Assignment of the gene for acid beta-glucosidase to human chromosome 1.

The structural gene for human acid beta-glucosidase (GBA) has been assigned to chromosome 1 using somatic cell hybridization techniques for gene mapping. The human enzyme was detected in mouse RAG cell-human fibroblast cell hybrids by a sensitive double antibody immunoprecipitation assay using a mouse antihuman GBA antibody. No cross-reactivity between mouse beta-glucosidase and human GBA or neutral beta-glucosidase (GBN) was observed. Fifty-two primary, secondary, and tertiary manmouse hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for the human chromosomes. Without exception, the presence of human GBA in these hybrid clones was correlated with the presence of human chromosome 1 or its enzymatic markers, phosphoglucomutase 1 (PGM1), and fumarate hydratase (FH). All other human chromosomes were eliminated by the independent segregation of GBA and their respective enzyme markers and/or chromosomes. Using a RAG X human fibroblast line with a mouse-human rearrangement of human chromosome 1, the locus for GBA was limited to the region 1p11 to 1qter.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene

It has been proposed that Ig gene rearrangement in the peritoneal cavity (Pc) B-1 cells might be involved in autoantibody generation. To study possible secondary B cell maturation, we prepared mice carrying a target integration of gfp gene into a rag1 locus (rag1/gfp mice). The GFP+ cells express rag1 mRNA and are undergoing Ig gene rearrangement. RAG1 expression was studied in Pc B-1 cells to detect cells during the stage of Ig gene rearrangement. In contrast to previous reports, Pc B-1 cells did not show RAG1 expression in adolescent or elderly mice. RAG1 expression was not induced in Pc B-1 cells in vivo after stimulation by oral or i.p. administration of LPS. Our results suggest that RAG1 expression in Pc B-1 cells is inhibited for a long period under normal condition and that this suppression is an essential state which maintains allelic exclusion of Ig genes.     

Role of interleukin 12 in hypercholesterolemia-induced inflammation

We have previously shown that T lymphocytes and interferon-gamma are involved in hypercholesterolemia-induced leukocyte adhesion to vascular endothelium. This study assessed the contribution of interleukin 12 (IL-12) to these hypercholesterolemia-induced inflammatory responses. Intravital videomicroscopy was used to quantify leukocyte adhesion and emigration and oxidant stress (dihydrorhodamine oxidation) in unstimulated cremasteric venules (wall shear rate > or =500 s-1) of wild-type (WT) C57Bl/6, lymphocyte-deficient [recombinase-activating gene knockout (RAG1-/-)], and IL-12-deficient (p35-/- and p40-/-; p35 and p40 are the two subunits of active IL-12) mice on either a normal (ND) or high-cholesterol (HC) diet for 2 wk. RAG1-/--HC mice received splenocytes from WT-HC (WT --> RAG1-/-), p35-/--HC (p35-/- --> RAG1-/-), or p40-/--HC (p40-/- --> RAG1-/-) mice. Compared with WT-ND mice, WT-HC mice exhibited exaggerated leukocyte adherence and emigration as well as increased dihydrorhodamine oxidation. The enhanced leukocyte recruitment was absent in the RAG1-/--ND, p35-/--ND, and p40-/--ND groups. Hypercholesterolemia-induced leukocyte adherence and emigration were attenuated in RAG1-/--HC vs. WT-HC mice but were similar to ND mice. Furthermore, compared with WT-HC animals, p35-/--HC and p40-/--HC mice showed significantly lower leukocyte adhesion and tissue oxidant stress responses, but these values were comparable to ND mice. Leukocyte adherence and emigration in WT --> RAG1-/- mice were similar to responses of WT-HC mice. However, p35-/- --> RAG1-/- mice had lower levels of adherence and emigration vs. the WT --> RAG1-/- and WT-HC groups. Elevated levels of leukocyte adherence and emigration were restored by approximately 50% toward WT-HC levels in p40-/- --> RAG1-/- mice. These findings implicate IL-12 in the inflammatory responses observed in the venules of hypercholesterolemic mice.     

Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.     

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.     

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.     

DNA cleavage activity of the V(D)J recombination protein RAG1 is autoregulated

RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.     

Self-association and conformational properties of RAG1: implications for formation of the V(D)J recombinase

RAG1 and RAG2 catalyze the initial DNA cleavage steps in V(D)J recombination. Fundamental properties of these proteins remain largely unknown. Here, self-association and conformational properties of murine core RAG1 (residues 384–1008) were examined. As determined by multi-angle laser light scattering measurements, the molecular masses of two predominant core RAG1 species corresponded to dimeric and tetrameric states. Similar results were obtained using a RAG1 fragment containing residues 265–1008, indicating that a non-core portion of RAG1 does not alter the oligomerization states observed for the core region. The fraction of core RAG1 in the tetrameric state increased significantly at lower ionic strengths (0.2 versus 0.5 M NaCl), indicating that this oligomeric form may factor into the physiological function of RAG1. In addition, the secondary structural content of core RAG1, obtained by circular dichroism spectroscopy, demonstrated a significant dependence on ionic strength with a 26% increase in α-helical content from 0.2 to 1.0 M NaCl. Together, these results indicate that structural and oligomerization properties of core RAG1 are strongly dependent on electrostatic interactions. Furthermore, the secondary structure of core RAG1 changes upon binding to DNA, with larger increases in α-helical content upon binding to the recombination signal sequence (RSS) as compared with non-sequence-specific DNA. As shown by electrophoretic mobility shift assays, higher order oligomeric forms of core RAG1 bound to the canonical RSS. Furthermore, core RAG2 (residues 1–387) formed complexes with multimeric RAG1 species bound to a single RSS, providing additional support for the physiological relevance of higher order oligomeric states of RAG1.     

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (K_D ∼0.4 μm) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the K_D value for their interaction, which could help limit off-target RAG activity.     

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.     

Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.     

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.     

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.     

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.     

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.     

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.     

Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex

During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG protein complex.