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Abstract:
The induction of localized DNA damage within a discrete nuclear volume is an important tool in DNA repair studies. Both charged particle irradiation and laser microirradiation (LMI) systems allow for such a localized damage induction, but the results obtained are difficult to compare, as the delivered laser dose cannot be measured directly. Therefore, we revisited the idea of a biological dosimetry based on the microscopic evaluation of irradiation-induced Replication Protein A (RPA) foci numbers. Considering that local dose deposition is characteristic for both LMI and charged particles, we took advantage of the defined dosimetry of particle irradiation to estimate the locally applied laser dose equivalent. Within the irradiated nuclear sub-volumes, the doses were in the range of several hundreds of Gray. However, local dose estimation is limited by the saturation of the RPA foci numbers with increasing particle doses. Even high-resolution 4Pi microscopy did not abrogate saturation as it was not able to resolve single lesions within individual RPA foci. Nevertheless, 4Pi microscopy revealed multiple and distinct 53BP1- and γH2AX-stained substructures within the lesion flanking chromatin domains. Monitoring the local recruitment of the telomere repeat-binding factors TRF1 and TRF2 showed that both proteins accumulated at damage sites after UVA–LMI but not after densely ionizing charged particle irradiation. Hence, our results indicate that the local dose delivered by UVA–LMI is extremely high and cannot be accurately translated into an equivalent ionizing radiation dose, despite the sophisticated techniques used in this study.
