Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of ...
regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80.
Mesh Terms:
Amino Acid Sequence, Antigens, Nuclear, Bacterial Proteins, DNA Helicases, DNA Repair, DNA, Complementary, DNA-Activated Protein Kinase, DNA-Binding Proteins, Gene Deletion, Gene Library, Glutathione Transferase, Hela Cells, Humans, Models, Genetic, Molecular Sequence Data, Nuclear Proteins, Peptides, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Sequence Homology, Amino Acid
Nucleic Acids Res.
Date: Sep. 01, 1999
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