Isolation of Ku70-binding proteins (KUBs).
DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and ... Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.
Mesh Terms:
Animals, Antigens, Nuclear, Base Sequence, Carrier Proteins, Cell Line, Cloning, Molecular, Clusterin, DNA Helicases, DNA Probes, DNA Repair, DNA-Activated Protein Kinase, DNA-Binding Proteins, Glycoproteins, Humans, Mice, Molecular Chaperones, Nuclear Proteins, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tumor Cells, Cultured
Animals, Antigens, Nuclear, Base Sequence, Carrier Proteins, Cell Line, Cloning, Molecular, Clusterin, DNA Helicases, DNA Probes, DNA Repair, DNA-Activated Protein Kinase, DNA-Binding Proteins, Glycoproteins, Humans, Mice, Molecular Chaperones, Nuclear Proteins, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tumor Cells, Cultured
Nucleic Acids Res.
Date: May. 15, 1999
PubMed ID: 10219089
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