Reconstitution of mitotic chromatids with a minimum set of purified factors
Reconstitution of mitotic chromatids with a minimum set of purified factors
复制标题
DOI：
10.1038/ncb3187
复制
发表时间：
2015-08-01
影响因子：
21.3
通讯作者：
Hirano, Tatsuya
中科院分区：
文献类型：
--
作者：
Shintomi, Keishi;Takahashi, Tatsuro S.;Hirano, Tatsuya
有丝分裂染色体的组装是细胞分裂过程中基因组准确传递的必要前提，每个染色体都由一对杆状染色单体组成。然而，人们对这一基本过程如何在细胞中实现和调节仍然知之甚少。在这里，我们报告了一个在体外系统中，有丝分裂染色单体可以通过混合一个简单的基板，只有六个纯化的因素：核心组蛋白，三个组蛋白伴侣（核质蛋白，Nap 1和FACT），拓扑异构酶II（topo II）和凝聚素I重建。我们发现含有胚胎变体H2 A.X-F的八聚体核小体对FACT高度敏感，并作为topo II和凝聚素I后续作用的最有效底物。凝聚素I的Cdk 1磷酸化是染色单体重建所需的唯一有丝分裂特异性修饰。这个实验系统将加强我们对单个因素的作用机制及其在此过程中的合作的理解。
The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.