Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20
| Autor: | B. K. Singleton, Maria C. Muhlmann-Diaz, Tracy Blunt, Graeme C. M. Smith, Stephen P. Jackson, Anne Priestley, P. A. Jeggo, Leonard C. Schalkwyk, Guillermo E. Taccioli, A. G. Amatucci, Joel S. Bedford, David A. Gell, Heather Beamish |
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| Rok vydání: | 1998 |
| Předmět: |
DNA Repair
Cell Survival DNA damage DNA repair Protein subunit Mutant CHO Cells DNA-Activated Protein Kinase Mice SCID Protein Serine-Threonine Kinases Biology Transfection Polymerase Chain Reaction Cell Line Mice Phosphatidylinositol 3-Kinases chemistry.chemical_compound Cricetinae Genetics Animals Humans Horses Nuclear protein Protein kinase A Chromosomes Artificial Yeast VDJ Recombinases Gene Library Kinase Nuclear Proteins Dose-Response Relationship Radiation DNA Molecular biology DNA-Binding Proteins enzymes and coenzymes (carbohydrates) chemistry Gamma Rays DNA Nucleotidyltransferases biological phenomena cell phenomena and immunity Research Article DNA Damage |
| Zdroj: | Nucleic Acids Research. 26:1965-1973 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/26.8.1965 |
| Popis: | The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity. |
| Databáze: | OpenAIRE |
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