Elia M C, Bradley M O
Department of Genetic and Cellular Toxicology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Cancer Res. 1992 Mar 15;52(6):1580-6.
Pulsed field gel electrophoresis was used to examine the influence of chromatin structure on the induction of DNA double strand breaks by gamma-irradiation in CHO-WBL cells, nuclei, and a series of protein-depleted chromatin substrates. We developed a method to isolate intact nuclei in agarose plugs that avoids DNA shearing and nucleolytic degradation during sample preparation, and facilitates nuclear protein extraction. Agarose plug-isolated nuclei are extracted with increasing concentrations of NaCl to selectively strip off: (a) nonhistone chromosomal proteins (NHP); (b) NHP and histone H1; (c) NHP, H1, and histone H2A-H2B dimers; or (d) NHP, H1, and H2A-H2B dimers and histone H3-H4 tetramers. Following treatment with up to 40 Gy of gamma-radiation, DNA from each sample is purified and the relative induction of DNA double strand breaks is assayed by asymmetric field inversion gel electrophoresis. At a dose of 20 Gy, removal of nonhistone proteins from nuclei results in a 3-fold increase in DNA double strand breaks, compared to intact CHO cells. Additional stripping of histone H1 results in an incremental increase in double strand break induction, whereas further removal of H2A-H2B dimers yields a greater than 10-fold increase in DNA double strand breaks compared to intact CHO cells. The dose-response profile for this latter sample is similar to that observed for purified DNA. These data indicate that distinct classes of chromosomal proteins afford the DNA with different levels of protection against gamma-ray-induced DNA double strand breaks. Thus, chromatin domains that differ in tertiary structure and protein composition may also differ in their susceptibility to DNA double strand breaks induced by ionizing radiation and, perhaps, other clastogens.
脉冲场凝胶电泳用于检测染色质结构对CHO-WBL细胞、细胞核以及一系列蛋白质缺失的染色质底物中γ射线诱导DNA双链断裂的影响。我们开发了一种在琼脂糖凝胶块中分离完整细胞核的方法，该方法可避免样品制备过程中的DNA剪切和核酸酶降解，并便于核蛋白提取。用浓度递增的NaCl提取琼脂糖凝胶块分离的细胞核，以选择性去除：(a)非组蛋白染色体蛋白（NHP）；(b) NHP和组蛋白H1；(c) NHP、H1和组蛋白H2A-H2B二聚体；或(d) NHP、H1和H2A-H2B二聚体以及组蛋白H3-H4四聚体。用高达40 Gy的γ射线处理后，纯化每个样品的DNA，并通过不对称场反转凝胶电泳检测DNA双链断裂的相对诱导情况。在20 Gy的剂量下，与完整的CHO细胞相比，从细胞核中去除非组蛋白会导致DNA双链断裂增加3倍。进一步去除组蛋白H1会使双链断裂诱导量逐渐增加，而与完整的CHO细胞相比，进一步去除H2A-H2B二聚体会使DNA双链断裂增加超过10倍。后一个样品的剂量反应曲线与纯化DNA观察到的相似。这些数据表明，不同类别的染色体蛋白为DNA提供了不同程度的保护，以抵御γ射线诱导的DNA双链断裂。因此，三级结构和蛋白质组成不同的染色质结构域对电离辐射及可能的其他致断裂剂诱导的DNA双链断裂的敏感性也可能不同。
