Isolation of Ku70-binding proteins (KUBs)

DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DN A double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRC C4 and DNA ligase IV, other proteins which play a role(s) in this repair pa thway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80), We isolated Ku70-binding proteins (KUB 1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XI P8), testosterone-repressed prostate message-2 (TRPM-5) and clusterin. KUB2 is Ku80, KUB3 and KUB4 are unknown, >10 kb transcripts. Interactions of ap oJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respect ively. 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 caus ed decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoc lonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/se nsitivity is under investigation.