Double‐strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions
Identifieur interne : 002360 ( Istex/Curation ); précédent : 002359; suivant : 002361Double‐strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions
Auteurs : Shengfang Jin [États-Unis] ; David T. Weaver [États-Unis]Source :
- The EMBO Journal [ 0261-4189 ] ; 1997-11-15.
English descriptors
- Teeft :
- Amino, Amino acid residues, Assay, Autoantigen, Bamhi, Binding, Binding activity, Binding properties, Biochemical features, Biol, Boubnov, Cdna, Cell extracts, Cell line, Cell lines, Cell lysates, Cells reconstituted, Chimera, Chimeric, Complementation, Core region, Derivative, Edta, Epitope, Expression vector, Hdf1, Heterodimerization, Higher mobility, Immunoprecipitation, Kinase, Kinase buffer, Lieber, Lysates, Lysis, Lysis buffer, Monoclonal, Monoclonal antibody, Mutagenesis, Mutant, Mutation, Mutational analysis, Nacl, Natl acad, Noti, Nucleic acids, Pefpuro, Peptide, Phosphoamino acid analysis, Phosphorylated, Phosphorylation, Primer, Protein kinase, Recombination, Reconstituted, Repair, Repair defects, Saccharomyces cerevisiae, Serine, Stably, Stably transfected, Subunit, Truncation, Truncation mutants, Yeast, Yeast expression vector.
Abstract
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.
Url:
DOI: 10.1093/emboj/16.22.6874
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<term>Amino acid residues</term>
<term>Assay</term>
<term>Autoantigen</term>
<term>Bamhi</term>
<term>Binding</term>
<term>Binding activity</term>
<term>Binding properties</term>
<term>Biochemical features</term>
<term>Biol</term>
<term>Boubnov</term>
<term>Cdna</term>
<term>Cell extracts</term>
<term>Cell line</term>
<term>Cell lines</term>
<term>Cell lysates</term>
<term>Cells reconstituted</term>
<term>Chimera</term>
<term>Chimeric</term>
<term>Complementation</term>
<term>Core region</term>
<term>Derivative</term>
<term>Edta</term>
<term>Epitope</term>
<term>Expression vector</term>
<term>Hdf1</term>
<term>Heterodimerization</term>
<term>Higher mobility</term>
<term>Immunoprecipitation</term>
<term>Kinase</term>
<term>Kinase buffer</term>
<term>Lieber</term>
<term>Lysates</term>
<term>Lysis</term>
<term>Lysis buffer</term>
<term>Monoclonal</term>
<term>Monoclonal antibody</term>
<term>Mutagenesis</term>
<term>Mutant</term>
<term>Mutation</term>
<term>Mutational analysis</term>
<term>Nacl</term>
<term>Natl acad</term>
<term>Noti</term>
<term>Nucleic acids</term>
<term>Pefpuro</term>
<term>Peptide</term>
<term>Phosphoamino acid analysis</term>
<term>Phosphorylated</term>
<term>Phosphorylation</term>
<term>Primer</term>
<term>Protein kinase</term>
<term>Recombination</term>
<term>Reconstituted</term>
<term>Repair</term>
<term>Repair defects</term>
<term>Saccharomyces cerevisiae</term>
<term>Serine</term>
<term>Stably</term>
<term>Stably transfected</term>
<term>Subunit</term>
<term>Truncation</term>
<term>Truncation mutants</term>
<term>Yeast</term>
<term>Yeast expression vector</term>
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<front><div type="abstract">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.</div>
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