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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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their functionality and underlying mechanisms of action. However, comparison and systemic interpretation of these data is challenging due to their scattered nature and differing experimental approaches. Here, we extracted, analyzed and compared the available results describing accumulation of 79 DSBR proteins at sites of DNA damage, which can be further explored using Cumulus (http://www.dna-repair.live/cumulus/)-the accompanying interactive online application. Despite large inter-study variability, our analysis revealed that the accumulation of most proteins starts immediately after damage induction, occurs in parallel and peaks within 15-20 min. Various DSBR pathways are characterized by distinct accumulation kinetics with major non-homologous end joining proteins being generally faster than those involved in homologous recombination, and signaling and chromatin remodeling factors accumulating with varying speeds. Our meta-analysis provides, for the first time, comprehensive overview of the temporal organization of the DSBR in mammalian cells and could serve as a reference for future mechanistic studies of this complex process., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)