Oestergaard Vibe H, Knudsen Birgitta R, Andersen Anni H
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, 8000 Aarhus C, Denmark.
J Biol Chem. 2004 Jul 2;279(27):28100-5. doi: 10.1074/jbc.M402119200. Epub 2004 Apr 28.
Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.
拓扑异构酶II是一种重要的酶，许多具有临床价值的抗癌药物都以它为靶点。一类被称为拓扑异构酶II毒剂的药物通过增加拓扑异构酶II介导的DNA断裂的细胞水平来发挥作用，从而导致细胞凋亡。另一类针对拓扑异构酶II的药物，即双二氧哌嗪类药物，可稳定酶的构象，使其达到无活性的盐稳定闭合钳结构。双二氧哌嗪类药物与拓扑异构酶II毒剂类似，可诱导细胞死亡，但其潜在机制目前尚不清楚。在本研究中，我们使用三种经过生物化学充分表征的不同人类拓扑异构酶IIα突变体酶，来剖析该酶在酵母中对双二氧哌嗪ICRF - 193和拓扑异构酶II毒剂m - AMSA产生显性敏感性所需的催化要求。我们发现，钳闭合活性、DNA切割活性，甚至这两种活性共同作用，都不足以使拓扑异构酶II在酵母中对ICRF - 193产生显性敏感性。相反，链通过事件本身是一个绝对必要条件，很可能是因为这涉及到酶与两个DNA片段的同时相互作用。此外，我们表明，人类拓扑异构酶IIα在酵母中对m - AMSA产生显性敏感性的能力并不取决于钳闭合或链通过，而是直接与该酶通过增加DNA切割复合物形成来响应m - AMSA的能力相关。
