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.

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Journal Nucleic Acids Research
Kochan, J.A. (Jakub A.), van den Belt, M. (Matthias), von der Lippe, J. (Julia), Desclos, E.C.B. (Emilie C B), Steurer, B, Hoebe, R.A, … Krawczyk, P.M. (2019). Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage. Nucleic Acids Research, 47(17). doi:10.1093/nar/gkz609