Molecular Mechanisms of DNA Sequence Selectivity in V(D)J Recombination.

Antigen receptor (AgR) diversity is central to the ability of adaptive immunity in jawed vertebrates to protect against pathogenic agents. The production of highly diverse AgR repertoires is initiated during B and T cell lymphopoiesis by V(D)J recombination, which assembles the receptor genes from component gene segments in a cut-and-paste recombination reaction. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank AgR gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence, and the molecular mechanism for semiselective V(D)J recombination specificity is unknown. The modulation of chromatin structure during V(D)J recombination is essential in the formation of diverse AgRs in adaptive immunity while also reducing the likelihood for off-target recombination events that can result in chromosomal aberrations and genomic instability. Here we review what is presently known regarding mechanisms that facilitate assembly of RAG1/2 with RSSs, the ensuing conformational changes required for DNA cleavage activity, and how the readout of the RSS sequence affects reaction efficiency.

An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

Mechanism of Anti-Inflammatory Activity of TLR4-Interacting SPA4 Peptide.

The TLR4-interacting SPA4 peptide suppresses inflammation. We assessed the structural and physicochemical properties and binding of SPA4 peptide to TLR4-MD2. We also studied the changes at the whole transcriptome level, cell morphology, viability, secreted cytokines and chemokines, and cell influx in cell systems and mouse models challenged with LPS and treated with SPA4 peptide. Our results demonstrated that the SPA4 peptide did not alter the cell viability and size and only moderately affected the transcriptome of the cells. Computational docking and rendering suggested that the SPA4 peptide intercalates with LPS-induced TLR4-MD2 complex. Results with alanine mutations of D-2 amino acid and NYTXXXRG-12-19 motif of SPA4 peptide suggested their role in binding to TLR4 and in reducing the cytokine response against LPS stimulus. Furthermore, therapeutically administered SPA4 peptide significantly suppressed the secreted levels of cytokines and chemokines in cells and bronchoalveolar lavage fluids of LPS-challenged mice. The results suggest that the SPA4 peptide intercalates with LPS-induced TLR4 complex and signaling for the suppression of inflammation.

Full length RAG2 expression enhances the DNA damage response in pre-B cells.

V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs. Each RAG2 construct was labeled with GFP at the N-terminus. Compared to the T490A mutant, cells expressing FL RAG2 exhibited elevated apoptosis by 24 h following irradiation, and this coincided with a greater amount of Caspase 3 cleavage measured in cell lysates. Pre-B cells expressing either RAG2 protein exhibited similar increases in phospho-p53 levels following irradiation. Interestingly, FL RAG2-expressing cells exhibited elevated division relative to the T490A clone beginning ~24 h following irradiation, as well as an increased percentage of cells proceeding through mitosis, suggesting an improved rate of recovery following the initial burst in apoptosis. Altogether, these data show that FL RAG2, but not its stable nuclear export-defective T490A mutant, participates in pre-B cell decisions between apoptosis versus DNA repair and cell cycle progression following DNA damage.

RAG2 localization and dynamics in the pre-B cell nucleus.

RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood. Here, we used cell imaging to measure the effect of mutating the RAG2 noncore region on properties of the full length protein. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant in the noncore region, which has unique regulatory properties. This showed that FL, T490A, and Core localized to nuclear domains that were adjacent to DAPI-rich heterochromatin, and that contained the active chromatin marker H3K4me3. Within the RAG2-enriched regions, T490A exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, colocalization of H3K4me3 with FL and T490A, but not Core, increased in conditions that increased H3K4me3 levels. Superresolution imaging showed H3K4me3 was distributed as puncta that RAG2 abutted, and mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating Trp453 of the T490A mutant (W453A,T490A), which blocks PHD-dependent interactions with H3K4me3, abolished the T490A-mediated increased colocalization with H3K4me3 and slower mobility compared to FL. Altogether, these data show that Thr490 in the noncore region modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting RAG2 interactions with H3K4me3.

Riches in RAGs: Revealing the V(D)J Recombinase through High-Resolution Structures.

Development of the adaptive immune system is dependent on V(D)J recombination, which forms functional antigen receptor genes through rearrangement of component gene segments. The V(D)J recombinase, comprising recombination-activating proteins RAG1 and RAG2, guides the initial DNA cleavage events to the recombination signal sequence (RSS), which flanks each gene segment. Although the enzymatic steps for RAG-mediated endonucleolytic activity were established over two decades ago, only recently have high-resolution structural studies of the catalytically active core regions of the RAG proteins shed light on conformational requirements for the reaction. While outstanding questions remain, we have a clearer picture of how RAG proteins function in generating the diverse repertoires of antigen receptors, the underlying foundation of the adaptive immune system.

The B3 Subunit of the Cone Cyclic Nucleotide-gated Channel Regulates the Light Responses of Cones and Contributes to the Channel Structural Flexibility.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3(-/-)/Nrl(-/-) mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3(-/-)/Nrl(-/-) mice compared with Nrl(-/-) mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors.

Structure of a TLR4-interacting SPA4 peptide.

We have recently identified a Toll-like receptor (TLR4)-interacting SPA4 peptide encoding amino acids: GDFRYSDGTPVNYTNWYRGE, a shorter region of human surfactant protein-A (SP-A). The SPA4 peptide suppressed lipopolysaccharide-induced inflammation (JPET 2011, Innate Immun 2013). In this report, we examined the structure of synthetic SPA4 peptide in solution by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The CD analysis revealed that the SPA4 peptide is composed of ∼35% beta sheet and <5% alpha helix. We used solution NMR to solve the structure of the SPA4 peptide. We calculated NMR structures using Nuclear Overhauser Enhancement (NOE) distance restraints. The superposition of the low energy structures indicated that the central 6-14 amino acids "SDGTPVNYT" of the 20mer SPA4 peptide form a turn, and amino acids on either side (GDFRY and NWYRGE) conform to flexible arms. Furthermore, thermal denaturation experiments demonstrated the structural flexibility of the peptide. The NMR structures of the SPA4 peptide align well with the homologous region within the available structure of rat SP-A and a computationally-docked model of SP-A-TLR4-MD2 protein complex. Together, our results support the structural adaptability of SPA4 peptide for binding to TLR4.

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.

Spatio-temporal regulation of RAG2 following genotoxic stress.

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr(490). In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.

Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein.

In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased ß-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.

Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS.

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild-type (WT) D2 loop consists of approximately 21% α-helix, approximately 20% ß-sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild-type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α-helicity. However, the thermal stability of the mutants was unchanged compared to wild-type, and all the mutants were capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.

A unique loop structure in oncostatin M determines binding affinity toward oncostatin M receptor and leukemia inhibitory factor receptor.

Oncostatin M (OSM) and leukemia inhibitory factor are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family. These cytokines play a crucial role in diverse biological events like inflammation, neuroprotection, hematopoiesis, metabolism, and development. The family is grouped together based on structural similarities and their ability to activate the transmembrane receptor glycoprotein 130 (gp130). The common structure among these cytokines defines the spacing and the orientation of binding sites for cell surface receptors. OSM is unique in this family as it can signal using heterodimers of gp130 with either leukemia inhibitory factor receptor (LIFR) (type I) or oncostatin M receptor (OSMR) (type II). We have identified a unique helical loop on OSM between its B and C helices that is not found on other IL-6 family cytokines. This loop is located near the "FXXK" motif in active site III, which is essential for OSM's binding to both LIFR and OSMR. In this study, we show that the BC loop does not play a role in OSM's unique ability to bind OSMR. Shortening of the loop enhanced OSM's interaction with OSMR and LIFR as shown by kinetic and equilibrium binding analysis, suggesting the loop may hinder receptor interactions. As a consequence of improved binding, these structurally modified OSMs exhibited enhanced biological activity, including suppressed proliferation of A375 melanoma cells.

Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotidegated channel by the tyrosine kinase adapter protein Grb14.

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged ß-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.

Elucidating the domain architecture and functions of non-core RAG1: the capacity of a non-core zinc-binding domain to function in nuclear import and nucleic acid binding.

BACKGROUND: The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction. RESULTS: Using a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain (CND; residues 87-217). CONCLUSIONS: We show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein.

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity.

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

The disease-causing mutations in the carboxyl terminus of the cone cyclic nucleotide-gated channel CNGA3 subunit alter the local secondary structure and interfere with the channel active conformational change.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransducton. Mutations in the channel subunits are associated with achromatopsia and progressive cone dystrophy in humans. More than 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them located in the carboxyl (C) terminus. This study investigates the defects of the two frequently occurring mutations, R377W and F488L, in the C-terminus of CNGA3. Ratiometric measurement of the intracellular Ca(2+) concentration and electrophysiological recordings showed the loss of functional activity of the mutant channels in an HEK293 heterologous expression system. Immunofluorescence labeling revealed an apparent cytosolic aggregation of the mutant channels compared to the wild type (WT). The R377W and F488L mutants, expressed and purified from Escherichia coli as glutathione S-transferase (GST) fused to the CNGA3 C-terminal domain, showed no negative effects on interactions with the channel subunits. Circular dichroism spectrum analyses were performed to examine the structural impact of the mutations. Although the R377W and F488L C-termini mutants retained stable, folded structures, the secondary structures of both mutants differed from the WT protein. Furthermore, the WT C-terminus exhibited a significant decrease in alpha-helical content in response to the channel ligands, while this allosteric transition was diminished in the two mutants. This is the first study showing the structural impact of the disease-causing mutations in the cone CNG channel subunit. The observed alterations in the local secondary structure and active conformational change may confer an adverse effect on the channel's activity and cellular processing.

A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity.

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn(2+) ions. Stripping the full complement of bound Zn(2+) ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn(2+) core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn(2+) ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn(2+) ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn(2+) coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn(2+) serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.

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.

Thermal dependency of RAG1 self-association properties.

BACKGROUND: Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed. RESULTS: Here, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37 degrees C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1. CONCLUSION: Together these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.