Roles of the “Dispensable” Portions of RAG-1 and RAG-2 in V(D)J Recombination

Abstract

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.Immunoglobulin and T-cell receptor gene segments are rearranged by V(D)J recombination to generate a diverse repertoire of antigen binding domains. The recombinase binds recombination signal sequences (hereafter termed signals) which flank the gene segments and introduces a double-stranded break (DSB) precisely between each signal and gene segment. This cleavage event produces two types of DNA termini, signal ends that terminate in signals and coding ends that contain the gene segment. Signal ends join to form a signal joint, whereas coding ends join to form a coding joint encoding the antigen binding domain (Fig. 1A) (24,27,28,33,38,40).The V(D)J recombinase minimally consists of the highly conserved, lymphoid-cell-specific proteins RAG-1 and RAG-2 (22, 32). Transfection of the genes encoding RAG-1 and RAG-2 into cultured fibroblasts renders these cells competent to rearrange extrachromosomal recombination substrates, indicating that the RAG proteins are the only lymphoid-cellspecific factors necessary for recombination (22). DSBs with the same characteristics as in vivo intermediates (27,28,33,35,40) are generated in cell-free reactions containing purified, truncated RAG-1 and RAG-2 and the appropriate divalent metal ion (20). After cleavage, the RAG proteins remain associated with the broken DNA ends. Stable complexes have been isolated that contain the RAG proteins and a pair of cleaved signal ends (1). More recently, complexes containing the RAG proteins and all four DNA ends (two signal ends and two coding ends) have been isolated (9). We and others have suggested that disassembly or remodelin...

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations

“…This is supported by a recent study 29 showing that the removal of dispensable regions of RAG-1 and RAG-2 impairs proper processing of recombination substrates.…”

Biochemical and Folding Defects in a RAG1 Variant Associated with Omenn Syndrome

“…We and others have observed that substitutions that disrupt the RING finger domain within the N-TR can virtually eliminate recombination despite the entire region's being dispensable for DNA cleavage (18 -20). It has also been demonstrated that cysteine-rich and arginine/lysinerich elements in the N-TR but outside the RING domain contribute to optimal levels of recombination activity (18,42), and that the N-TR as a whole promotes SJ formation (43). Taken together, these data strongly suggest that the N-TR plays a regulatory role.…”

Postcleavage Sequence Specificity in V(D)J Recombination

“…In contrast, these three nucleotides have a clear in vivo function during stage 2 of V(D)J joining. It is possible that the interaction in question is fairly weak and/or transient, and it is of particular interest that nonamer-proximal nucleotides of the heptamer have been highlighted as a possible contact site for RAG1/2 in one modification interference study (43).…”