Lavin M F, Davidson M
J Cell Sci. 1981 Apr;48:383-91. doi: 10.1242/jcs.48.1.383.
A number of different assay methods have been used to study repair of strand breaks in DNA after exposure of cells to ionizing radiation. Use of these methods indicates that fibroblasts from patients with ataxia telangiectasia (AT), a multiform genetic disease exhibiting high sensitivity to ionizing radiation, have a normal ability to repair strand breaks in DNA. All of these methods determine the extent of breakage of DNA and the resealing of these breaks but do not provide information on restoration of DNA configuration in the nucleus. In this report we have used a sensitive technique to investigate restoration of the 3-dimensional structure of DNA in AT lymphoblastoid cells after exposure to ionizing radiation. This technique provides a means of lysing cells using a high concentration of salt and a non-ionic detergent, giving rise to structures called nucleoids which contain nuclear RNA and DNA, are depleted in protein, and sediment in a manner characteristic of supercoiled DNA. We have shown that the degree of supercoiling is the same in control and AT lymphoblastoid cells using sedimentation in the presence of ethidium bromide. The extent of breakage after exposure of cells to gamma-radiation, and the rate of repair of these breaks are similar in both cell types. Rate of repair of strand breaks is dose dependent and the restructured rapidly sedimenting complex behaves similarly, on sucrose gradients containing ethidium bromide, to that extracted from unirradiated cells.
已使用多种不同的检测方法来研究细胞暴露于电离辐射后DNA链断裂的修复情况。这些方法的使用表明，患有共济失调毛细血管扩张症（AT）的患者的成纤维细胞，一种对电离辐射表现出高敏感性的多形遗传病，具有修复DNA链断裂的正常能力。所有这些方法都能确定DNA的断裂程度以及这些断裂的重新封闭情况，但无法提供关于细胞核中DNA构象恢复的信息。在本报告中，我们使用了一种灵敏的技术来研究AT淋巴母细胞暴露于电离辐射后DNA三维结构的恢复情况。该技术提供了一种使用高浓度盐和非离子去污剂裂解细胞的方法，产生称为类核的结构，其包含核RNA和DNA，蛋白质含量低，并以超螺旋DNA特有的方式沉降。我们已经表明，使用溴化乙锭存在下的沉降法，对照和AT淋巴母细胞中的超螺旋程度相同。两种细胞类型在暴露于γ辐射后断裂的程度以及这些断裂的修复速率相似。链断裂的修复速率是剂量依赖性的，并且在含有溴化乙锭的蔗糖梯度上，重组的快速沉降复合物的行为与从未受辐射细胞中提取的复合物相似。
