Roufa D J, Marchionni M A
Proc Natl Acad Sci U S A. 1982 Mar;79(6):1810-4. doi: 10.1073/pnas.79.6.1810.
When animal cells replicate chromatin under conditions precluding new histone biosynthesis, half of the daughter DNAs are devoid of nucleosomes and are sensitive to staphylococcal nuclease. DNA sequences resistant to nuclease are associated with preexisting nucleosomes, which redistribute to progeny DNA duplexes during replication. We labeled newly replicated DNA sequences in a simian virus 40 (SV40)-transformed Chinese hamster cell clone with 5-bromodeoxyuridine (BrdUrd) in the presence and absence of a protein biosynthesis inhibitor, emetine. We resolved single-stranded BrdUrd- and dT-DNA sequences protected from nuclease digestion by nucleosomes and determined from which strands of the integrated viral DNA parental template (dT) and newly replicated progeny (BrdUrd) sequences were derived. Because we knew that the cell clone studied contained all of its integrated SV40 DNA at a single chromosomal site, we were able to determine that preexisting nucleosomes segregated to only one of the two daughter duplexes containing the integrated viral sequence. Additionally, in the presence of emetine, the integrated viral origin of replication, ORIsv, appeared not to function as a chromosomal replication origin, perhaps reflecting the drug's effect on synthesis of SV40 large tumor antigen.
当动物细胞在阻止新组蛋白生物合成的条件下复制染色质时，一半的子代DNA不含核小体，并且对葡萄球菌核酸酶敏感。对核酸酶有抗性的DNA序列与预先存在的核小体相关联，这些核小体在复制过程中重新分布到子代DNA双链体上。我们在存在和不存在蛋白质生物合成抑制剂依米丁的情况下，用5-溴脱氧尿苷（BrdUrd）标记了猿猴病毒40（SV40）转化的中国仓鼠细胞克隆中新复制的DNA序列。我们解析了受核小体保护而免受核酸酶消化的单链BrdUrd和dT-DNA序列，并确定了整合病毒DNA亲本模板（dT）和新复制的子代（BrdUrd）序列来自哪条链。因为我们知道所研究的细胞克隆在单个染色体位点包含其所有整合的SV40 DNA，所以我们能够确定预先存在的核小体仅分离到包含整合病毒序列的两个子代双链体中的一个。此外，在存在依米丁的情况下，整合的病毒复制起点ORIsv似乎没有作为染色体复制起点起作用，这可能反映了该药物对SV40大肿瘤抗原合成的影响。
