Thermophilic topoisomerase I on a single DNA molecule
Thermophilic topoisomerase I on a single DNA molecule
复制标题
DOI：
10.1016/s0022-2836(03)00320-6
复制
发表时间：
2003-05-30
影响因子：
5.6
通讯作者：
Croquette, V
中科院分区：
文献类型：
--
作者：
Dekker, NH;Viard, T;Croquette, V
DNA 拓扑结构的控制对于嗜热生物体至关重要，因为环境温度升高会威胁双螺旋的稳定性。拓扑异构酶 I 在这种控制中发挥着重要作用，它是 IA 型拓扑异构酶家族的成员。我们使用单分子技术研究了来自超嗜热细菌海栖热袍菌的拓扑异构酶在双链 DNA 上的结合和活性，并为其提供了各种底物，例如 (+) plectonemes、(-) plectonemes 和变性气泡。我们发现拓扑异构酶对两种类型的 plectoneme 均无活性，但对缠绕下 DNA 拉伸力增加时产生的变性气泡有活性。松弛速率敏感地取决于所施加的力和蛋白质浓度。这些观察结果可以从拓扑异构酶对单链 DNA 而非双链 DNA 的偏好来理解，并且可以更好地理解在环状质粒的批量实验中拓扑异构酶的活性。使用无法进行切割的突变体对单个双链体分子进行的结合实验证实了这一解释，并表明 T. maritima 拓扑异构酶 I 通过降低缠绕下 DNA 的变性阈值而表现得像 SSB。最后，使用独特的单链 DNA 进行的实验表明，切割的 DNA 的两端都被酶紧密地维持着，支持了这种拓扑异构酶的酶桥机制。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix. An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases. We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles. We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA. The relaxation rate depended sensitively on the applied force and the protein concentration. These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids. Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T. maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA. Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase. (C) 2003 Elsevier Science Ltd. All rights reserved.