Williams N L, Howells A J, Maxwell A
Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK.
J Mol Biol. 2001 Mar 9;306(5):969-84. doi: 10.1006/jmbi.2001.4468.
DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the binding and hydrolysis of ATP. The N-terminal domains of the gyrase B dimer constitute an ATP-operated clamp that is proposed to capture the T segment during the DNA supercoiling reaction. We have locked this clamp in the closed conformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta,gamma-imidodiphosphate). The clamp-locked enzyme is able to bind and cleave DNA, albeit at a reduced level. Although the locked enzyme is not capable of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzyme molecule via entry of the T segment through the exit gate of the enzyme. The DNA-protein complex of the clamp-locked enzyme has a conformation that differs from the normal positively wrapped conformation of the gyrase-DNA complex. These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibrium across the DNA gate can be achieved via T-segment entry through the ATP-operated clamp or through the exit gate.
DNA 促旋酶通过在与 ATP 的结合和水解偶联的反应中使一段 DNA（T 段）穿过另一段 DNA（G 段）来催化 DNA 超螺旋化。促旋酶 B 二聚体的 N 末端结构域构成一个由 ATP 驱动的夹子，有人提出该夹子在 DNA 超螺旋化反应期间捕获 T 段。我们使用不可水解的 ATP 类似物 ADPNP（5'-腺苷β，γ-亚氨基二磷酸）将这个夹子锁定在闭合构象。尽管水平有所降低，但夹子锁定的酶仍能够结合并切割 DNA。虽然锁定的酶不能进行 DNA 超螺旋化，但它可以催化有限的 DNA 松弛，这与每个酶分子通过 T 段通过酶的出口门进入而完成一次链通过事件的能力一致。夹子锁定的酶的 DNA-蛋白质复合物具有与促旋酶-DNA 复合物正常的正向包裹构象不同的构象。这些实验证实了由 ATP 驱动的夹子在促旋酶的链通过反应中的作用，并提出了一个 DNA 与促旋酶相互作用的模型，其中通过 T 段通过由 ATP 驱动的夹子或通过出口门进入，可以实现 T 段在 DNA 门两侧处于平衡状态的构象。
