Mah Li-Jeen, Vasireddy Raja S, Tang Michelle M, Georgiadis George T, El-Osta Assam, Karagiannis Tom C
Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct.
J Vis Exp. 2010 Apr 6(38):1957. doi: 10.3791/1957.
DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX(1). Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)(2). Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB(2,3). This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning approximately 2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete gammaH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy(2). The loss of gammaH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary(4-8). The disappearence of gammaH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C(5,6). Further, removal of gammaH2AX by redistribution involving histone exchange with H2A.Z has been implicated(7,8). Importantly, the quantitative analysis of gammaH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of gammaH2AX foci in gamma-irradiated adherent human keratinocytes(9).
DNA双链断裂（DSBs）可由内源性代谢过程或外源性因素诱导产生，是关乎生存及基因组完整性维持的最关键的DNA损伤之一。对DSBs诱导的早期反应是组蛋白变体H2A的H2AX在丝氨酸139残基处发生磷酸化，该残基位于高度保守的C端SQEY基序中，形成γH2AX(1)。DSBs诱导后，H2AX会被磷脂酰肌醇3激酶（PIKK）家族的蛋白质——共济失调毛细血管扩张突变蛋白（ATM）、DNA蛋白激酶催化亚基以及ATM和RAD3相关蛋白（ATR）迅速磷酸化(2)。通常情况下，一个DSB仅涉及少数几个碱基对（bp），然而，鉴于染色质修饰在DNA损伤信号传导和修复中的重要性，会有显著的信号放大。主要由ATM介导的H2AX磷酸化会扩散至染色质的相邻区域，每个DSB会影响约0.03%的细胞总H2AX(2,3)。这相当于约2000个H2AX分子发生磷酸化，跨越围绕DSB位点的约2 Mbp染色质区域，导致形成离散的γH2AX焦点，可通过免疫荧光显微镜轻松观察和定量(2)。DSB处γH2AX的消失反映了修复过程，然而，对于如何定义DSBs的完全修复存在一些争议；有人提出DNA两条链重新连接就足够了，然而也有人认为恢复原始紧密染色质状态是必要的(4 - 8)。γH2AX的消失至少部分涉及磷酸酶（磷酸酶2A和磷酸酶4C）的去磷酸化作用(5,6)。此外，有人认为通过与H2A.Z进行组蛋白交换的重新分布来去除γH2AX也有作用(7,8)。重要的是，γH2AX焦点的定量分析已在医学和核研究中得到广泛应用。在此，我们展示了通过检测和定量γ射线照射的贴壁人角质形成细胞中的γH2AX焦点来评估初始DNA损伤的最常用免疫荧光方法(9)。
