Fig. 2: Mutations in globular domain residues of Mre11-Rad50 partially destabilize complex formation and interaction with Xrs2.

A The boxed panels show two views rotated by 90° of protein-protein interactions between the Mre11 capping domain and Rad50 head domain in the MR-dsDNA complex. Conserved Rad50 E1155, D1167 and E1243 residues and Mre11 N387, R389, R390, K410 and R412 residues are labeled in the boxed segments. B Evolutionary conservation of Rad50 (top) and Mre11 (bottom) residues mediating protein-protein interactions. Tm, Thermotoga maritima; Ct, Chaetomium thermophilum; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Mm, Mus musculus; Hs, Homo sapiens. Rad50 and Mre11 tumor alleles and their number of instances (in brackets) are depicted below the alignments. C CPT-survival of rad50 (top) and mre11 (bottom) mutants in the Mre11-Rad50 interaction interface. Plates were either incubated at 30 °C (left panel) or 37 °C (right panel). The following abbreviations were used: rad50-MRI-3A (rad50-E1155A D1167A E1243A); mre11-MRI-5A1 (K322A N387A R389A R390A R412A); mre11-MRI-5A2 (K322A N387A R389A R390A K410A); mre11-MRI-6A (K322A N387A R389A R390A K410A R412A). D Assessment of the Mre11-Rad50 and Mre11-Xrs2 interaction by co-immunoprecipitation. Cells were either grown at 30 °C (left panel) or 37 °C (right panel) and Rad50, Mre11 or Xrs2 were immunoprecipitated from the prepared cell extracts (labeled on the left) and probed with anti-Rad50, anti-Mre11 or anti-Xrs2 antibodies (labeled on the right) by Western blot. Molecular size markers in kDa are indicated on the left. Images shown are representative of three independent experiments with comparable results. Uncropped Western blots are provided as a source data file. E Assessment of the Mre11-Rad50 and Mre11-Xrs2 interaction by the yeast 2-hybrid assay. Rad50, Mre11 and Xrs2 were expressed as a fusion with the Gal4-DNA Binding Domain (pGBD; TRP1) or the Gal4-Activation Domain (pGAD; LEU2) as indicated. Reporter activation assessed by growth in the absence of histidine (Do-TLH) and in the absence of adenine and histidine (Do-TLAH) were assessed in WT, mre11-MRI-6A and mre11-E38K mutants. Empty plasmids were included as a negative control.