Topoisomerase II etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes
Topoisomerase II etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes
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DOI：
10.1074/jbc.271.46.29238
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发表时间：
1996-11-15
影响因子：
4.8
通讯作者：
Osheroff, N
中科院分区：
文献类型：
--
作者：
Burden, DA;Kingma, PS;Osheroff, N
拓扑异构酶II是几种高活性抗癌药物的靶标，这些药物通过增强酶介导的DNA断裂诱导细胞死亡。尽管这些试剂以位点特异性的方式显著增加了核酸切割水平，但人们对它们改变拓扑异构酶II的DNA位点选择性的机制知之甚少。因此，我们进行了一系列的动力学和结合实验，以确定抗癌药物依托泊苷促进22个特定核酸序列切割复合物形成的机制基础。总的来说，最大DNA断裂水平（即C-max）的变化范围比依托泊苷对这些位点的表观亲和力（即K-m）的变化范围要大得多，而且这两个动力学参数之间没有相关性。此外，酶的药物结合和加成顺序实验表明，依托泊苷和拓扑异构酶II在没有DNA的情况下形成了一个动力学上有效的复合物。这些发现表明依托泊苷拓扑异构酶II（而不是依托泊苷DNA）的相互作用介导了卵裂复合体的形成。最后，特定位点的虔诚率与C-max值呈负相关，表明依托泊苷诱导的最大程度的断裂反映了药物抑制特定序列虔诚的能力，而不是药物对位点特异性酶- dna复合物的亲和力。
Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. C-max) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. K-m) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide topoisomerase II (rather than etoposide DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with C-max values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.