A model for Ku heterodimer assembly and interaction with DNA. Implications for the function of Ku antigen.
Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371-510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179-510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179-732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases.
Wang
J
J
Departments of Medicine, Microbiology and Immunology, Thurston Arthritis Research Center, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280, USA.
Dong
X
X
Reeves
W H
WH
D034961:Antigens, Nuclear; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D000939:Epitopes; D006801:Humans; D000072200:Ku Autoantigen; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D001324:Autoantigens; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen