Tyler J K, Bulger M, Kamakaka R T, Kobayashi R, Kadonaga J T
Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla 92093-0347, USA.
Mol Cell Biol. 1996 Nov;16(11):6149-59. doi: 10.1128/MCB.16.11.6149.
To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.
为了更好地理解与DNA复制偶联的染色质组装过程，我们分离出了编码果蝇染色质组装因子1（dCAF-1）最小亚基（表观分子量为55 kDa，称为p55）的cDNA。dCAF-1是一种多亚基蛋白，在体外将核小体组装到新复制的DNA上是必需的。p55多肽包含七个WD重复基序，与哺乳动物RbAp48蛋白同源，该蛋白与HD1组蛋白去乙酰化酶相关。通过使用针对p55的亲和纯化抗体对dCAF-1进行免疫纯化；所得的dCAF-1制剂具有dCAF-1的四个假定亚基（p180、p105、p75和p55），并且对与DNA复制偶联的染色质组装具有活性。此外，dCAF-1活性被针对p55的抗体特异性耗尽。因此，p55是dCAF-1的一个组成部分。p55定位于细胞核，并且在果蝇发育过程中全程存在。与p55和与HD1相关的RbAp48蛋白之间的同源性一致，观察到组蛋白去乙酰化酶活性可从果蝇核提取物中与p55特异性共免疫沉淀。此外，一部分p55蛋白在DNA复制后与新组装的染色质结合。这些发现共同表明，p55可能作为与DNA复制偶联的染色质组装和组蛋白修饰之间的联系发挥作用。
