Gruss C, Wu J, Koller T, Sogo J M
Institute of Cell Biology, Swiss Federal Institute of Technology, Zürich.
EMBO J. 1993 Dec;12(12):4533-45. doi: 10.1002/j.1460-2075.1993.tb06142.x.
The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt-treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T-antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross-linking of the DNA and by micrococcal nuclease digestion. A 5- and 10-fold excess of protein-free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.
利用含有完整SV40基因组的体外组装染色质以及经盐处理的天然SV40微型染色体，在体外研究了染色质复制过程中亲代核小体的命运。当DNA在染色质组装前与T抗原、胞质S100提取物和三种脱氧核苷三磷酸预孵育时，体外组装的微型染色体能够在体外高效复制，这表明复制起始点必须没有核小体。通过DNA的补骨脂素交联和微球菌核酸酶消化分析了复制分子中新合成子链的染色质结构。微型染色体复制过程中存在5倍和10倍过量的无蛋白质竞争DNA会捕获分离的组蛋白。与已发表的数据相反，这表明亲代组蛋白在复制叉区域仅松散地或未附着于DNA。在假定没有游离组蛋白的情况下进行复制表明，一个亚核小体颗粒随机组装在子链上。这些数据与在这些条件下形成H3/H4四聚体复合物相一致，支持了在生理条件下新合成子链上的核小体核心组装是通过H2A/H2B二聚体与H3/H4四聚体复合物结合发生的这一观点。
