Assembly of the RAG1/RAG2 Synaptic Complex

Mundy, Cynthia L.; Patenge, Nadja; Matthews, Adam G. W.; Oettinger, Marjorie A.

doi:10.1128/mcb.22.1.69-77.2002

Cited by 88 publications

(113 citation statements)

References 37 publications

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“…In one model of the PC, RAG-1 and RAG-2 are bound as a mixed tetramer containing two molecules of each protein, based on the following lines of evidence: (i) RAG-1 alone forms a stable dimer in solution (4) and retains this configuration when bound to DNA (37), regardless of whether RAG-2 is present (48); (ii) RAG-1 and RAG-2 associate as a tetramer in solution (in a 1:1 ratio) (4); and (iii) two active sites are present in each dimer of RAG-1, one contributed from each RAG-1 subunit (22,46). However, recent studies provide indirect evidence suggesting that the PC contains a larger number of RAG-1 protomers, possibly a tetramer (22,31). Discriminating between these models necessarily requires a direct approach that compares the RAG stoichiometries within the SC and PC side by side.…”

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

Swanson

2002

Molecular and Cellular Biology

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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...

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

Swanson

2002

Molecular and Cellular Biology

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“…The noncore domains of RAG2 could facilitate localization of the protein to specific sites within the nucleus of progenitor lymphocytes where IgH and TCR␤ V-to-DJ joining takes place. The recently noted order of synaptic complex assembly, with RAG proteins initially assembling on one RSS (preferably a 12-RSS) then searching for the second site (35,36), might also be affected by the absence of the C terminus of RAG2 if, for example, the complex is less stable. In this context, the PHD theoretically could associate with chromatin over IgH and V␤ segments to facilitate synaptic complex assembly between V RSSs and RAG-bound 5Ј-D RSSs (1).…”

Section: Discussionmentioning

confidence: 99%

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice

Akamatsu

Monroe

Dudley

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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During lymphocyte development, V(D)J recombination is highly regulated in the contexts of lineage specificity, developmental-stage specificity, and allelic exclusion (4, 5). In addition, developing lymphocytes use strategies that require productive V(D)J rearrangements for continued developmental progression (4, 5). For example, Ig heavy chain (IgH) variable-region genes are assembled in progenitor (pro) B cells via an ordered process in which D H -to-J H rearrangement occurs on both alleles before V H -to-DJ H rearrangement. Productive V H -to-DJ H rearrangements produce cell-surface expression of IgH chains that signal expansion and differentiation of pro-B cells to the pre-B cell stage. This expression of IgH chains also signals the cessation of further V H -to-DJ H rearrangement via feedback regulation. Pro-B cells that first assemble nonproductive V H -to-DJ H rearrangements proceed to rearrange their second allele.Both RAG1 and RAG2 are required for V(D)J recombination, because RAG1-or RAG2-deficient mice exhibit a complete block in lymphocyte development at the progenitor stage (6, 7). In addition, RAG1 and RAG2 together are sufficient to initiate V(D)J recombination in vitro (8). Nearly all in vitro studies of RAG function have used the minimal regions of RAG1 (amino acids 384-1,008 of 1,040) and RAG2 (amino acids 1-383 of 527) required for activity, because full-length RAG1 and RAG2 are largely insoluble. However, the activities of these mutant ''core'' proteins differ from those of the full-length RAGs when assayed with extrachromosomal substrates in transfected cells. In this context, the mutant core RAG proteins support V(D)J recombination with reduced efficiency and with different levels and types of recombination products (9-15).Although not required for the biochemistry of V(D)J recombination, the noncore regions of RAG1 and RAG2 are conserved throughout evolution. For example, sequence conservation across the entire RAG2 protein from pufferfish to humans is Ϸ60%, with conservation of the noncore C-terminal region slightly higher than the core domain (16). Therefore, the noncore regions of RAG1 and RAG2 may serve important accessory and͞or regulatory functions in chromosomal V(D)J recombination. Consistent with this notion, studies of Abelson murine leukemia virus-transformed pre-B cells have suggested that the noncore regions of RAG1 are important for IgH locus D H -to-J H rearrangement (17), whereas the C terminus of RAG2 is more important for V H -to-DJ H rearrangement than for D H -to-J H rearrangement (18). The C terminus of RAG2 is predicted to fold into a plant homeodomain (PHD) (19,20), a motif found in a number of chromatin-associated proteins including some that have chromatin-modifying activities (21-24).To investigate potential in vivo function of the RAG2 noncore region in chromosomal V(D)J recombination and normal lymphocyte development, we have generated and characterized mice containing specific replacement of the full-length endogenous RAG2 gene with a gene encoding the mouse core...

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“…The stoichiometry of pre-cleavage RAG1⅐RAG2⅐RSS complexes is controversial, with two reports suggesting that these complexes contain more than two (presumably four) monomers of RAG1 (25,26), and another suggesting that they contain only a single dimer of RAG1 (24). If the former model is correct, then since DNA-bound StrRAG1 dimers show no propensity to associate with other StrRAG1 dimers, it is likely that stable association of two dimers of the RAG1 core with an RSS requires RAG2.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

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Assembly of the RAG1/RAG2 Synaptic Complex

Cited by 88 publications

References 37 publications

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

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

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice

RAG1-DNA Binding in V(D)J Recombination

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