Koster Daniel A, Palle Komaraiah, Bot Elisa S M, Bjornsti Mary-Ann, Dekker Nynke H
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Nature. 2007 Jul 12;448(7150):213-7. doi: 10.1038/nature05938. Epub 2007 Jun 24.
Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.
对化疗药物作用机制的认识有限，常常阻碍了提高其杀死癌细胞能力的进程。喜树碱类药物，如拓扑替康，通过毒害DNA拓扑异构酶I诱导细胞死亡，该酶能够消除DNA超螺旋。拓扑替康被认为可稳定共价拓扑异构酶-DNA复合物，使其成为DNA复制叉的障碍。在此，我们使用单分子纳米操纵技术监测拓扑替康存在时人类拓扑异构酶I的动力学。这使我们能够实时检测单个拓扑替康分子的结合和解离，并量化药物诱导的拓扑异构酶在DNA上的滞留。出乎意料的是，我们的研究结果还表明，拓扑替康显著阻碍拓扑异构酶介导的DNA解旋，对去除正（过度缠绕）超螺旋比对负超螺旋的影响更为明显。在芽殖酵母中的体内实验验证了由此产生的预测，即由于拓扑异构酶I被喜树碱毒害，正超螺旋在转录和复制过程中会积累。然而，用表达具有催化活性、对喜树碱耐药的拓扑异构酶I突变体的细胞进行药物处理，并未诱导产生正超螺旋。单分子数据和体内数据的这种结合提示了喜树碱类药物的一种细胞毒性机制，其中复制机器前方正超螺旋的积累会诱导潜在致命的DNA损伤。
