Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently Tagged Macrodomains.
Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently Tagged Macrodomains.
复制标题
DOI：
10.1007/978-1-4939-8588-3_2
复制
发表时间：
2018
影响因子：
--
通讯作者：
Rebecca Smith;Gyula Timinszky
中科院分区：
文献类型：
--
作者：
Rebecca Smith;Gyula Timinszky
聚(ADP-核糖基化)是一种动态的翻译后修饰，在DNA损伤部位快速添加和移除。PAR化对DNA修复的许多方面都很重要，包括染色质解缩和蛋白质募集。DNA损伤诱导后PAR化水平的可视化通常是使用传统的免疫荧光技术在固定细胞上获得的，这导致时间分辨率有限。在这里，我们描述了一种基于显微镜的方法来跟踪断裂位点的ADP-核糖化。这种方法依赖于用405 nm FRAP激光对表达GFP标记的PAR结合蛋白的Hoechst处理的细胞进行DNA损伤诱导，例如，在DNA损伤部位，PAR结合蛋白的募集提供了PAR化水平的指示。
Poly(ADP-ribosyl)ation (PARylation) is a dynamic posttranslational modification that is added and removed rapidly at sites of DNA damage. PARylation is important for numerous aspects of DNA repair including chromatin decondensation and protein recruitment. Visualization of PARylation levels after DNA damage induction is generally obtained using traditional immunofluorescent techniques on fixed cells, which results in limited temporal resolution. Here, we describe a microscopy-based method to track ADP-ribosylation at break sites. This method relies on DNA damage induction using a 405 nm FRAP laser on Hoechst-treated cells expressing GFP-tagged PAR-binding proteins, such as macrodomains where the recruitment of the PAR-binder to sites of DNA damage gives an indication of PARylation levels.