A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Abstract

Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.The antigen binding regions of immunogobulin and T-cell receptors are encoded in arrays of noncontiguous coding segments, called variable (V), diversity (D), and joining (J), that are assembled during lymphocyte development to generate the variable-region exon of the mature antigen receptor gene (5). The assembly process, termed V(D)J recombination, involves a series of site-specific DNA rearrangements mediated by recombination signal sequences (RSSs) abutting the coding segments. Each RSS contains conserved heptamer and nonamer elements, separated by nominally conserved spacer DNA that is either 12 or 23 bp long (12-RSS and 23-RSS, respectively). The RSS facilitates the generation of functional antigen receptors via the "12/23 rule," a restriction that limits recombination to a pair of coding segments whose flanking RSSs bear spacer DNAs of different lengths.The products of recombination-activating genes 1 and 2, RAG-1 and RAG-2 (35, 42), initiate V(D)J recombination by introducing a DNA double-strand break (DSB) at a 12/23 pair of RSSs (at the heptamer-coding segment border). The cleavage reaction generates two distinct DNA intermediates: blunt, 5Ј-phosphorylated signal ends (SE) and coding ends terminating in covalently sealed DNA hairpin structures (38,39,43). DSB intermediates are generated in two biochemical steps: first-strand nicking at the 5Ј end of the heptamer, followed by a direct transesterification reaction in which the 3Ј-OH exposed in the nicking step attacks the phosphodiester on the antiparallel DNA strand (26, 52). The RAG proteins also mediate other reactions in vitro, including a disintegratio...

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

“…As a negative control, the cleavage activity of catalytically inactive D600A cMR1/ cMR2 was examined in parallel, showing a profile of products identical to samples containing no RAG proteins. As expected from our previous studies (50,51), incubation of an intact 12-RSS with WT cMR1/cMR2 in the presence of Mg 2ϩ for 1 h at 37°C yields predominantly the nicked product and a small amount of hairpin product. In contrast, E649A cMR1/cMR2 yields about 20-fold more hairpin product under the same conditions and about 2.5-fold less nicked product, likely because the nicked DNA is converted to hairpin products ( Fig.…”

“…We find that in the absence of HMGB1, both WT and E649A cMR1/cMR2 form two distinct protein-DNA complexes, called SC1 and SC2, similarly when incubated with an isolated RSS substrate, both with intact and with nicked 23-RSS substrates. In a previous study (51), both complexes were shown to contain a RAG-1 dimer, but the RAG-2 stoichiometries differed between SC1 and SC2, with the former containing monomeric RAG-2 and the latter containing two RAG-2 molecules. For both RAG preparations, the SC1 and SC2 complexes are comparably supershifted in the presence of HMGB1, forming HSC1 and HSC2, respectively.…”

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

“…As expected from previous studies (28), no cleavage of intact RSS or cRSS substrates was observed by the D600A RAGs (Fig. 1, A-D).…”

“…As expected from previous studies (28), two major RAG-RSS complexes are detected by EMSA when the RAGs are incubated with a consensus RSS in the absence of HMGB1, termed SC1 and SC2. Previous work in our laboratory suggests that SC1 and SC2 contain a RAG1 dimer and either one (SC1) or two (SC2) subunits of RAG2 (28). The addition of HMGB1 supershifts both complexes (HSC1 and HSC2, respectively).…”

RAG1-DNA Binding in V(D)J Recombination

“…Some experiments indicated the presence of a dimer of RAG1 (18,22,23,24), whereas others argue for the presence of three or four RAG1 subunits (25,26). RAG2 is present in the SC either as a monomer (18) or as a dimer (23,26).…”