Introduction DNA damage and in particular double-strand breaks (DSBs) pose a major threat to the preservation of genome integrity. Mammalian cells have developed efficient DNA repair mechanisms in response to DNA damage. In the non-homologous end-joining (NHEJ), one major pathway of DSB-repair, the DNA-dependent protein kinase (DNAPK) is supposed to participate in the early stages of DNA end detection and processing. The DNA-PK comprises the Ku subunits (Ku70 and Ku80) and the catalytic subunit DNAPKcs. The aim of this study was to visualize and analyse the recruitment of DNAPKcs to ionizing radiation induced DNA DSBs.

Biological effects in unirradiated cells located near to irradiated cells are called bystander effects. Targeted irradiation of single cells by means of the heavy ion microprobe developed at GSI [1] was used to improve our understanding of these effects on a molecular and cellular basis. The protein of our interest was the cyclindependent kinase inhibitor CDKN1A (p21). Based on previous results obtained by broadbeam irradiation, we investigated the spatial distribution of the induction of p21 protein on the level of single bystander cells. We targeted single cells (3%) of a confluent monolayer of normal human fibroblasts (AG1522C) with 5 particles of carbon ions in 2 crossed lines per nucleus. Three hours after exposure we performed immunofluorescence (IF) staining for CDKN1A and 53BP1 protein. 53BP protein is recruited to sites of DNA damage immediately after irradiation by forming foci and is used to identify the targeted cells unambiguously (see figure 1a: the irradiated cell indicated with an arrow and enlarged in the box).