Biotin-tag affinity purification of a centromeric nucleosome assembly complex
Biotin-tag affinity purification of a centromeric nucleosome assembly complex
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DOI：
10.4161/cc.5.12.2889
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发表时间：
2006-06-15
期刊：
影响因子：
4.3
通讯作者：
Henikoff, Steven
中科院分区：
文献类型：
--
作者：
Furuyama, Takehito;Henikoff, Steven
着丝粒是微管结合的染色体位点，其确保染色体在有丝分裂中正确分离到子细胞。这个过程是由一个特殊的着丝粒特异性组蛋白H3变体（CenH 3）介导的，它包装着丝粒染色质并在表观遗传学上将着丝粒保持在一个不同的染色体位置。然而，CenH 3相对于典型组蛋白以低丰度存在，这对将CenH 3组装成着丝粒处的核小体的分子伴侣机制的分离和表征提出了挑战。为了解决这一挑战，我们使用了控制过表达的果蝇CenH 3（CID）和一个有效的生化纯化策略提供的体内生物素化CID成功地纯化和表征可溶性CID核小体组装复合物。它由单一的伴侣蛋白RbAp 48与CID和组蛋白H4复合组成。RbAp 48也存在于组装典型组蛋白H3和替换组蛋白H3.3的蛋白质复合物中。在这里，我们强调了我们改进的生物素介导的纯化方法的好处，并解决了简单的CID/H4-RbAp 48伴侣复合物如何在着丝粒特异性介导核小体组装的问题。
Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, we used controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. Here, we highlight the benefits of our improved biotin-mediated purification method, and address the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres.