生物谷報道： 人們對抗癌藥物Topotecan（拓撲替康）的分子機制一直不甚了解,。最近,，荷蘭Delft工業(yè)大學Kavli納米科學研究所的研究人員觀測了Topotecan對DNA單分子的作用，對Topotecan的工作機制有了新的認識,。詳細研究過程和結(jié)果刊登于本周Nature雜志,。文章第一作者為6月1日剛拿到博士學位的Daniel Koster。
Topotecan與重要蛋白（TopoIB）相互作用,，引起（癌）細胞故障,。TopoIB蛋白負責拆除DNA的loop環(huán)，其與DNA分子結(jié)合,，夾住DNA并切開DNA雙鏈中的一條,，然后使DNA鏈伸展，最終將斷裂端連接起來,。
人們一直認為Topotecan只是促進TopoIB蛋白停留在DNA分子上的時間更久,，干擾細胞分裂，損傷（癌）細胞,。但Delft研究人員最近發(fā)現(xiàn),，Topotecan還能夠明顯阻止伸展和促進DNA loop聚集。這些DNA loop 的聚集為研發(fā)治癌藥物提供了參考,。
實驗過程中,，研究人員將單個DNA分子固定在玻璃盤和磁珠之間,，在兩塊磁鐵的幫助下，拉伸和扭曲DNA分子,。當向處于扭曲狀態(tài)的DNA分子添加TopoIB后,，研究人員發(fā)現(xiàn)loop慢慢分開。奇怪的是,，TopoIB的一種酶在DNA分子上仍有活性,。另注：St．Jude兒童研究醫(yī)院的同事在活體酵母細胞中觀察到了相同的機制。
原始出處:
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: [email protected]).
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.
