VprBP (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation.

The assembly of Ig genes in developing B lymphocytes by V(D)J recombination is initiated by the RAG1-RAG2 endonuclease complex. We previously identified an interaction between RAG1 and viral protein R binding protein (VprBP) (also known as DNA damage binding protein 1 cullin 4-associated factor 1 [DCAF1]), a substrate receptor for the cullin 4-really interesting new gene (RING) E3 ubiquitin ligase (CRL4). We report in this article that in mice, B cell-intrinsic loss of VprBP increases RAG1 protein levels and disrupts expression of the endoribonuclease Dicer, which is essential for microRNA maturation. Rag1/2 transcription is known to be derepressed by loss of microRNA-mediated suppression of phosphatase and tensin homolog, raising the possibility that the elevated level of RAG1 observed in VprBP-deficient B cells is caused indirectly by the loss of Dicer. However, we show that VprBP restrains RAG1 expression posttranscriptionally and independently of Dicer. Specifically, loss of VprBP stabilizes RAG1 protein, which we show is normally degraded via a mechanism requiring both 20S proteasome and cullin-RING E3 ubiquitin ligase activity. Furthermore, we show that RAG1 stabilization through small molecule inhibition of cullin-RING E3 ubiquitin ligase activation promotes V(D)J recombination in a murine pre-B cell line. Thus, in addition to identifying a role for VprBP in maintaining Dicer levels in B cells, our findings reveal the basis for RAG1 turnover and provide evidence that the CRL4VprBP(DCAF1) complex functions to maintain physiological levels of V(D)J recombination.

V(D)J recombination: mechanisms of initiation.

V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.

IL10 restrains autoreactive B cells in transgenic mice expressing inactive RAG1.

IL10 plays a dual role in supporting humoral immunity and inhibiting inflammatory conditions. B cells producing IL10 are thought to play a key regulatory role in maintaining self-tolerance and suppressing excessive inflammation during autoimmune and infectious diseases, primarily by inhibiting associated T cell responses. The extent to which B cells, through the provision of IL10, might function to sustain or inhibit autoantibody production is less clear. We previously described transgenic mice expressing catalytically inactive RAG1 (dnRAG1 mice), which show expansion of an IL10-compentent CD5+ B cell subset that phenotypically resembles B10 B cells, hypogammaglobulinemia, and a restricted B cell receptor repertoire with features indicative of impaired B cell receptor editing. We show here that B10-like B cells in dnRAG1 mice bind the membrane-associated autoantigen phosphatidylcholine (PtC), and that in vitro lipopolysaccharide (LPS) stimulation of dnRAG1 splenocytes induces a robust IgM response enriched in reactivity toward lupus-associated autoantigens. This outcome was correlated with detection of sIgMhi B cell populations that were distinct from, but in addition to, sIgMint populations observed after similar treatment of wild-type splenocytes. Loss of IL10 expression in dnRAG1 mice had no significant effect on B10-like B cell expansion or the frequency of PtC+ B cells. Compared to IL10+/+ dnRAG1 mice, levels of serum IgM, but not serum IgG, were highly elevated in some naïve IL10-/- dnRAG1 mice, and was correlated with a significant increase in serum BAFF levels. Differentiation of sIgMint B cells from LPS-stimulated dnRAG1 splenocytes was enhanced by loss of IL10 expression and IL10 blockade, but was suppressed by treatment with recombinant IL10. In vitro LPS-induced differentiation and antibody production was inhibited by treatment with JAK/STAT inhibitors or a synthetic corticosteroid, independent of IL10 expression and genotype. Taken together, these data suggest that IL10 expression in dnRAG1 mice maintains suppression of IgM levels in part by inhibiting BAFF production, and that regulatory B10-like B cells, through the provision of IL10, constrains B cell differentiation in response to mitogenic stimuli. Furthermore, autoantibody profiling raises a possible link between CD5+ B cell expansion, mitogenic stimulation, and autoantibodies associated with autoimmune complications observed in lupus and lupus-related disorders.

A long noncoding RNA, lincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3, located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 (Tnfaip3) gene, is an early-primary response gene controlled by nuclear factor-κB (NF-κB) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-κB-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-κB/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-κB/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Tnfaip3 to act as a coactivator of NF-κB for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.-Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

VprBP Is Required for Efficient Editing and Selection of Igκ+ B Cells, but Is Dispensable for Igλ+ and Marginal Zone B Cell Maturation and Selection.

B cell development past the pro-B cell stage in mice requires the Cul4-Roc1-DDB1 E3 ubiquitin ligase substrate recognition subunit VprBP. Enforced Bcl2 expression overcomes defects in distal VH-DJH and secondary Vκ-Jκ rearrangement associated with VprBP insufficiency in B cells and substantially rescues maturation of marginal zone and Igλ(+) B cells, but not Igκ(+) B cells. In this background, expression of a site-directed Igκ L chain transgene increases Igκ(+) B cell frequency, suggesting VprBP does not regulate L chain expression from a productively rearranged Igk allele. In site-directed anti-dsDNA H chain transgenic mice, loss of VprBP function in B cells impairs selection of Igκ editor L chains typically arising through secondary Igk rearrangement, but not selection of Igλ editor L chains. Both H and L chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together, these data argue that VprBP is required for the efficient receptor editing and selection of Igκ(+) B cells, but is largely dispensable for Igλ(+) B cell development and selection, and that VprBP is necessary to rescue autoreactive B cells from anergy induction.

VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity.

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.

A biochemically defined system for coding joint formation in V(D)J recombination.

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

Cd1d regulates B cell development but not B cell accumulation and IL10 production in mice with pathologic CD5(+) B cell expansion.

BACKGROUND: CD1d is a widely expressed lipid antigen presenting molecule required for CD1d-restricted invariant natural killer T (iNKT) cell development. Elevated CD1d expression is detected in CD5(+) IL10-producing B cells, called B10 B cells, and is correlated with poorer prognosis in chronic lymphocytic leukemia (CLL), a CD5(+) B cell malignancy with B10-like functional properties. Whether CD1d expression regulates CD5(+) B cell accumulation, IL10 competence, and antibody production in naïve mice with pathologic CD5(+) B cell expansion remains untested. RESULTS: Using three different transgenic mouse models of benign or leukemic CD5(+) B cell expansion, we found that CD1d was differentially expressed on CD5(+) B cells between the three models, but loss of CD1d expression had no effect on CD5(+) B cell abundance or inducible IL10 expression in any of the models. Interestingly, in the CLL-prone Eµ-TCL1 model, loss of CD1d expression suppressed spontaneous IgG (but not IgM) production, whereas in the dnRAG1xEµ-TCL1 (DTG) model of accelerated CLL, loss of CD1d expression was associated with elevated numbers of splenic CD4(+) and CD8(+) T cells and an inverted CD4(+):CD8(+) T cell ratio. Unexpectedly, before leukemia onset, all three transgenic CD1d-deficient mouse strains had fewer splenic transitional B cells than their CD1d-proficient counterparts. CONCLUSIONS: The results show that CD1d expression and iNKT cells are dispensable for the development, accumulation, or IL10 competence of CD5(+) B cells in mice prone to benign or leukemic CLL-like B cell expansion, but reveal a novel role for iNKT cells in supporting B cell progression through the transitional stage of development in these animals. These results suggest CD1d-directed therapies to target CLL could be evaded by downregulating CD1d expression with little effect on continued leukemic CD5(+) B cell survival. The data also imply that iNKT cells help restrain pro-leukemic CD8(+) T cell expansion in CLL, potentially explaining a reported correlation in human CLL between disease progression, the loss of NKT cells, and a paradoxical increase in CD8(+) T cells.

Accelerated progression of chronic lymphocytic leukemia in Eµ-TCL1 mice expressing catalytically inactive RAG1.

Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eµ-TCL1 mice to determine whether dnRAG1 expression in Eµ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eµ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.

Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex.

During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg(2+). By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

VprBP (DCAF1): a promiscuous substrate recognition subunit that incorporates into both RING-family CRL4 and HECT-family EDD/UBR5 E3 ubiquitin ligases.

The terminal step in the ubiquitin modification system relies on an E3 ubiquitin ligase to facilitate transfer of ubiquitin to a protein substrate. The substrate recognition and ubiquitin transfer activities of the E3 ligase may be mediated by a single polypeptide or may rely on separate subunits. The latter organization is particularly prevalent among members of largest class of E3 ligases, the RING family, although examples of this type of arrangement have also been reported among members of the smaller HECT family of E3 ligases. This review describes recent discoveries that reveal the surprising and distinctive ability of VprBP (DCAF1) to serve as a substrate recognition subunit for a member of both major classes of E3 ligase, the RING-type CRL4 ligase and the HECT-type EDD/UBR5 ligase. The cellular processes normally regulated by VprBP-associated E3 ligases, and their targeting and subversion by viral accessory proteins are also discussed. Taken together, these studies provide important insights and raise interesting new questions regarding the mechanisms that regulate or subvert VprBP function in the context of both the CRL4 and EDD/UBR5 E3 ligases.

Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.

Smoke-induced signal molecules in bone marrow cells from altered low-density lipoprotein receptor-related protein 5 mice.

Mechanism underlying smoke-induced loss of bone mass is unknown. In this study, we hypothesized that protein signals induced by smoking in bone marrow may be associated with the loss of bone mass. Using a proteomics approach, we identified 38 proteins differentially expressed in bone marrow cells from low-density lipoprotein receptor-related protein 5 (Lrp5) mice exposed to cigarette smoking. Smoking effects on protein expression in bone marrow among three genotypes (Lrp5(+/+), Lrp5(G171V), and Lrp5(-/-)) varied. On the basis of the ratio of protein expression induced by smoking versus nonsmoking, smoke induced protein expression significantly in wild-type mice compared to the other two genotypes (Lrp5(G171V) and Lrp5(-/-)). These proteins include inhibitors of ß-catenin and proteins associated with differentiation of osteoclasts. We observed that S100A8 and S100A9 were overexpressed in human smokers compared to nonsmokers, which confirmed the effect of smoking on the expression of two proteins in Lrp5 mice, suggesting the role of these proteins in bone remodeling. Smoke induced expression of S100A8 and S100A9 in a time-dependent fashion, which was opposite of the changes in the ratio of OPG/RANKL in bone marrow cells, suggesting that the high levels of S100A8 and S100A9 may be associated with smoke-induced bone loss by increasing bone resorption.

Receptor editing constrains development of phosphatidyl choline-specific B cells in VH12-transgenic mice.

B1 B cells reactive to phosphatidyl choline (PtC) exhibit restricted immunoglobulin heavy chain (HC) and light chain (LC) combinations, exemplified by VH12/Vκ4/5H. Two checkpoints are thought to focus PtC+ B cell maturation in VH12-transgenic mice (VH12 mice): V-J rearrangements encoding a "permissive" LC capable of VH12 HC pairing are selected first, followed by positive selection based on PtC binding, often requiring LC receptor editing to salvage PtC- B cells and acquire PtC reactivity. However, evidence obtained from breeding VH12 mice to editing-defective dnRAG1 mice and analyzing LC sequences from PtC+ and PtC- B cell subsets instead suggests that receptor editing functions after initial positive selection to remove PtC+ B cells in VH12 mice. This offers a mechanism to constrain natural, polyreactive B cells to limit their frequency. Sequencing also reveals occasional in-frame hybrid LC genes, reminiscent of type 2 gene replacement, that, testing suggests, arise via a recombination-activating gene (RAG)-independent mechanism.

Accumulation of B1-like B cells in transgenic mice over-expressing catalytically inactive RAG1 in the periphery.

During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild-type mice. The dnRAG1 mice accumulate splenic B cells with a B1-like phenotype that exhibit defects in B-cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B-cell development at the immature-to-mature transition, immunoglobulin deficiency, and poorer immune responses to thymus-independent antigens. Interestingly, dnRAG1 mice expressing the anti-dsDNA 3H9H56R heavy chain fail to accumulate splenic B1-like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1-like B cells in the spleen.

Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis.

The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein-DNA complexes that support this process have mainly been studied using RAG-RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's '12/23 rule' and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the post-cleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.

The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line.

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.

Molecular mechanism underlying RAG1/RAG2 synaptic complex formation.

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complex.

A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex.

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex--which is the normal enzyme for DNA cleavage at V, D or J segments--nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20-30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.