U2OS cells were transiently transfected with pEYFP-MRE11 (WT), MRE11S676AS678A (MUT), MRE11S676DS678D (DD), MRE11S676A (S676A) or MRE11S678A (S678A) plasmids respectively using electroporation (Amaxa Nucleofector, Germany) 24 h prior to irradiation. Charged particle irradiation was done at the UNILAC accelerator at GSI using 9.8 MeV/u carbon ions (LET 170 keV/μm) or 4.7 MeV/u uranium ions (LET 15000 keV/μm) under a low angle as described previously (43 (link)). Cells were fixed with 2% paraformaldehyde unirradiated or 10 min, 1, 4 or 12 h post-irradiation. Immunostaining with γH2AX (Millipore) was done according to Jakob et al. (44 (link)). DNA was counterstained with 1 μg per ml DAPI. Microscopic imaging was performed using a spinning disc confocal microscope (Nikon Eclipse Ti with Yokogawa CSU_X1) utilizing a Plan APO 100 × 1.4 NA oil immersion lens or a Leica SPE laser scanning confocal (Planapo 63× 1.3 NA). Optical sections were recorded in increments of 300 μm across the thickness of cells. The total magnification of the systems yielded pixels corresponding to 72 × 72 nm in lateral dimensions. Real time recruitment kinetics of wild-type (WT) and mutant YFP-MRE11 after carbon or uranium ion irradiation was evaluated using the GSI beamline microscope as described previously (45 (link)). About 32 to 55 nuclei were analysed for each plasmid (8 samples for each condition).