RESUMEN

The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.

Asunto(s)

Antígenos Nucleares/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Antígenos Nucleares/aislamiento & purificación , Fraccionamiento Celular , Línea Celular , ADN/metabolismo , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/aislamiento & purificación , Proteína HMGB1/metabolismo , Proteínas de Homeodominio/genética , Humanos , Autoantígeno Ku , Proteínas Nucleares , Conformación de Ácido Nucleico , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Tiorredoxinas/metabolismo