Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity

Abstract

Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs). These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity. These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.