Wang Degui, Tian Yingxia, Wei Dong, Jing Yuhong, Niu Haitao, Xie Kun, Song Yanfeng
Department of Anatomy and Histology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000, China.
Department of Internal Medicine, Gansu Provincial Academic Institute for Medical Research, Lanzhou, 730050, China.
Biochem Genet. 2016 Apr;54(2):147-57. doi: 10.1007/s10528-015-9707-8. Epub 2015 Dec 30.
DNA damage repair elements accumulate at DNA damage sites to form ionizing radiation-induced foci (IRIF) for damage repair. IRIF, which represent direct evidence of DNA damage response activity, which are conveniently to be observed via immunofluorescence staining. Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, the substrate binding protein E3 ubiquitin-ligases enzymes are recruited to DNA damage sites, then the E2 ubiquitin-conjugating enzymes are recruited to these sites by the E3 where they catalyze protein ubiquitination. However, IRIF of E2 enzymes are relatively transient and unstable in vivo and difficult to detect. Here, we present a new method for the observation of E2 IRIF. This method is based on the co-transfection of interacting E2 and E3 enzymes into cells and identifies IRIF via immunofluorescence following DNA damage.
DNA损伤修复元件在DNA损伤位点聚集，形成电离辐射诱导灶（IRIF）以进行损伤修复。IRIF是DNA损伤反应活性的直接证据，通过免疫荧光染色便于观察。蛋白质泛素化在启动DNA损伤反应中起重要作用。DNA损伤后，底物结合蛋白E3泛素连接酶被招募到DNA损伤位点，然后E2泛素结合酶被E3招募到这些位点，在那里它们催化蛋白质泛素化。然而，E2酶的IRIF在体内相对短暂且不稳定，难以检测。在这里，我们提出了一种观察E2 IRIF的新方法。该方法基于将相互作用的E2和E3酶共转染到细胞中，并在DNA损伤后通过免疫荧光鉴定IRIF。
