Histone H2AX phosphorylation: a marker for DNA damage.
Histone H2AX phosphorylation: a marker for DNA damage.
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DOI：
10.1007/978-1-61779-998-3_40
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发表时间：
2012-01-01
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影响因子：
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通讯作者：
Almasan, Alexandru
中科院分区：
文献类型：
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作者：
Sharma, Arishya;Singh, Kamini;Almasan, Alexandru
DNA损伤反应可以响应于生理过程中遇到的各种应激信号或响应于外源性线索（例如电离辐射或DNA损伤治疗剂）而启动。已经开发了许多方法来检查DNA损伤反应期间发生的形态学、生物化学和分子变化。当细胞暴露于电离辐射或DNA损伤化疗剂时，产生双链断裂（DSB），其迅速导致组蛋白H2 A变体H2 AX的磷酸化。由于H2 AX在Ser 139（γ-H2 AX）处的磷酸化是丰富的、快速的，并且与每个DSB良好相关，因此它是可用于检查所产生的DNA损伤和随后的DNA损伤修复的最灵敏的标记物。γ-H2 AX可通过免疫印迹和免疫染色使用显微镜或流式细胞术检测来检测。由于γ-H2 AX也可以在DNA复制过程中产生，作为细胞凋亡的结果，或者因为它被发现与残留的DNA损伤相关，所以确定γ-H2 AX相关病灶的动力学、数量、大小和形态是重要的。本章介绍了一些标准协议，我们已经成功地在我们的实验室中使用了一些实验系统，主要是血液和上皮细胞培养。
The DNA damage response can be initiated in response to a variety of stress signals that are encountered during physiological processes or in response to exogenous cues, such as ionizing radiation or DNA-damaging therapeutic agents. A number of methods have been developed to examine the morphological, biochemical, and molecular changes that take place during the DNA damage response. When cells are exposed to ionizing radiation or DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of histone H2A variant H2AX. Because phosphorylation of H2AX at Ser 139 (gamma-H2AX) is abundant, fast, and correlates well with each DSB, it is the most sensitive marker that can be used to examine the DNA damage produced and the subsequent repair of the DNA lesion. gamma-H2AX can be detected by immunoblotting and immunostaining using microscopic or flow cytometric detection. Since gamma-H2AX can be also generated during DNA replication, as a consequence of apoptosis, or as it is found associated with residual DNA damage, it is important to determine the kinetics, number, size, and morphology of gamma-H2AX-associated foci. This chapter describes a few standard protocols that we have successfully used in our laboratory for a number of experimental systems, primarily hematologic and epithelial cells grown in culture.