γH2AX foci analysis for monitoring DNA double-strand break repair Strengths, limitations and optimization
γH2AX foci analysis for monitoring DNA double-strand break repair Strengths, limitations and optimization
复制标题
DOI：
10.4161/cc.9.4.10764
复制
发表时间：
2010-02-15
期刊：
影响因子：
4.3
通讯作者：
Jeggo, Penny A.
中科院分区：
文献类型：
--
作者：
Loebrich, Markus;Shibata, Atsushi;Jeggo, Penny A.
DNA双链断裂（DSB）是一种重要的辐射诱导的损伤，受损的DSB修复提供了与辐射敏感性的最佳相关性。用于监测DSB修复的物理技术需要高的非生理剂量，并且不能可靠地检测细微缺陷。对DNA损伤反应的广泛研究的一个结果是观察到H2AX（组蛋白H2A的变体形式）在DSB处经历广泛的磷酸化，产生可以通过免疫荧光可视化的γ H2AX病灶。γ H2AX病灶和DSB数量之间以及病灶丢失率和DSB修复之间存在密切相关性，提供了使用生理剂量监测单个细胞中DSB修复的灵敏测定。然而，γ H2AX形成可以发生在复制或修复过程中出现的单链DNA区域，因此不仅仅与DSB形成相关。在这里，我们提出并讨论的证据表明，暴露于电离辐射，γ H2AX焦点分析可以提供一个敏感的监测DSB的形成和修复，并描述技术，以优化分析。我们讨论了该技术的局限性和好处，使程序得到最佳利用，但不滥用。
DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating gamma H2AX foci that can be visualized by immunofluorescence. There is a close correlation between gamma H2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, gamma H2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionizing radiation, gamma H2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimize the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.