Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity.
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.
Han
Z
Z
Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.
Johnston
C
C
Reeves
W H
WH
Carter
T
T
Wyche
J H
JH
Hendrickson
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001483:Base Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004265:DNA Helicases; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D014644:Genetic Variation; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D000072200:Ku Autoantigen; D008322:Mammals; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D015342:DNA Probes; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen