Supercoiled loops and eucaryotic DNA replication
Supercoiled loops and eucaryotic DNA replication
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超螺旋环和真核 DNA 复制
DOI：
10.1016/0092-8674(80)90156-7
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发表时间：
1980
期刊：
影响因子：
64.5
通讯作者：
D. S. Coffey
中科院分区：
文献类型：
--
作者：
B. Vogelstein;D. Pardoll;D. S. Coffey
最近的研究表明，真核细胞核的DNA是以超螺旋环的形式组织的。我们在这里表明，耗尽组蛋白和其他核蛋白的间期核后，通过处理与非离子去污剂和高盐，完整的（无切口的）循环的DNA可以被视为一个光环周围的核骨架或矩阵。使用不同浓度的溴化乙锭可以可逆地缠绕DNA环的这种环，并且当DNA用DNA酶I轻轻地切口或暴露于紫外线时不可逆地解绕。这些结构由锚定在核基质上的解开的DNA环组成，为我们提供了一种检查DNA复制与DNA环之间关系的方法。用~ 3 H-胸苷脉冲标记3 T3细胞不同时间，并在基质晕结构上进行放射自显影。发现在1分钟脉冲后，在核基质内发现了> 80%的代表新复制的DNA的放射自显影颗粒，即使周围的晕圈区域含有80%的总核DNA。随着脉冲时间的延长，放射自显影颗粒逐渐从核基质向外移动到晕圈区域。超过70%的放射自显影颗粒可以被赶出中央矩阵到周围的晕，如果1分钟的脉冲后，1小时的孵育过量的未标记的胸苷。这些发现表明，DNA复制发生在固定的网站在该基地的循环，并建议复制的DNA环是能动的，相对于他们的核基质锚定位点。
Recent studies have indicated that the DNA of the eucaryotic nucleus is organized in the form of supercoiled loops. We show here that after depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact (unnicked) loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix. This halo of DNA loops can be reversibly wound using various concentrations of ethidium bromide and is irreversibly unwound when the DNA is gently nicked with DNAase I or exposure to ultraviolet light. These structures, comprising a halo of unwound DNA loops anchored to a nuclear matrix, provided us with a way in which to examine the relationship of DNA replication to the DNA loops. 3T3 cells were pulse-labeled with 3H-thymidine for various periods and autoradiography was performed on the matrix-halo structures. It was found that after a 1 min pulse,> 80% of the autoradiographic grains representing newly replicated DNA were found within the nuclear matrix even though the surrounding halo region contained 80% of the total nuclear DNA. With longer pulse times, the autoradiographic grains could be seen to move progressively outward from the nuclear matrix into the halo region. Over 70% of the autoradiographic grains could be chased out of the central matrix into the surrounding halo if the 1 min pulse was followed by a 1 hr incubation with excess unlabeled thymidine. These findings indicate that DNA replication occurs at fixed sites at the base of the loops and suggests that the replicating DNA loops are motile with respect to their nuclear matrix anchorage sites.