Formation of clustered DNA damage in vivo upon irradiation with ionizing radiation: Visualization and analysis with atomic force microscopy

Clustered DNA damage is related to the biological effects of ionizing radiation. However, its precise yield and complexity (i.e., number of lesions per damaged site) in vivo remain unknown. To better understand the consequences of clustered DNA damage, a method was established to evaluate its yield and complexity in irradiated cells by atomic force microscopy. This was achieved by isolating and concentrating damaged DNA fragments from purified genomic DNA. It was found that X-rays and Fe ion beams caused clustered DNA damage in human TK6 cells, whereas Fenton's reagents did it less efficiently, highlighting clustered DNA damage as a signature of ionizing radiation. Moreover, Fe ion beams produced clustered DNA damage with high complexity. Remarkably, Fe ion beam–induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying the lethal effect of complex DSBs.