生物谷报道： 人们对抗癌药物Topotecan（拓扑替康）的分子机制一直不甚了解。最近，荷兰Delft工业大学Kavli纳米科学研究所的研究人员观测了Topotecan对DNA单分子的作用，对Topotecan的工作机制有了新的认识。详细研究过程和结果刊登于本周Nature杂志。文章第一作者为6月1日刚拿到博士学位的Daniel Koster。
Topotecan与重要蛋白（TopoIB）相互作用，引起（癌）细胞故障。TopoIB蛋白负责拆除DNA的loop环，其与DNA分子结合，夹住DNA并切开DNA双链中的一条，然后使DNA链伸展，最终将断裂端连接起来。
人们一直认为Topotecan只是促进TopoIB蛋白停留在DNA分子上的时间更久，干扰细胞分裂，损伤（癌）细胞。但Delft研究人员最近发现，Topotecan还能够明显阻止伸展和促进DNA loop聚集。这些DNA loop 的聚集为研发治癌药物提供了参考。
实验过程中，研究人员将单个DNA分子固定在玻璃盘和磁珠之间，在两块磁铁的帮助下，拉伸和扭曲DNA分子。当向处于扭曲状态的DNA分子添加TopoIB后，研究人员发现loop慢慢分开。奇怪的是，TopoIB的一种酶在DNA分子上仍有活性。另注：St．Jude儿童研究医院的同事在活体酵母细胞中观察到了相同的机制。
原始出处:
Nature advance online publication 24 June 2007 | doi:10.1038/nature05938; Received 6 January 2007; Accepted 15 May 2007; Published online 24 June 2007
Antitumour drugs impede DNA uncoiling by topoisomerase I
Daniel A. Koster1, Komaraiah Palle2, Elisa S. M. Bot1, Mary-Ann Bjornsti2 & Nynke H. Dekker1
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Department of Molecular Pharmacology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105, USA
Correspondence to: Nynke H. Dekker1 Correspondence and requests for materials should be addressed to N.H.D. (Email: n.h.dekker@tudelft.nl).
Abstract
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 supercoils1, 2, 3, 4. Topotecan is thought to stabilize a covalent topoisomerase–DNA complex5, 6, 7, rendering it an obstacle to DNA replication forks2, 3, 8, 9. 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.
