Department of Physics, University of Illinois at Chicago, Chicago, IL 60607, USA.
J Cell Biol. 2010 Mar 8;188(5):653-63. doi: 10.1083/jcb.200910085. Epub 2010 Mar 1.
We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II-specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.
我们分析了有丝分裂染色体内部染色质的拓扑组织。我们通过实验表明，拓扑异构酶（topo） II 可减少有丝分裂染色体的弹性刚度，从而使有丝分裂染色质发生严重的自身缠绕。通过添加外源性拓扑异构酶 II，单条染色体的松弛度可降低 35%，这种松弛度依赖于可水解的三磷酸腺苷（ATP），而无活性的拓扑异构酶 II 切割突变体则不会改变染色体的硬度。此外，使用 I 型拓扑异构酶的实验产生的松弛效果比拓扑异构酶 II 小得多，这表明拓扑异构酶 II 引起的染色体松弛是由于双链 DNA 的解缠结和/或去纽结。在进一步的实验中，染色体首先暴露于蛋白酶中以部分释放对染色质的蛋白质约束，仅 ATP 即可使有丝分裂染色体松弛。拓扑异构酶 II 特异性抑制剂 ICRF-187 阻断了这种效应，表明这是由与染色体结合的内源性拓扑异构酶 II 引起的。我们的实验表明，DNA 缠绕与蛋白质介导的压缩协同作用，将染色质折叠成有丝分裂染色体。
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