Fig. 4. Essential domain of 53BP1 for targeting to the nuclear foci induced by IR. (A) Schematic diagram of GFP-fusion constructs. The various portions of mouse 53BP1 were fused at their N-terminus (in some cases at their C-terminus) to GFP and tested for their ability to target (+) or not (–) to the IR-induced 53BP1 foci. The positions of the RG stretch (RG), Tudor motifs, nuclear localization signal (NLS) and BRCT motifs are boxed. (B) Amino acid sequence alignment of the RG-stretch in mouse, human and Xenopus 53BP1. (C-M) HeLa cells were transfected with the GFP-fusion constructs indicated, treated with (+) or without (–) irradiation of 10 Gy X rays and observed under a fluorescent microscope. (N) Endogenous 53BP1 was visualized with an antibody to the N-terminus of 53BP1 (red) that does not recognize the M (minimum) domain, in NIH3T3 cells expressing GFP-M (green). (O,P) GFP-M fusion protein (green) was transiently expressed in the mutant MEF (–/–), irradiated with 10 Gy X rays, fixed, and stained with anti-H2AX antibodies (Cy3 in red) or with anti-N-terminus m53BP1 (Cy3 in red). Bars, 10 µm.