Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.
Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.
Jin
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Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
Weaver
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D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D002460:Cell Line; D016384:Consensus Sequence; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004307:Dose-Response Relationship, Radiation; D005816:Genetic Complementation Test; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D017386:Sequence Homology, Amino Acid
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; 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