度依赖性，在低至50 ℃时仍保持显著的活性。在71摄氏度下的无负载反应速率超过每秒5转，这比迄今为止所指出的高>10（2）倍，但低于测量的20 s（-1）的ATP酶速率，表明松散偶联。当扭转应力在DNA中积累时，过缠绕反应急剧减慢，并且在仅仅类似于5pN.nm的应力时停止，其中多一圈将仅消耗六倍的热能。因此，这种酶会使DNA保持在轻微的过度缠绕状态，以保护但不是过度保护超嗜热菌的基因组免受热熔化。过缠绕活性对DNA张力也高度敏感，有效相互作用长度超过反向促旋酶的大小，这意味着需要松弛的DNA。所有的结果都指向这样的机制，其中链通道依赖于热运动，如在拓扑异构酶IA中，是积极的，但松散地偏向于过度卷绕。
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.