RESUMO
The DNA-dependent protein kinase is a mammalian protein complex composed of Ku70, Ku80, and DNA-PKcs subunits that has been implicated in DNA double-strand break repair and V(D)J recombination. Here, by gene targeting, we have constructed a mouse with a disruption in the kinase domain of DNA-PKcs, generating an animal model completely devoid of DNA-PK activity. Our results demonstrate that DNA-PK activity is required for coding but not for signal join formation in mice. Although our DNA-PKcs defective mice closely resemble Scid mice, they differ by having elevated numbers of CD4+CD8+ thymocytes. This suggests that the Scid mice may not represent a null phenotype and may retain some residual DNA-PKcs function.
Assuntos
Proteínas de Ligação a DNA , Marcação de Genes , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Tolerância a Radiação/genética , Imunodeficiência Combinada Severa/genética , Animais , Linfócitos B/citologia , Catálise , Diferenciação Celular/genética , Células Cultivadas , Proteína Quinase Ativada por DNA , Embrião de Mamíferos , Fibroblastos/efeitos da radiação , Genes Codificadores dos Receptores de Linfócitos T/genética , Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Recombinação Genética/genética , Linfócitos T/citologiaRESUMO
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.