Covalent attachment of ethidium to DNA results in enhanced topoisomerase II-mediated DNA cleavage.
Covalent attachment of ethidium to DNA results in enhanced topoisomerase II-mediated DNA cleavage.
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DOI：
10.1021/bi971858c
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发表时间：
1997-12
期刊：
影响因子：
2.9
通讯作者：
G. Marx;H. Zhou;D. Graves;N. Osheroff
中科院分区：
文献类型：
--
作者：
G. Marx;H. Zhou;D. Graves;N. Osheroff
使用经典的DNA嵌入剂乙锭来探测（i）嵌入和（ii）DNA的共价修饰对拓扑异构酶II的催化活性的影响。溴化乙锭通过嵌入可逆地结合DNA，在高达100 μ M的浓度下不刺激拓扑异构酶II介导的DNA切割，表明该分子与DNA的嵌入结合不足以改变酶的活性。与此相反，共价连接的光反应性乙锭类似物的DNA拓扑异构酶II介导的单链和双链DNA切割的显着增强。与其他拓扑异构酶II毒物如依托泊苷或m-AMSA相比，在对应于纳摩尔浓度的非常低的药物结合密度（每10-80个碱基对<1个药物）下观察到DNA切割的这种增加，所述其他拓扑异构酶II毒物如依托泊苷或m-AMSA需要微摩尔浓度来引发可比较的DNA切割水平。在过去的十年中，拓扑异构酶II已经成为各种临床相关抗癌剂的重要靶标，因为这些药物能够将这种酶转化为细胞毒素，导致酶介导的DNA断裂水平增加。通过共价连接DNA靶向嵌入剂（即，溴化乙锭）导致酶的切割/再连接平衡向切割状态“毒物”拓扑异构酶II的显著移动，如通过单链和双链切割的增强所观察到的;因此，获得了对DNA结合剂可能影响拓扑异构酶II的催化性质的机制的关键认识。这些数据表明，可逆的乙锭-DNA复合物转化为不可逆的加合物导致无效的嵌入药物转化为有效的拓扑异构酶II靶向剂。最后，他们提供了支持最近提出的“位置中毒模型”的DNA损伤和抗癌药物对II型酶的作用。
The classic DNA intercalator, ethidium, was used to probe the effects of (i) intercalation and (ii) covalent modification of the DNA on the catalytic activity of topoisomerase II. Ethidium bromide, which binds reversibly to DNA via intercalation, does not stimulate topoisomerase II-mediated DNA cleavage at concentrations up to 100 microM, indicating that the intercalative binding of this molecule to DNA is not sufficient to alter the activity of the enzyme. In contrast, covalent attachment of the photoreactive ethidium analog to DNA resulted in marked enhancement of topoisomerase II-mediated single- and double-stranded DNA cleavage. This increase in DNA cleavage was observed at very low drug binding densities (<1 drug per 10-80 base pairs) which correspond to nanomolar concentrations, as compared with other topoisomerase II poisons such as etoposide or m-AMSA which require micromolar concentrations to elicit comparable DNA cleavage levels. Over the past decade, topoisomerase II has been an important target for a variety of clinically relevant anticancer agents due to the abilities of these agents to convert this enzyme to a cellular toxin resulting in an increase in the levels of enzyme-mediated DNA breaks. Modification of DNA by covalently attaching a DNA-targeting intercalating agent (i.e., ethidium bromide) resulted in a marked shift of the cleavage/religation equilibrium of the enzyme toward the cleaved state "poison" topoisomerase II as observed by the enhancement in single- and double-stranded cleavage; thus, key insight was gained into the mechanism(s) through which DNA binding agents may influence the catalytic properties of topoisomerase II. These data demonstrate that conversion of a reversible ethidium-DNA complex to an irreversible adduct results in the transformation of an ineffective intercalating drug into a potent topoisomerase II-targeted agent. Finally, they provide support for the recently proposed "positional poisoning model" for the actions of DNA lesions and anticancer drugs on the type II enzyme.