Every day, genomes are affected by genotoxic factors that create multiple DNA lesions. Several DNA repair systems have evolved to counteract the deleterious effects of DNA damage. These systems include a set of DNA repair mechanisms, damage tolerance processes, and activation of cell-cycle checkpoints. This study describes selected confocal microscopy techniques that investigate DNA damage-related nuclear events after UVA- and γ-irradiation and compare the DNA damage response (DDR) induced by the two experimental approaches. In both cases, we observed induction of the nucleotide excision repair (NER) pathway and formation of localized double-strand breaks (DSBs). This was confirmed by analysis of cyclobutane pyrimidine dimers (CPDs) in the DNA lesions and by increased levels of γH2AX and 53BP1 proteins in the irradiated genome. DNA damage by UVA-lasers was potentiated by either BrdU or Hoechst 33342 pre-sensitization and compared to non-photosensitized cells. DSBs were also induced without BrdU or Hoechst 33342 pre-treatment. Interestingly, no cyclobutane pyrimidine dimers (CPDs) were detected after 405 nm UVA laser micro-irradiation in non-photosensitized cells. The effects of UVA and γ-irradiation were also studied by silver staining of nucleolar organizer regions (AgNORs). This experimental approach revealed changes in the morphology of nucleoli after genome injury. Additionally, to precisely characterize DDR in locally induced DNA lesions, we analysed the kinetics of the 53BP1 protein involved in DDR by fluorescence recovery after photobleaching (FRAP).

Institute of Biophysics Academy of Sciences of the Czech Republic v v i Brno Czech Republic

Institute of Cellular Biology and Pathology 1st Faculty of Medicine Charles University Prague Czech Republic