Amino acid residues in RAG1 responsible for the interaction with RAG2 during the V(D)J recombination process

The V(D)J recombinase, a complex of RAG1 and RAG2, carries out a gene rearrangement process that is required for the achievement of diverse antigen receptor repertoires during the early developmental stage of lymphocytes. It recognizes a specific site spanning the coding DNA region of antigen receptor genes and produces double-stranded DNA breaks at the board between coding and signal sequences. Two broken DNA ends are joined by a double-stranded break repair system. Both RAG (recombination activation gene) 1 and RAG2 proteins are absolutely required for this process although the catalytic residues of V(D)J recombinase are exclusively located at RAG1 according to recent mutational analyses. In this study we identified some acidic amino acid residues in RAG1 responsible for the interaction with RAG2. Mutation on these residues caused a decrease of cleavage activity in vitro and failure of RAG-RSS DNA synaptic complex formation. This result is complementary to previous reports in which positively charged amino acids in RAG2 play an important role in RAG1 binding.     

The C-terminal portion of RAG2 protects against transposition in vitro

The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.     

DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins

The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.     

Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.     

Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.     

Ordered DNA release and target capture in RAG transposition

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.     

Assembly of the RAG1/RAG2 Synaptic Complex

Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.     

An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex

V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.     

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.     

Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro

The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination.     

DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.     

The central domain of core RAG1 preferentially recognizes single-stranded recombination signal sequence heptamer

RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528-760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.     

Cell cycle-dependent accumulation in vivo of transposition-competent complexes between recombination signal ends and full-length RAG proteins

V(D)J recombination is initiated by a specialized transposase consisting of RAG-1 and RAG-2. Because full-length RAG proteins are insoluble under physiologic conditions, most previous analyses of RAG activity in vitro have used truncated core RAG-1 and RAG-2 fragments. These studies identified an intermediate in V(D)J recombination, the signal end complex (SEC), in which core RAG proteins remain associated with recombination signal sequences at the cleaved signal ends. From transfected cells expressing affinity-tagged RAG proteins, we have isolated in vivo assembled SECs containing full-length RAG proteins and cleaved recombination substrates. SEC formation in vivo did not require the repair proteins DNA-dependent protein kinase, Ku80, or XRCC4. In the presence of full-length RAG-2, SEC formation in vivo was cell cycle-regulated and restricted to the G(0)/G(1) phases. In contrast, complexes accumulated throughout cell cycle in cells expressing a RAG-2 CDK2 phosphorylation site mutant. Both core and full-length SECs supported transposition in vitro with similar efficiencies. Intracellular SECs, which are likely to persist in the absence of coding ends, represent potential donors whose transposition is not suppressed by the non-core regions of the RAG proteins.     

The RAG1/RAG2 complex constitutes a 3' flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination

During V(D)J recombination, processing of branched coding end intermediates is essential for generating junctional diversity. Here, we report that the RAG1/ RAG2 recombinase is a 3' flap endonuclease. Substrates of this nuclease activity include various coding end intermediates, suggesting a direct role for RAG1/ RAG2 in generating junctional diversity during V(D)J recombination. Evidence is also provided indicating that site-specific RSS nicking involves RAG1/RAG2-mediated processing of a localized flap-like structure, implying 3' flap nicking in multiple DNA processing reactions. We have also demonstrated that the bacterial transposase Tn10 contains a 3' flap endonuclease activity, suggesting a mechanistic parallel between RAG1/RAG2 and other transposases. Based on these data, we propose that numerous transposases may facilitate genomic evolution by removing single-stranded extensions during the processing of excision site junctions.     

Effect of CpG methylation on RAG1/RAG2 reactivity: implications of direct and indirect mechanisms for controlling V(D)J cleavage

It has been suggested that DNA methylation/demethylation is involved in regulating V(D)J rearrangement. Although methylated DNA is thought to induce an inaccessible chromatin structure, it is unclear whether DNA methylation can directly control V(D)J recombination independently of chromatin structure. In this study, we tested whether DNA methylation directly affects the reactivity of the RAG1/RAG2 complex. Specific methylation within the heptamer of the recombination signal sequences (RSS) markedly reduced V(D)J cleavage without inhibiting RAG1/RAG2–DNA complex formation. By contrast, methylation at other positions around the RSS did not affect the reactivity of the RAG proteins. The presence of a methyl-CpG binding-domain protein inhibited the binding of the RAG1/RAG2 complex to all the methylated CpG sites that were tested. Our findings suggest that DNA methylation around the RSS may have a previously unexpected function in regulating V(D)J recombination by directly inhibiting V(D)J cleavage, in addition to its general function of inducing an inaccessible chromatin configuration.     

Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex.

In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction.     

A Non-Sequence-Specific DNA Binding Mode of RAG1 Is Inhibited by RAG2

RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, with consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction under physiological conditions is masked by a high-affinity non-sequence-specific DNA binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence-specific DNA, resulting in a large differential in binding affinity for the RSS versus the non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS and that, therefore, association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.     

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

RAG1 and RAG2 proteins are key components in V(D)J recombination. The core region of RAG1 is capable of catalyzing the recombination reaction; however, the biological function of non-core RAG1 remains largely unknown. Here, we show that in a murine-model carrying the RAG1 ring-finger conserved cysteine residue mutation (C325Y), V(D)J recombination was abrogated at the cleavage step, and this effect was accompanied by decreased mono-ubiquitylation of histone H3. Further analyses suggest that un-ubiquitylated histone H3 restrains RAG1 to the chromatin by interacting with the N-terminal 218 amino acids of RAG1. Our data provide evidence for a model in which ubiquitylation of histone H3 mediated by the ring-finger domain of RAG1 triggers the release of RAG1, thus allowing its transition into the cleavage phase. Collectively, our findings reveal that the non-core region of RAG1 facilitates chromosomal V(D)J recombination in a ubiquitylation-dependent pathway.     

Three-dimensional clustering of human RAG2 gene mutations in severe combined immune deficiency

The V(D)J recombination, which leads to the somatic rearrangement of variable, diversity, and joining segments, is the mechanism accountable for the diversity of T cell receptor- and Ig-encoding genes. The products of the RAG1 and RAG2 genes are the lymphoid-specific factors responsible for the initiation of the V(D)J recombination through the generation of a DNA double strand break. RAG1 or RAG2 gene inactivation in the mouse leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and, ultimately, to severe combined immune deficiency (SCID). A human SCID condition is also characterized by an absence of mature T and B lymphocytes and is associated with mutations in either RAG1- or RAG2-encoding genes. Based on the predicted beta-propeller three-dimensional structure model for RAG2, we found that six out of the seven mutations described to date in T-B-SCID patients are clustered on one side of the propeller, in regions exposed to solvent. This finding reinforces the biological significance of this predicted model and suggests that RAG1 interacts with RAG2 on one of the side of the scaffold formed by the beta-propeller.     

RAG2 is down-regulated by cytoplasmic sequestration and ubiquitin-dependent degradation

Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cell-cycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.