ON THE MECHANISM OF TOPOISOMERASE-I INHIBITION BY CAMPTOTHECIN - EVIDENCE FOR BINDING TO AN ENZYME DNA COMPLEX
ON THE MECHANISM OF TOPOISOMERASE-I INHIBITION BY CAMPTOTHECIN - EVIDENCE FOR BINDING TO AN ENZYME DNA COMPLEX
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DOI：
10.1021/bi00437a018
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发表时间：
1989-05-30
期刊：
影响因子：
2.9
通讯作者：
HECHT, SM
中科院分区：
文献类型：
--
作者：
HERTZBERG, RP;CARANFA, MJ;HECHT, SM
喜树碱是一种细胞毒性抗肿瘤化合物，已被证明可产生由哺乳动物拓扑异构酶I介导的蛋白质连接的DNA断裂。我们研究了喜树碱破坏DNA加工拓扑异构酶I的机制，并研究了某些结构相关的化合物对DNA-拓扑异构酶I共价复合物形成的影响。在喜树碱存在下酶介导的超螺旋血浆DNA的切割在加入外源线性DNA或稀释反应混合物后完全逆转。喜树碱和拓扑异构酶I从超螺旋DNA或松弛DNA产生相同量的切割。此外，生物碱降低了由催化量的拓扑异构酶I介导的超螺旋DNA松弛的初始速度。抑制发生在有利于进行性催化的条件下，以及有利于分布催化的条件下。通过使用[3 H]喜树碱和平衡透析试验，表明该生物碱与DNA-拓扑异构酶I复合物可逆结合，但不与分离的酶或分离的DNA结合。这些结果是一致的模型，其中喜树碱可逆地陷阱的中间体参与DNA解链的拓扑异构酶I，从而扰乱了一组平衡，导致增加的DNA裂解。通过检查某些与喜树碱结构相关的化合物，发现已被证明对抗肿瘤活性至关重要的20-羟基对于DNA和拓扑异构酶I之间的共价复合物的稳定也是必需的。与此相反，没有这样的相关性存在的UV-光诱导的DNA裂解铜（II）-喜树碱衍生物。
Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasma DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.