Krude T, de Maddalena C, Knippers R
Fakultät für Biologie, Universität Konstanz, Germany.
Mol Cell Biol. 1993 Feb;13(2):1059-68. doi: 10.1128/mcb.13.2.1059-1068.1993.
Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.
通过体外复制分析，我们比较了从感染细胞核中分离出的天然的和经盐处理的猴病毒40微小染色体。两种制备物中的微小染色体都含有完整的核小体，但盐处理会去除组蛋白H1和一部分非组蛋白染色质蛋白。两种类型的微小染色体都能很好地作为体外复制的模板，但复制产物的结构却显著不同。如先前所示（T. Krude和R. Knippers，《分子细胞生物学》11:6257 - 6267，1991），经盐处理后复制的微小染色体平均含有正常核小体数量的约一半。相比之下，未处理的复制微小染色体被发现核小体紧密堆积，这表明在体外复制过程中发生了新核小体的组装。生化和免疫学数据表明，与天然微小染色体相关的非组蛋白染色质蛋白部分包括一种核小体组装活性，该活性似乎与染色质组装因子I密切相关（S. Smith和B. W. Stillman，《细胞》58:15 - 25，1989）。此外，这种与微小染色体结合的核小体组装因子能够对正在复制的无蛋白竞争DNA发挥反式活性。因此，天然染色质本身含有在复制过程中有序组装核小体所需的活性。
