Induction of DNA double strand breaks by long wavelength UV-A exposure and their repair

Full text: UV-A irradiation has been assumed to be a relatively harmless genotoxic compound that should not induce severe DNA double strand breaks (dsbs). Recent findings, however, have indicated that UV irradiation at lambda>320nm induces dsbs, too. These findings are based on the detection of DNA fragmentation. In this work we additionally show the activation of the double strand break repair pathways after UV-A (365nm, 160-1280 kJ/m2) exposure in situ. By immunohistochemical detection of Rad51 foci formation, the involvement of the homologous recombination repair (HRR) is shown. Also the formation of gamma-H2AX foci was observed, which is a specific phosphorylation at ser139 of histone 2A. This phosphorylation is an early event in the recognition of dsbs and is correlated to the non homologous end joining (NHEJ) pathway. Close spatial proximity of the Rad51 and the gamma-H2AX foci is visualised by 3D fluorescence microscopy. Western blotting shows a more than 7 fold increase of the gamma-H2AX protein in the first 20 minutes during irradiation and a quick decrease after exposure. In contrast the amount of Rad51 protein is only increased by 20%. A time course of the formation of Rad51 and gamma-H2AX foci during irradiation suggests that the detected dsbs are induced directly and are not a result of stalled replication in S-phase. Micronucleus induction during the first mitosis after irradiation has proven that chromosomal aberrations are also induced by UV-A exposure. RNA expression profiles of 164 DNA damage and repair related genes have been analysed using a custom made DNA gene array. The data obtained at 2 and 5 hours after acute exposure to 700 kJ/m2 UV-A as well as after an chronicle exposure during 3 days, support the findings from western blotting analysis, that only a weak up-regulation of repair proteins from the NHEJ and HRR pathways is observed