A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination

Bailin, Tu; Mo, Xianming; Sadofsky, Moshe J.

doi:10.1128/mcb.19.7.4664

Cited by 68 publications

(100 citation statements)

References 31 publications

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“…Both cleavage steps are minimally supported in vitro in a protein-DNA complex, termed a stable or single complex (SC), containing a RAG-1 dimer and one or two subunits of RAG-2 bound to an isolated RSS (4,46,48). HMG-1 and HMG-2 can be stably incorporated into the SC (30,47,51).…”

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confidence: 99%

“…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).…”

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confidence: 99%

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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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confidence: 99%

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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“…It has been shown that the recombination intermediates are held in a postcleavage complex that contains RAG1 and RAG2 proteins (27,28,46). The purified RAG1/2 proteins were found to mediate the reverse reaction of the cleavage to generate hybrid joints and open-shut products (31).…”

Section: Figurementioning

confidence: 99%

“…RAG1 and RAG2 proteins have been shown to possess end-processing activities, including binding of recombination intermediates (27,28), nicking synthetic hairpin ends (29,30), and rejoining cleaved signal ends to coding ends (31). Recently, it has been demonstrated that the Mre11 protein, when complexed with Rad50 and the Nijmegen breakage syndrome gene product, Nbs1, exhibits several nuclease activities in vitro, such as hairpin nicking and processing of opened ends (32).…”

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Metabolism of Recombination Coding Ends in scid Cells

Brown

Chang

2000

The Journal of Immunology

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V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3′ overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.

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“…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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A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination

Cited by 68 publications

References 31 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

Metabolism of Recombination Coding Ends in scid Cells

RAG1-DNA Binding in V(D)J Recombination

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