In addition to an essential role in initiating V(D)J rearrangements, RAG1/2 has been proposed to have important functions subsequent to the generation of DSBs. 7 Hence, the efficiency of binding and cleavage of individual RSSs is significantly affected by interactions of the RAG1/2 endonuclease with the heptamer, nonamer, 12 or 23 spacer, and coding sequences. Sequence elements within the nonconserved 12 and 23 spacers 7 and coding flanks 8 – 11 contribute to RAG1/2 interaction with the recombining DNA and strongly influence coding segment utilization. The products of RAG1/2 cleavage are blunt, 5′ phosphorylated signal ends and covalently closed, hairpin coding ends. RAG1/2 nicks the duplex DNA at the junction between the RSS heptamer and coding sequences then uses the resulting 3′ OH in a direct transesterification reaction with the phosphodiester backbone of the opposing strand. 4 Efficient cleavage requires recombination between one 12 RSS and one 23 RSS in the context of a synaptic, paired complex mediated by RAG1/2. The RAG1/2 endonuclease initially binds to RSSs that are composed of a conserved heptamer and nonamer separated by relatively nonconserved spacer sequences of either 12 or 23 nucleotides. 4 The broken ends are then processed and joined by ubiquitously expressed nonhomologous end-joining (NHEJ) DNA repair factors, including Ku70, Ku80, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, DNA ligase IV, XRCC4 and Cernunnos/XLF. 2, 3 DNA double-strand breaks (DSBs) are generated by RAG1/2 at specific recombination signal sequences (RSSs) adjacent to the numerous rearranging V, D, and J coding exons. 1 These site-specific chromosomal rearrangements are initiated in progenitor lymphocytes by the recombination activating gene 1 (RAG1) and RAG2 proteins that comprise a lymphoid-specific endonuclease, RAG1/2. The immense diversity of genes encoding the variable regions of antigen receptors is generated through rearrangement of component V, D, and J segments via a cut-and-paste mechanism known as V(D)J recombination. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences.

Leaky scid