RESUMO
Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , DNA/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.
Assuntos
Antígenos Nucleares , DNA Helicases , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética/genética , Animais , Células CHO , Sobrevivência Celular/genética , Células Clonais/metabolismo , Células Clonais/efeitos da radiação , Cricetinae , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Expressão Gênica/genética , Humanos , Autoantígeno Ku , Proteínas Nucleares/metabolismo , Mutação Puntual/genética , Deleção de Sequência/genética , TransfecçãoRESUMO
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.
