Figure 5.

MAD2L2 is recruited at DSBs by protein interaction with the 53BP1-RIF1 complex and suppresses BRCA1 accumulation. a) HeLa cells were irradiated with 10 Gy and 53BP1 was immuno-precipitated after 2 h recovery. H4K20me2 and MAD2L2 were co-purified with 53BP1 exclusively upon irradiation. b) Cells expressing FLAG-tagged MAD2L2 were irradiated and FLAG immunoprecipitation was performed after 2 h recovery. Western blot analysis of inputs, supernatant and elution shows that FLAG-MAD2L2 is efficiently and equally purified from both non-irradiated and irradiated cells. c) 53BP1, RIF1 and ATM were identified by mass spectrometry analysis as novel protein interactors of MAD2L2 together with 7 previously known MAD2L2 protein interactors. PSM (peptide spectrum matches) counts are shown for 2 independent experiments. Specificity of the interaction is addressed in a smaller scale FLAG-MAD2L2 immunoprecipitation (Supplementary Figure 1). d) U2OS cells transfected with control GFP vector (EV) or GFP-MAD2L2 where irradiated with 10 Gy at 48 h after transfection, fixed at 1 h post IR and stained for Cyclin A (serving as S/G2 marker) and BRCA1 to assess BRCA1 accumulation to DSBs in S/G2 (n = 2 independent replicates).