Abramenkovs Andris, Stenerlöw Bo
Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
Radiat Res. 2017 Dec;188(6):597-604. doi: 10.1667/RR14693.1. Epub 2017 Sep 27.
Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or γ-H2AX) or flow cytometric analysis of γ-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, γ-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than γ-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of γ-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of γ-H2AX.
细胞中DNA双链断裂（DSB）的失控产生被视为一种严重威胁细胞存活的毒性事件。辐射诱导的DNA DSB通常通过脉冲场凝胶电泳、对积累的DNA损伤反应蛋白（如53BP1或γ-H2AX）进行显微镜评估或对γ-H2AX进行流式细胞术分析来测量。流式细胞术分析的优点是可以研究DSB的形成和修复与细胞周期阶段或其他蛋白质表达的关系。然而，γ-H2AX无法监测照射后最初60分钟内的修复动力学，而这一时期大多数DSB会进行修复。非同源末端连接修复中的一个关键蛋白是DNA依赖性蛋白激酶的催化亚基。在DNA依赖性蛋白激酶的几个磷酸化位点中，第2609位的苏氨酸（T2609）由共济失调毛细血管扩张症突变蛋白（ATM）或DNA依赖性蛋白激酶催化亚基本身磷酸化，可激活DSB的末端加工。我们在此通过流式细胞术表明，T2609处的磷酸化对DSB的反应比γ-H2AX更快。此外，对T2609进行流式细胞术分析比分析γ-H2AX能更好地反映快速修复动力学。在连接酶IV活性降低的细胞以及DNA依赖性蛋白激酶活性受到抑制的野生型细胞中，通过对T2609进行流式细胞术评估观察到DSB修复能力降低。总之，对DNA依赖性蛋白激酶T2609进行流式细胞术评估可作为早期DSB修复的标志物，并且比分析γ-H2AX能更好地反映早期修复事件。
