Rogakou E P, Pilch D R, Orr A H, Ivanova V S, Bonner W M
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1998 Mar 6;273(10):5858-68. doi: 10.1074/jbc.273.10.5858.
When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. gamma-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes gamma-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 x 10(6) base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, gamma-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.
当哺乳动物细胞培养物或小鼠受到可存活剂量或致死剂量的电离辐射时，在二维凝胶的组蛋白H2A区域会发现新的质谱成分。这些成分统称为γ，它们在体内通过几种能在DNA中引入双链断裂的过程形成。γ成分似乎是细胞暴露于电离辐射后，在乙酸-尿素- Triton X - 100 -乙酸-尿素-十六烷基三甲基溴化铵或SDS -乙酸-尿素-十六烷基三甲基溴化铵凝胶上通过质谱或32P04掺入检测到的唯一主要新成分，结果表明它们是在丝氨酸139处发生特异性磷酸化的组蛋白H2AX种类。细胞培养物暴露于电离辐射后，γ-H2AX迅速出现；1分钟时达到最大量的一半，10分钟时达到最大量。在最大值时，每格雷电离辐射约1%的H2AX发生γ磷酸化，这一发现表明，每格雷引入哺乳动物G1基因组6×10^9个碱基对中的35个DNA双链断裂，会导致分布在1%染色质上的H2AX发生γ磷酸化。因此，每个DNA双链断裂似乎涉及约0.03%的染色质。该值对应于每个双链断裂约2×10^6个碱基对的DNA，表明每个DNA双链断裂涉及大量染色质。因此，γ-H2AX的形成是细胞对DNA双链断裂存在的快速且敏感的反应，这种反应可能有助于深入了解高阶染色质结构。
