Froelich-Ammon S J, Patchan M W, Osheroff N, Thompson R B
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
J Biol Chem. 1995 Jun 23;270(25):14998-5004. doi: 10.1074/jbc.270.25.14998.
Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.
尽管目前用于治疗人类癌症的一些药物通过刺激拓扑异构酶II介导的DNA断裂发挥作用，但对于这些药物与该酶之间的相互作用却知之甚少。为了进一步明确药物作用机制，对椭圆玫瑰树碱（一种具有临床相关性的嵌入性药物）与酵母拓扑异构酶II之间的相互作用进行了表征。通过利用酵母遗传系统，确定拓扑异构酶II是该药物的主要细胞靶点。此外，椭圆玫瑰树碱并不抑制酶介导的DNA重新连接，这表明它通过提高切割的正向速率来刺激DNA断裂。最后，通过稳态和频域荧光光谱法评估了椭圆玫瑰树碱与DNA、拓扑异构酶II以及酶-DNA复合物的结合情况。根据荧光强度和发射最大波长的变化以及寿命分析确定，只有椭圆玫瑰树碱的质子化形式与含有拓扑异构酶II切割位点的双链40聚体寡核苷酸结合（解离常数约为65 nM）。相比之下，主要是去质子化的椭圆玫瑰树碱与酶-DNA复合物（解离常数约为1.5 μM）或在无核酸情况下与酶结合（解离常数约为160 nM）。这些发现表明椭圆玫瑰树碱直接与拓扑异构酶II相互作用，并且该酶决定了三元复合物中药物的离子状态。提出了一个模型，其中拓扑异构酶II-椭圆玫瑰树碱-DNA复合物是通过药物首先与酶或DNA结合形成的。
