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Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.
Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.
- Source :
-
Journal of molecular biology [J Mol Biol] 2003 Feb 07; Vol. 326 (1), pp. 93-103. - Publication Year :
- 2003
-
Abstract
- Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.
- Subjects :
- Antigens, Nuclear chemistry
DNA genetics
DNA Damage
DNA Ligase ATP
DNA-Activated Protein Kinase
DNA-Binding Proteins chemistry
HeLa Cells
Humans
Ku Autoantigen
Macromolecular Substances
Nuclear Proteins metabolism
Phosphorylation
Precipitin Tests
Protein Binding
Protein Subunits
Substrate Specificity
Tumor Cells, Cultured
Antigens, Nuclear metabolism
DNA metabolism
DNA Helicases
DNA Ligases metabolism
DNA Repair
DNA-Binding Proteins metabolism
Protein Serine-Threonine Kinases chemistry
Protein Serine-Threonine Kinases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-2836
- Volume :
- 326
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 12547193
- Full Text :
- https://doi.org/10.1016/s0022-2836(02)01328-1