Mechanochemical analysis of DNA gyrase using rotor bead tracking
Mechanochemical analysis of DNA gyrase using rotor bead tracking
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DOI：
10.1038/nature04319
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发表时间：
2006-01-05
期刊：
影响因子：
64.8
通讯作者：
Bustamante, C
中科院分区：
文献类型：
--
作者：
Gore, J;Bryant, Z;Bustamante, C
DNA 旋转酶是一种分子机器，它利用 ATP 水解的能量将必需的负超螺旋引入 DNA(1-3)。超螺旋的方向性是通过在链通过之前将 DNA(4,5) 手性缠绕在酶的特定结构域 (6-9) 周围来确保的。在这里，我们通过跟踪附着在拉伸 DNA 分子侧面的亚微米珠子的旋转来实时观察旋转酶的活性 (10)。在存在旋转酶和 ATP 的情况下，我们观察到旋转的爆发，对应于严格的二倍数（11）中负超螺旋的持续、逐步引入。 DNA 张力的变化对超螺旋速度没有可检测到的影响，但当张力仅在零点皮牛顿范围内增加时，酶的持续性明显降低。这种行为可以通过一个简单的机械化学模型进行定量解释，其中持续合成能力取决于解离和快速、张力敏感的 DNA 包裹之间的动力学竞争。在我们的检测的高分辨率变体中，我们直接检测对应于两个动力学子步骤的旋转暂停：反应周期结束时的 ATP 独立步骤，以及循环中间的 ATP 结合步骤，随后 DNA 包裹。
DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA(1-3). The directionality of supercoiling is ensured by chiral wrapping of the DNA(4,5) around a specialized domain(6-9) of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule(10). In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two(11). Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.