RESUMEN
The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.
Asunto(s)
Linfocitos B/citología , Proteínas Portadoras/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Serina-Treonina Quinasas , TransgenesRESUMEN
V(D)J recombination is initiated by the synapsis and cleavage of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins. Our understanding of these processes has been greatly aided by the development of in vitro biochemical assays of RAG binding and cleavage activity. Accumulating evidence suggests that synaptic complex assembly occurs in a step-wise manner and that the RAG proteins catalyze RSS cleavage by mechanisms similar to those used by bacterial transposases. In this chapter we will review the molecular mechanisms of RAG synaptic complex assembly and 12/23-regulated RSS cleavage, focusing on recent advances that shed new light on these processes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Proteínas de Homeodominio/metabolismo , Recombinación Genética , Emparejamiento Cromosómico , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Señales de Clasificación de Proteína , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismoRESUMEN
V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.
Asunto(s)
Reordenamiento Génico de Linfocito B/genética , Recombinación Genética/genética , Animales , Secuencia de Bases , Línea Celular , ADN/química , ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Activación Enzimática , Humanos , Insectos , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.