Single-molecule analysis of DNA uncoiling by a type II topoisomerase
Single-molecule analysis of DNA uncoiling by a type II topoisomerase
复制标题
DOI：
10.1038/35009144
复制
发表时间：
2000-04-20
期刊：
影响因子：
64.8
通讯作者：
Bensimon, D
中科院分区：
文献类型：
--
作者：
Strick, TR;Croquette, V;Bensimon, D
II型DNA拓扑异构酶是普遍存在的ATP依赖性酶，能够通过第二个DNA片段中的瞬时双链断裂转运DNA（1）。这使它们能够解开DNA（2-6）并放松扭曲DNA中出现的相互缠绕的超螺旋（plectonemes）（7）。在体内，它们负责解开复制的染色体，并且它们在有丝分裂或减数分裂中的缺失最终导致细胞死亡（8，9）。在这里，我们描述了一个显微操作实验，在这个实验中，我们在真实的时间内跟踪单个黑腹果蝇拓扑异构酶II作用于机械拉伸和超螺旋的线性DNA分子（10-13）。通过在ATP存在下监测DNA的延伸，我们直接观察到在单个催化周转期间两个超螺旋的弛豫。通过控制作用在分子上的拉力，我们确定了反应速率随外加应力的变化。最后，在没有ATP的情况下，我们观察到一个单一的拓扑异构酶至少在两个不同的时间尺度（配置）的DNA交叉钳位。这些结果表明，单分子实验是研究拓扑异构酶的一个强有力的新工具。
Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment(1). This enables them to untangle DNA(2-6) and relax the interwound supercoils (plectonemes) that arise in twisted DNA(7). In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death(8,9). Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled(10-13). By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.