Department of Medical Biology, Amsterdam University Medical Centers (location AMC), Cancer Center Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
Nucleic Acids Res. 2019 Sep 26;47(17):e100. doi: 10.1093/nar/gkz609.
The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.
大多数参与双链 DNA 断裂 (DSB) 处理的蛋白质在损伤部位积累。使用所谓的 UV 激光或重粒子束进行微照射，实时成像和分析这些过程，为深入了解潜在的 DSB 修复机制提供了有价值的见解。为了研究由更具临床相关性的 DNA 损伤剂引发的 DSB 修复反应的时间组织，我们开发了一种称为 X 射线多微束显微镜 (XM3) 的系统，能够同时用超软 X 射线对大量细胞进行高剂量率（微）照射，并对随后的细胞反应进行成像。使用该装置，我们分析了参与哺乳动物 DSB 修复信号通路的 MRE11、MDC1、RNF8、RNF168 和 53BP1 蛋白在非癌细胞和癌细胞中的实时动力学变化，以及在存在或不存在光敏剂的情况下，对 X 射线和 UV 激光诱导的 DNA 损伤的反应。我们的结果首次揭示了 X 射线微照射引发的 DSB 信号转导的动力学，并确立了 XM3 作为实时分析细胞 DSB 修复反应的强大平台。
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