Cusick M E, DePamphilis M L, Wassarman P M
J Mol Biol. 1984 Sep 15;178(2):249-71. doi: 10.1016/0022-2836(84)90143-8.
The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.
在用环己酰亚胺处理以防止新生组蛋白组装成核小体的猿猴病毒40（SV40）感染细胞中，分析了预制（“旧”）组蛋白八聚体在DNA复制叉双臂之间的分布。在环己酰亚胺存在下合成的病毒染色质显示缺乏核小体。复制型SV40 DNA（野生型800和衣壳组装突变体tsB11）在完整细胞或补充有胞质溶胶的核提取物中进行放射性标记。然后通过用微球菌核酸酶广泛消化分离的细胞核、核提取物或分离的病毒染色体来释放新生核小体单体。纯化标记的核小体DNA，发现其与取自DNA复制起点两侧的SV40 DNA限制片段的两条链杂交，而冈崎片段仅与代表逆行DNA模板的链杂交。此外，用两种链特异性外切核酸酶消化分离的复制型SV40染色体，这些酶从复制叉的正向或逆向切除新生DNA。用环己酰亚胺预处理细胞不会导致复制叉两侧的前核小体DNA过量，但会增加核小体间DNA的量。这些数据与核小体分离的分散模型一致，即“旧”组蛋白八聚体分布在DNA复制叉的双臂上。
