Foci in focus Visualizing and analyzing the effects of DNA damage at subcellular and multicellular scales

Double-strand breaks (DSBs) represent one of the most cytotoxic forms of DNA damage, posing a significant threat to genomic stability while also serving as a central mechanism exploited in cancer therapy. This thesis investigates how DSB complexity, DNA-damaging agents, and cellular context influence the DNA damage response, with a focus on repair protein dynamics and methodological innovation in live-cell imaging and functional assays. Complex DSBs generated by high linear energy transfer radiation present clustered lesions that challenge repair machinery yet enhance therapeutic effectiveness, particularly in particle therapy. To enable investigation of these events, a portable live-cell irradiation imaging platform was developed, allowing real-time fluorescence microscopy at accelerator facilities. Proof-of-concept studies revealed asynchronous recruitment of key DDR proteins following α-particle exposure, highlighting the distinct repair kinetics of complex damage.
Super-resolution imaging further demonstrated agent-specific nanoscale organisation of repair proteins, with MDC1 forming diffuse chromatin-associated scaffolds, while RNF168 and 53BP1 displayed more focal distributions reflecting specialised functional roles. To extend analysis beyond mechanistic observation, the LeGO-CSA platform combined fluorescent barcoding with machine-learning segmentation to enable high-throughput assessment of clonal heterogeneity and treatment response. Finally, systematic analysis of DDR dynamics showed that repair kinetics vary with chromatin state, radiation quality, temperature, and dose, with high-LET damage eliciting rapid and robust recruitment of repair factors. Collectively, these findings reveal the multifactorial nature of DSB repair and underscore the importance of damage complexity and cellular context in shaping DDR outcomes, providing insights relevant to both fundamental biology and the optimisation of DNA damage-based cancer therapies.