Recruitment Kinetics of XRCC1 and RNF8 Following MeV Proton and α-Particle Micro-Irradiation

SIMPLE SUMMARY: We used the charged-particle microbeam installed at the AIFIRA facility to perform micro-irradiation experiments and measure the recruitment kinetics of DNA signaling and repair proteins. We developed and validated image acquisition and processing methods to enable a systematic study of the recruitment kinetics of two GFP-tagged proteins (GFP-XRCC1 and GFP-RNF8) after irradiation with protons and α-particles. Online measurement of fluorescence intensity and recruitment time as a function of particle type and number allowed us to characterize the differences in behavior between the two proteins. ABSTRACT: Time-lapse fluorescence imaging coupled to micro-irradiation devices provides information on the kinetics of DNA repair protein accumulation, from a few seconds to several minutes after irradiation. Charged-particle microbeams are valuable tools for such studies since they provide a way to selectively irradiate micrometric areas within a cell nucleus, control the dose and the micro-dosimetric quantities by means of advanced detection systems and Monte Carlo simulations and monitor the early cell response by means of beamline microscopy. We used the charged-particle microbeam installed at the AIFIRA facility to perform micro-irradiation experiments and measure the recruitment kinetics of two proteins involved in DNA signaling and repair pathways following exposure to protons and α-particles. We developed and validated image acquisition and processing methods to enable a systematic study of the recruitment kinetics of GFP-XRCC1 and GFP-RNF8. We show that XRCC1 is recruited to DNA damage sites a few seconds after irradiation as a function of the total deposited energy and quite independently of the particle LET. RNF8 is recruited to DNA damage sites a few minutes after irradiation and its recruitment kinetics depends on the particle LET.