Fig. 5: H2AX C-terminal linker region interacts with 53BP1 via the oligomerization and tudor domains.
From: Evolved histone tail regulates 53BP1 recruitment at damaged chromatin
a Domain organization of 53BP1. Oligomerization, GAR, Tudor, UDR, and BRCT domains are indicated and the minimal IRIF forming region is denoted as containing the oligomerization, GAR, Tudor, and UDR domains. b Representative immunofluorescence images of Myc-53BP1 IRIF fragment (a.a.1220–1639) localization with endogenous 53BP1 in H2AX KO with stable reconstitution of SFB-H2AX Y142L at 1 h after 10 Gy radiation. Experiment was performed independently three times with similar results. c Pulldown assay with purified recombinant proteins MBP or MBP-53BP1 IRIF formation fragment a.a. 1220–1639 using biotinylated phosphorylated S139 H2AX C-terminal tail peptide. Repeated three times independently with similar results. d Pulldown assay with cell lysates expressing Myc-53BP1 IRIF formation fragment a.a. 1220-1639, MDC1 tandem BRCT ___domain or MDC1 BRCT ___domain with K1936M mutation using biotinylated phosphorylated S139 H2AX C-terminal tail peptide. Repeated three times independently with similar results. e Pulldown assay with cell lysates expressing Myc-53BP1 IRIF formation fragment using biotinylated phosphorylated S139 H2AX C-terminal tail peptide with or without the 18 linker residues. Repeated three times independently with similar results. f Pulldown assay with cell lysates expressing Myc-53BP1 IRIF formation fragment or Myc-Crb2 a.a. 108–508 using biotinylated phosphorylated S139 H2AX C-terminal tail peptide. Repeated three times independently with similar results. Source data are provided as Source Data file. g Pulldown assay with cell lysates expressing Myc-53BP1 IRIF formation fragment using biotinylated phosphorylated S139 H2AX C-terminal tail peptide or biotinylated phosphorylated S139 H2AX C-terminal tail with Y142L mutation. Repeated three times independently with similar results. h Schematic diagram of 53BP1 fragments used in (i). i Pulldown assay of Myc-53BP1 IRIF wildtype or deletion mutants using biotinylated phosphorylated S139 H2AX C-terminal tail peptide. Repeated three times independently with similar results. Source data are provided as Source Data file.
