DISPERSIVE SEGREGATION OF NUCLEOSOMES DURING REPLICATION OF SIMIAN VIRUS-40 CHROMOSOMES
DISPERSIVE SEGREGATION OF NUCLEOSOMES DURING REPLICATION OF SIMIAN VIRUS-40 CHROMOSOMES
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DOI：
10.1016/0022-2836(84)90143-8
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发表时间：
1984-01-01
影响因子：
5.6
通讯作者：
WASSARMAN, PM
中科院分区：
文献类型：
--
作者：
CUSICK, ME;DEPAMPHILIS, ML;WASSARMAN, PM
在用放线菌酮处理以防止新生组蛋白组装核小体后，对 SV40 感染的细胞中 DNA 复制叉的 2 个臂之间预先形成的（旧）组蛋白八聚体的分布进行了分析。在放线菌酮存在下合成的病毒染色质被证明缺乏核小体。在完整细胞或补充有细胞质的核提取物中对复制的 SV40 DNA（野生型 800 和衣壳组装突变体，tsB11）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 限制性片段的两条链杂交。冈崎片段仅与代表逆行 DNA 模板的链杂交。分离的复制 SV40 染色体用 2 条链特异性核酸外切酶消化，从复制叉的正向或逆行侧切除新生 DNA。用放线菌酮预处理细胞不会导致复制叉两侧的核小体前 DNA 过量，但确实增加了核小体间 DNA 的量。这些数据与核小体分离的分散模型一致，其中旧的组蛋白八聚体分布到 DNA 复制叉的两个臂上。
The distribution of preformed (old) histone octamers between the 2 arms of DNA replication forks was analyzed in 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. Okazaki fragments hybridized only to the strand representing the retrograde DNA template. Isolated, replicating SV40 chromosomes were digested with 2 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.