過度螺旋的電話線會(huì)使總長度變短,限制通話人移動(dòng)的距離,。St.Jude兒童研究醫(yī)院Delft大學(xué)研究人員說,喜樹堿(camptothecins,可用以治療癌癥)殺滅癌細(xì)胞的機(jī)理與此相似,。拓?fù)洚悩?gòu)酶(topotecan poisoning)的拓?fù)涮婵?topotecan,TPT)中毒,,是通過迫使DNA堆積超螺旋激發(fā)細(xì)胞自殺的。
利用名為磁性鑷子(magnetic tweezers)和酵母細(xì)胞,,研究人員發(fā)現(xiàn)喜樹堿類藥物拓?fù)涮婵担╰opotecan)通過抑制DNA拓?fù)洚悩?gòu)酶I解螺開DNA雙螺旋,,殺滅癌細(xì)胞。緊密扭曲的DNA雙鏈被稱作超螺旋(supercoils),,從過度纏繞的DNA分子上突出出來,,如同過度纏繞的電話線的突出部份。如果這些超螺旋積聚并“頑固不化”,,那么細(xì)胞在準(zhǔn)備分裂而欲將DNA雙鏈分開時(shí)會(huì)死亡,。
在這項(xiàng)突破性研究中,研究人員利用微型磁性鑷子監(jiān)控拓?fù)洚悩?gòu)酶I引起的單個(gè)DNA分子的長度變化,,研究單個(gè)拓?fù)涮婵捣肿优c酶-DNA復(fù)合體相結(jié)合,,改變DNA解鏈狀態(tài)的方式。根據(jù)這些結(jié)果,,研究人員發(fā)展了超螺旋理論,,解釋藥物殺滅癌細(xì)胞的機(jī)理,然后在酵母細(xì)胞中檢測該理論,。文章刊登于近期Nature雜志,。
在細(xì)胞分裂之前,一個(gè)細(xì)胞機(jī)器拉開DNA雙鏈,,將兩條連分為叉骨狀的復(fù)制叉,。雙鏈分離是染色體復(fù)制的關(guān)鍵步驟,但這會(huì)增加復(fù)制叉頭部DNA的張力,,引起其彎曲為雙螺旋,。為保證復(fù)制叉持續(xù)拆開雙鏈DNA,細(xì)胞利用拓?fù)洚悩?gòu)酶I在超螺旋DNA兩條鏈中的一條的核酸骨架上開出一個(gè)臨時(shí)性的刻痕,,這樣消除了超螺旋,,使復(fù)制叉能夠繼續(xù)分開雙鏈。拓?fù)涮婵登∏∈抢昧思?xì)胞準(zhǔn)備分開雙鏈時(shí)拓?fù)洚悩?gòu)酶I與雙鏈DNA結(jié)合的性質(zhì),。
已知道拓?fù)涮婵低ㄟ^與拓?fù)洚悩?gòu)酶和DNA鏈上的刻痕結(jié)合,,捕獲拓?fù)洚悩?gòu)酶。拓?fù)涮婵?拓?fù)洚悩?gòu)酶-DNA復(fù)合體改變?yōu)檎系K物,,抑制復(fù)制叉前進(jìn),。目前的流行觀點(diǎn)認(rèn)為,拓?fù)涮婵禋绨┘?xì)胞很容易,,因?yàn)閺?fù)制叉與捕獲的拓?fù)洚悩?gòu)酶沖突,。Bjornsti的研究結(jié)果顯示,復(fù)制叉頭部積聚的正超螺旋才是細(xì)胞自殺的主要原因。
研究人員利用磁性鑷子技術(shù),,將磁珠上的雙鏈DNA分子的一端綁定在玻璃表面,,另一端保持靜止,然后旋轉(zhuǎn)一個(gè)罩在磁珠上部的微型磁鐵,,結(jié)果攜帶DNA的磁珠跟著旋轉(zhuǎn),,將DNA扭曲為超螺旋,將其長度縮減到不到原來的1/7,。
向DNA添加拓?fù)洚悩?gòu)酶,,超螺旋會(huì)在幾秒鐘內(nèi)解開,恢復(fù)原長,。這說明酶在超螺旋上形成了一個(gè)缺口,,消除了張力,使DNA伸展到原長,。但存在拓?fù)涮婵禃r(shí),,拓?fù)洚悩?gòu)酶解開DNA的速度會(huì)下降20倍。然而,,奇怪的是藥物結(jié)合減慢拓?fù)洚悩?gòu)酶解開過度纏繞的DNA(正超螺旋)比DNA鏈重新纏繞為負(fù)超螺旋還要多,。
研究人員發(fā)現(xiàn)拓?fù)涮婵灯蛴跍p慢正超螺旋DNA的解開速度,提示DNA超螺旋實(shí)際上是抑制復(fù)制叉前進(jìn),,刺激細(xì)胞死亡,。Bjornsti等決定通過研究喜樹堿(拓?fù)涮婵凳窍矘鋲A的一個(gè)類似物或相關(guān)藥物)對處于基因表達(dá)過程中的酵母細(xì)胞的效果,檢測這個(gè)模型,。Bjornsti小組首先將雙鏈DNA環(huán)(質(zhì)粒)插入酵母細(xì)胞中,,得到一個(gè)研究喜樹堿效果的模型。
基因表達(dá)過程中,,DNA鏈?zhǔn)欠珠_的,,細(xì)胞能夠?qū)⑦z傳信息拷貝到RNA中。與DNA復(fù)制一樣,,基因表達(dá)需要DNA鏈的伸展,,不同的是,轉(zhuǎn)錄機(jī)器產(chǎn)生的是mRNA,。隨著基因轉(zhuǎn)錄,,DNA的解開在轉(zhuǎn)錄機(jī)器的前方形成正超螺旋,后方形成負(fù)超螺旋,。
當(dāng)研究人員添加拓?fù)洚悩?gòu)酶后,,正超螺旋和負(fù)超螺旋以相同的速度消失,似乎是正超螺旋的消失是由與之相似的負(fù)超螺旋的消失平衡的,。當(dāng)再加入藥物喜樹堿后,拓?fù)洚悩?gòu)酶解開正超螺旋的速度比負(fù)超螺旋消失的速度慢,。這種現(xiàn)象證明拓?fù)洚悩?gòu)酶的喜樹堿(或拓?fù)涮婵担┲卸?,偏愛于刺激正超螺旋在解開DNA的復(fù)合體(如轉(zhuǎn)錄機(jī)器和復(fù)制叉)的頭部聚積,。
然而,喜樹堿不會(huì)引起正超螺旋在酵母細(xì)胞中聚集,,酵母細(xì)胞表達(dá)的拓?fù)洚悩?gòu)酶對這種藥物有抗性,。這進(jìn)一步證明喜樹堿類物質(zhì)如拓?fù)涮婵担ㄟ^抑制拓?fù)洚悩?gòu)酶解開正超螺旋而將細(xì)胞殺滅,。
原始出處:
Nature 448, 213-217 (12 July 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]).
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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.