Bojanowski K, Maniotis A J, Plisov S, Larsen A K, Ingber D E
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Cell Biochem. 1998 May 1;69(2):127-42. doi: 10.1002/(sici)1097-4644(19980501)69:2<127::aid-jcb4>3.0.co;2-u.
Topoisomerase II has been suggested to play a major role in chromosome organization based on its DNA decatenating activity and its ability to mediate direct binding interactions between DNA and nuclear matrix. However, this latter point remains controversial. Here we address the question of whether the chromatin binding activity of Topoisomerase II is sufficient to modify chromosome form using whole mammalian chromosomes in vitro. Intact chromosomes were microsurgically removed from living cells and disassembled by treatment with protease or heparin. When these disassembled chromosomes were incubated with recombinant human Topoisomerase II, the enzyme became incorporated into chromatin and reassembly resulted, leading to almost complete restoration of pre-existing chromosome shape and position within minutes. Chromosome reconstitution by Topoisomerase II was dose-dependent, saturable, and appeared to be controlled stoichiometrically, rather than enzymatically. Similar reassembly was observed in the absence of ATP and when a catalytically inactive thermosensitive Topoisomerase II mutant was used at the restrictive temperature. Chromosome recondensation also could be induced after the strand-passing activity of Topoisomerase II was blocked by treatment with an inhibitor of its catalytic activity, amsacrine. When a non-hydrolyzable beta,gamma-imido analog of ATP (AMP-PNP) was used to physiologically fix bound Topoisomerase II enzyme in a closed form around DNA, subsequent chromosome disassembly was prevented in the presence of high salt. These data suggest that Topoisomerase II may control higher order chromatin architecture through direct binding interactions, independently of its well-known catalytic activity.
基于拓扑异构酶II的DNA解连环活性及其介导DNA与核基质之间直接结合相互作用的能力，有人提出它在染色体组织中起主要作用。然而，后一点仍存在争议。在这里，我们探讨了拓扑异构酶II的染色质结合活性是否足以在体外使用完整的哺乳动物染色体来改变染色体形态这一问题。完整的染色体通过显微手术从活细胞中取出，并用蛋白酶或肝素处理使其解体。当这些解体的染色体与重组人拓扑异构酶II一起孵育时，该酶会整合到染色质中并导致重新组装，从而在几分钟内几乎完全恢复先前存在的染色体形状和位置。拓扑异构酶II介导的染色体重建是剂量依赖性的、可饱和的，并且似乎是按化学计量控制的，而不是通过酶促作用。在没有ATP的情况下，以及在限制温度下使用催化无活性的热敏拓扑异构酶II突变体时，也观察到了类似的重新组装。在用拓扑异构酶II的催化活性抑制剂安吖啶处理阻断其链通过活性后，也可以诱导染色体再凝聚。当使用ATP的不可水解的β,γ-亚氨基类似物（AMP-PNP）以封闭形式在DNA周围生理固定结合的拓扑异构酶II酶时，在高盐存在下可防止随后的染色体解体。这些数据表明，拓扑异构酶II可能通过直接结合相互作用来控制高级染色质结构，而与其众所周知的催化活性无关。
