Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair.
The Chinese hamster ovary (CHO) mutant cell line xrs-6C is highly sensitive to radiation and is deficient in DNA double-strand break (DSB) repair. The repair defect of xrs-6C is complemented by the human DSB repair gene designated as XRCC5. This gene was recently identified as Ku80, which encodes the human autoantigen protein Ku p80. Ku80 protein forms heterodimer with the Ku70 subunit to form a complex that possesses a DNA end-binding activity. Ku70/Ku80 heterodimer can recruit the catalytic p350 subunit of the DNA-dependent protein kinase. It is demonstrated here that, while the Ku70 mRNA expression is normal in the xrs-6C mutant, Ku70 protein is undetectable. However, introduction of human Ku80 gene into the mutant lead to increased expression of Ku70 protein and restored Ku70 binding to DNA ends, suggesting that mutation of the Ku80 gene affected the formation of Ku70/Ku80 dimers and the stability of the Ku70 protein. We also demonstrated that, although p350 protein expression in the mutants was unaffected, the capacity of p350 to bind to DNA ends was impaired in the mutants. After introduction of the human Ku80 into the mutant, the association of p350 with DNA end was restored, accompanied by recovery in cell survival and DNA double-strand break repair. The results in this report show that mutation of the Ku80 gene disrupts formation of the Ku70/Ku80 dimer and compromises the ability of Ku protein to recruit the DNA-PK p350 subunit to DNA double-strand breaks, causing a dysfunction of DNA DSB repair in the cell.
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
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D000818:Animals; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D006224:Cricetinae; D004265:DNA Helicases; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015345:Oligonucleotide Probes; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D034961:Antigens, Nuclear; D001324:Autoantigens; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; 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