Assuntos
Antígenos Nucleares , Dano ao DNA , DNA Helicases , Reparo do DNA , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Animais , Linhagem Celular , Cricetinae , DNA/genética , DNA/efeitos da radiação , Células HeLa , Humanos , Indicadores e Reagentes , Autoantígeno Ku , Proteínas Nucleares/genética , Proteínas Recombinantes/biossíntese , Transfecção , Raios Ultravioleta , Xeroderma Pigmentoso/genéticaRESUMO
V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.
Assuntos
DNA Nucleotidiltransferases/genética , DNA/metabolismo , Proteínas Serina-Treonina Quinases/química , Recombinação Genética/genética , Animais , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/genética , Ativação Enzimática/genética , Camundongos , Camundongos SCID , Mutação/genética , Conformação de Ácido Nucleico , VDJ RecombinasesRESUMO
V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.
Assuntos
Antígenos Nucleares , DNA Helicases , Receptores de Antígenos/genética , Recombinação Genética , Animais , Diversidade de Anticorpos , Evolução Biológica , DNA/genética , DNA/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku , Modelos Genéticos , Mutação , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/genéticaRESUMO
X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.
Assuntos
Antígenos Nucleares , Autoantígenos/genética , DNA Helicases , DNA Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Tolerância a Radiação , Sequência de Aminoácidos , Animais , Sequência de Bases , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Teste de Complementação Genética , Autoantígeno Ku , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , VDJ RecombinasesRESUMO
Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.