title: Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation.
abstract: The Mre11.Rad50.nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11.Rad50.nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, we have performed site-directed mutagenesis of conserved residues in these motifs. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11.Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently.
authors.affiliation: Molecular Genetics Program, Virginia Mason Research Center, University of Washington School of Medicine, Seattle, WA 98101-2795, USA.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Cerosaletti
authors.forename: Patrick
authors.initials: P
authors.lastname: Concannon
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry*; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012190:Retroviridae / Q000235:genetics; D016196:S Phase
chemical_list: D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C112156:NBN protein, human; D009687:Nuclear Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases