Hertzberg R P, Busby R W, Caranfa M J, Holden K G, Johnson R K, Hecht S M, Kingsbury W D
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
J Biol Chem. 1990 Nov 5;265(31):19287-95.
Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.
喜树碱（CPT）可逆地结合并稳定DNA与拓扑异构酶I之间形成的可裂解复合物。通过使用两种与喜树碱结构相关的亲和标记试剂，研究了CPT与DNA-拓扑异构酶I二元复合物的相互作用性质：10-溴乙酰氨基甲基喜树碱（BrCPT）和7-甲基-10-溴乙酰氨基甲基喜树碱（BrCPTMe）。这些化合物已被证明能不可逆地捕获DNA-拓扑异构酶I复合物。尽管用高盐或过量竞争DNA处理后，由拓扑异构酶I和喜树碱介导的DNA质粒切割显著减少，但经BrCTPMe稳定的酶介导的DNA切割在类似处理后至少持续4小时。不可逆的拓扑异构酶I-DNA切割的产生是时间依赖性的，这表明BrCPTMe首先非共价结合到酶-DNA复合物上，然后在第二步较慢的过程中，以阻止DNA连接的方式使酶或DNA烷基化。采用[3H]BrCPT的实验支持了共价键的形成，该实验表明[3H]BrCPT可在酶-DNA复合物中标记拓扑异构酶I。DNA的存在极大地增强了[3H]BrCPT对拓扑异构酶I的标记；对分离的拓扑异构酶I或分离的DNA几乎没有标记。即使在有DNA存在的情况下，喜树碱也会抑制[3H]BrCPT对拓扑异构酶I的标记，这表明CPT和BrCPT都结合在DNA-拓扑异构酶I二元复合物的同一位点上。这些研究提供了进一步的证据，表明在DNA-拓扑异构酶I复合物形成时会产生一个喜树碱结合位点，并表明喜树碱的A环靠近一个酶残基。
