Knab A M, Fertala J, Bjornsti M A
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1993 Oct 25;268(30):22322-30.
The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. Saccharomyces cerevisiae cells expressing yeast DNA topoisomerase I mutant top1 vac (I725R, N726A) or top1N726L, in which the amino acid residues N terminus to the active site tyrosine Tyr-727 were changed as indicated, were found to be camptothecin-resistant even though the mutant proteins expressed in Escherichia coli were previously shown to be active. Assays of enzyme-catalyzed relaxation of supercoiled DNA in vitro and in vivo in yeast showed that the camptothecin resistance of these mutants arises by entirely different mechanisms. Top1N726L-catalyzed DNA relaxation was not detected in yeast. The Top1 vac protein was catalytically active; however, camptothecin was inefficient in trapping the covalent intermediate formed between the Top1 vac enzyme and DNA. Yeast cells expressing human mutant htop1 vac, with similar substitutions near the active site tyrosine Tyr-723, were also camptothecin-resistant. Surprisingly, in the absence of camptothecin, yeast rad52 mutants defective in the repair of double-stranded DNA breaks were nonviable when top1N726L or top1 vac was overexpressed but viable when htop1 vac was overexpressed. These results suggest differences between yeast and human enzyme function in vivo.
抗癌药物喜树碱通过捕获催化活性酶与DNA之间形成的共价复合物来靶向真核生物DNA拓扑异构酶I。表达酵母DNA拓扑异构酶I突变体top1 vac（I725R，N726A）或top1N726L的酿酒酵母细胞，其中活性位点酪氨酸Tyr-727之前的氨基酸残基N端如所示发生了变化，尽管之前显示在大肠杆菌中表达的突变蛋白具有活性，但这些细胞被发现对喜树碱具有抗性。在酵母中对超螺旋DNA进行体外和体内酶催化松弛的测定表明，这些突变体对喜树碱的抗性是由完全不同的机制产生的。在酵母中未检测到Top1N726L催化的DNA松弛。Top1 vac蛋白具有催化活性；然而，喜树碱在捕获Top1 vac酶与DNA之间形成的共价中间体方面效率低下。表达人突变体htop1 vac的酵母细胞，在活性位点酪氨酸Tyr-723附近有类似的取代，也对喜树碱具有抗性。令人惊讶的是，在没有喜树碱的情况下，当top1N726L或top1 vac过表达时，在双链DNA断裂修复方面有缺陷的酵母rad52突变体无法存活，但当htop1 vac过表达时则可以存活。这些结果表明酵母和人酶在体内功能上存在差异。
