Affinage

RAG1

V(D)J recombination-activating protein 1 · UniProt P15918

Length
1043 aa
Mass
119.1 kDa
Annotated
2026-06-10
100 papers in source corpus 40 papers cited in narrative 39 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 9/9 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RAG1 is the catalytic engine of the V(D)J recombinase, the enzyme that initiates antigen-receptor gene assembly in developing lymphocytes by introducing site-specific DNA breaks at recombination signal sequences (RSS) (PMID:2360047, PMID:10678172). RAG1 alone has only weak recombinase activity, but co-expression with RAG2 increases recombination frequency by at least 1000-fold, and the two proteins assemble into a Y-shaped heterotetramer (two copies each) in which intertwined RAG1 N-terminal domains form a central stalk and each RAG1-RAG2 heterodimer composes an arm, with the catalytic center between them (PMID:2360047, PMID:25707801). Catalysis depends on a single DDE-type active site built from RAG1 aspartates D600 and D708, structurally related to transposase/integrase active sites and responsible for all DNA cleavage (PMID:10678172); a conserved RAG1 tryptophan (W956) supports the base-flip required for hairpin formation, while a dedicated nonamer-binding domain dimerizes to synapse two RSS elements (PMID:17307873, PMID:19396172). RAG1 binds and recognizes both the nonamer and heptamer of the RSS, but its intrinsic high-affinity non-sequence-specific DNA binding masks RSS recognition; RAG2 suppresses this non-specific binding to confer specificity, disrupts inactive RAG1 nuclear aggregates, and—through PHD-domain engagement of H3K4me3—relieves an autoinhibition of the hairpinning step and links the complex to active chromatin (PMID:9671477, PMID:19232525, PMID:34644584, PMID:21149691). After cleavage, RAG1/2 holds signal ends in a stable synaptic complex together with HMG1/2 and DNA-PK components, and full-length RAG1 contacts Ku70/Ku80, coupling cleavage to end-joining repair (PMID:9094713, PMID:23325855, PMID:18281312). The non-core N-terminus of RAG1 is a RING-finger E3 ubiquitin ligase that auto-ubiquitylates RAG1 and mono-ubiquitylates histone H3 to release RAG1 from chromatin and license the cleavage phase, and it recruits the VprBP/DDB1/Cul4A/Roc1 (CRL4-VprBP) complex that controls RAG1 protein stability and recombination fidelity (PMID:12629039, PMID:14671314, PMID:25572281, PMID:22157821, PMID:29925675). Additional layers of negative regulation include nucleolar sequestration of RAG1 via an internal motif (aa 216-383), DNA-damage feedback through the ATM-FOXO1 axis that downregulates RAG1, and direct post-transcriptional repression by miR-29c (PMID:32047031, PMID:27559048, PMID:34260911). In vitro the same complex catalyzes transposition, disintegration, hybrid-joint formation, and hairpin opening, reactions held in check by GTP and full-length RAG2 to limit genome-destabilizing transposition in cells (PMID:9723614, PMID:9727489, PMID:10892649, PMID:12682024, PMID:9535663, PMID:10330156). An S723C RAG1 mutation that is cleavage-proficient but defective in post-cleavage complex formation impairs lymphocyte development and predisposes to translocation-bearing thymic lymphomas, illustrating the danger of uncoupling cleavage from repair (PMID:19126872).

Mechanistic history

Synthesis pass · year-by-year structured walk · 19 steps
  1. 1990 High

    Established that the V(D)J recombinase is not a single activity but requires two genes acting together, defining RAG1's obligate partnership with RAG2.

    Evidence Co-transfection of RAG1 and/or RAG2 into fibroblasts with a recombination-frequency readout

    PMID:2360047

    Open questions at the time
    • Did not define which protein carries catalysis
    • No biochemical reconstitution of the cleavage chemistry
  2. 1997 High

    Showed that RAG1/2 do not release DNA after cutting but form a stable post-cleavage synaptic complex incorporating repair factors, linking cleavage to downstream joining.

    Evidence In vitro cleavage with nuclease protection, EMSA, and Co-IP detecting HMG-1/2 and DNA-PK

    PMID:9094713

    Open questions at the time
    • Stoichiometry of the complex not resolved
    • Direct role of DNA-PK components in handoff unclear
  3. 1998 High

    Defined RAG1's DNA-recognition and reaction repertoire: it distorts the RSS with RAG2 for stable specific binding, can run the reaction backward to form hybrid joints, and together with RAG2 acts as a bona fide transposase.

    Evidence EMSA/chemical footprinting, in vitro joining assays, and in vitro transposition with purified proteins and product sequencing

    PMID:9535663 PMID:9671477 PMID:9723614 PMID:9727489

    Open questions at the time
    • Why transposition is rare in vivo not addressed
    • Active-site residues not yet mapped
  4. 1999 High

    Localized core RAG1 as the minimal dimeric RSS-binding, zinc-containing species recognizing both heptamer and nonamer, and identified intrinsic hairpin-tip nicking activity relevant to coding-end processing.

    Evidence EMSA, zinc analysis, and in vitro cleavage/nuclease assays with recombinant core RAG1; HMG2 incorporation tests

    PMID:10330156 PMID:10390537

    Open questions at the time
    • Catalytic residues still undefined
    • Mechanism of HMG2 stimulation not structural
  5. 2000 High

    Pinpointed the catalytic active site to RAG1 D600/D708 and dissected how transposition intermediates resolve, explaining the in vivo rarity of RAG transposition via Mg2+-favored disintegration.

    Evidence Structure prediction plus site-directed mutagenesis with recombination assays; in vitro transposition/disintegration at varying Mg2+

    PMID:10678172 PMID:10892649

    Open questions at the time
    • No atomic structure of the active site yet
    • Cellular regulators of disintegration not identified
  6. 2003 High

    Revealed a second, non-recombinase function of RAG1: its N-terminal RING finger is an active E3 ubiquitin ligase that auto-ubiquitylates RAG1, and showed that GTP and full-length RAG2 suppress transposition by blocking target capture.

    Evidence In vitro ubiquitylation with RAG1 N-terminal/RING fragments and CDC34, cell-based ubiquitylation, RING mutants; in vitro transposition with defined GTP/Ca2+/RAG2 conditions

    PMID:12629039 PMID:12682024 PMID:14671314

    Open questions at the time
    • Physiological ubiquitylation substrates not yet identified
    • Functional consequence of auto-ubiquitylation in vivo unknown
  7. 2004 High

    Demonstrated intramolecular autoregulation: RAG1's central domain has intrinsic cleavage activity that its own C-terminal domain inhibits in a substrate-dependent manner.

    Evidence In vitro ssDNA/dsDNA cleavage with isolated RAG1 domain fragments

    PMID:15254250

    Open questions at the time
    • Structural basis of C-terminal inhibition unresolved
    • How RAG2 modulates this autoregulation not addressed
  8. 2007 High

    Provided a mechanistic model for hairpin formation by identifying RAG1 W956 as a base-flip residue analogous to Tn5 transposase, unifying RAG chemistry with the transposase superfamily.

    Evidence Site-directed mutagenesis with abasic-substrate rescue in in vitro cleavage assays

    PMID:17307873

    Open questions at the time
    • Direct structural visualization of the flipped base lacking
    • Coordination with nicking step not fully resolved
  9. 2008 Medium

    Linked the cleavage and joining phases biochemically by showing full-length (non-core) RAG1 associates with the NHEJ Ku70/Ku80 heterodimer.

    Evidence Co-IP/pulldown comparing full-length vs core RAG1

    PMID:18281312

    Open questions at the time
    • Single Co-IP without reciprocal validation
    • Interaction interface and in vivo relevance undefined
  10. 2009 High

    Resolved the architecture and binding logic of RAG1: the nonamer-binding domain dimerizes to synapse two RSS, the synaptic complex contains paired heterotetramers in side-by-side configuration, RSS binding triggers RAG1 conformational change, and RAG2 confers specificity by suppressing RAG1 non-specific DNA binding; an S723C knockin established a discrete post-cleavage/joining function.

    Evidence Crystallography of the NBD-nonamer complex with FRET; AFM mass measurement; intrinsic fluorescence/CD; fluorescence anisotropy/EMSA; in vivo RAG1-S723C knockin mouse

    PMID:12488446 PMID:19126872 PMID:19232525 PMID:19396172 PMID:19502597 PMID:19647518

    Open questions at the time
    • Full-complex high-resolution structure still missing at this stage
    • How conformational change is transmitted to catalysis unclear
  11. 2010 High

    Defined how RAG1 is targeted in vivo: it binds focally to RSS-containing recombination centers within active chromatin, with binding controlled by enhancers globally and promoters/transcription locally, confirming the accessibility model.

    Evidence ChIP-seq in primary lymphoid cells and ChIP on enhancer/promoter mutant Tcra/Tcrb alleles

    PMID:20398922 PMID:21115692

    Open questions at the time
    • Chromatin marks driving binding not yet dissected (resolved later)
    • Mechanism coupling binding to cleavage timing unclear
  12. 2010 High

    Explained the trigger for cleavage: C-terminal regions of RAG1 and RAG2 autoinhibit hairpinning, and RAG2 PHD-domain binding to H3K4me3 relieves this inhibition, coupling catalysis to active chromatin marks.

    Evidence In vitro cleavage with truncated/full-length RAG proteins plus H3K4me3 peptide binding assays

    PMID:21149691

    Open questions at the time
    • Structural mechanism of inhibition relief not defined
    • Quantitative link to in vivo timing absent
  13. 2011 High

    Connected RAG1's E3 ligase activity to a cellular ubiquitin machine by showing the CRL4-VprBP complex binds full-length RAG1, assembles into RAG-DNA complexes, and is required for normal B-cell development and recombination fidelity.

    Evidence Co-IP, in vitro ubiquitylation, and B-lineage VprBP conditional knockout with V(D)J junction sequencing

    PMID:22157821

    Open questions at the time
    • Relevant ubiquitylation substrate of CRL4-VprBP at this stage unclear
    • Relationship to RAG1 RING activity not yet reconciled
  14. 2013 High

    Clarified how the architectural factor HMGB1 is incorporated, showing DNA cooperatively strengthens the otherwise weak RAG1-HMGB1 interaction.

    Evidence Fluorescence anisotropy and pulldown with/without DNA

    PMID:23325855

    Open questions at the time
    • Structural placement of HMGB1 in the complex not defined
    • Functional contribution to synapsis quantification incomplete
  15. 2014 Medium

    Identified locus-specific direction of RAG1 deposition by a sequence-specific transcription factor, RUNX1, which binds a TCR-delta RSS and recruits RAG1 to enforce ordered gene assembly.

    Evidence ChIP, Co-IP of RUNX1 and RAG1, and recombination assays with RSS mutants

    PMID:25135298

    Open questions at the time
    • Single-lab study
    • Generality to other loci untested
  16. 2015 High

    Delivered the definitive heterotetramer architecture, mapped the RAG1-RAG2 binding interface, and revealed that RAG1 E3 activity ubiquitylates histone H3 to release RAG1 from chromatin and license the cleavage phase.

    Evidence 3.2 Å crystal structure of RAG1-RAG2; biolayer interferometry/mutagenesis of the interface; C325Y RING knockin mouse with in vitro ubiquitylation and ChIP

    PMID:25572281 PMID:25707801 PMID:25745109

    Open questions at the time
    • Structure is mouse and partly core; full regulatory non-core regions not resolved
    • How H3 ubiquitylation is spatially coordinated with cleavage unclear
  17. 2016 High

    Dissected genome-wide RAG1 targeting into two chromatin modes (RAG2-PHD/H3K4me3 promoter mode and non-core-RAG1/H3K27Ac enhancer mode), traced the evolutionary origin of RAG1 to transposases, and identified ATM-FOXO1 damage feedback downregulating RAG1.

    Evidence Deep RAG1 ChIP-seq with core/full-length comparison; ancestral RAG1/Transib reconstitution assays; genotoxic stress with FOXO1-Erag ChIP and ATM inhibition

    PMID:27056670 PMID:27436288 PMID:27559048

    Open questions at the time
    • Relative contribution of the two binding modes to productive recombination unclear
    • How damage feedback integrates with E3-driven release not resolved
  18. 2020 High

    Established nucleolar sequestration (via aa 216-383) as a negative regulatory reservoir for RAG1, with nucleolar egress regions (aa 1-215) required for activity.

    Evidence BioID interactome, fluorescence microscopy, and V(D)J assays in pre-B cell lines with localization mutants

    PMID:32047031

    Open questions at the time
    • Signal triggering nucleolar release not identified
    • Nucleolar partners' mechanistic roles undefined
  19. 2021 Medium

    Added two further control layers: RAG2 actively disrupts nuclear RAG1 aggregates to enable activity (species-specific), and miR-29c directly represses RAG1 to tune recombination during B-cell development.

    Evidence Fluorescence imaging with cross-species RAG2 complementation; CRISPR editing of the miR-29c site in RAG1 with overexpression/knockout and recombination assays

    PMID:34260911 PMID:34644584

    Open questions at the time
    • Aggregate-disruption mechanism not reconstituted in vitro
    • Interplay between miRNA and protein-level regulation unquantified

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the structural transitions of the heterotetramer—conformational change on RSS binding, autoinhibition relief by H3K4me3, E3-driven H3 ubiquitylation and chromatin release, and post-cleavage handoff to NHEJ—are temporally ordered and integrated with damage and miRNA feedback into a single regulated reaction cycle remains unresolved.
  • No time-resolved structure of the catalytic cycle in chromatin context
  • Direct linkage between RING E3 activity and NHEJ recruitment uncharacterized
  • Quantitative hierarchy among the negative regulators (nucleolar, ATM-FOXO1, miR-29c, CRL4-VprBP) unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140097 catalytic activity, acting on DNA 5 GO:0003677 DNA binding 4 GO:0016874 ligase activity 4 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005694 chromosome 3 GO:0005634 nucleus 2 GO:0005730 nucleolus 2
Pathway
R-HSA-1266738 Developmental Biology 3 R-HSA-168256 Immune System 3 R-HSA-73894 DNA Repair 3
Partners
HMGB1KU70KU80RAG2RCH1RUNX1SRP1VPRBP
Complex memberships
CRL4-VprBP (VprBP/DDB1/Cul4A/Roc1) E3 ligaseRAG1-RAG2 heterotetramer (V(D)J recombinase)post-cleavage signal-end synaptic complex (with HMG1/2 and DNA-PK)

Evidence

Reading pass · 39 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1990 RAG1 alone inefficiently induces V(D)J recombinase activity in fibroblasts, but co-transfection with the adjacent gene RAG2 results in at least a 1000-fold increase in recombination frequency, demonstrating that RAG1 and RAG2 synergistically activate V(D)J recombination. Transfection of fibroblasts with RAG1 and/or RAG2 expression constructs followed by recombination frequency assay Science High 2360047
1998 RAG1 and RAG2 together form a transposase capable of excising DNA containing recombination signals from a donor site and inserting it into a target DNA molecule, producing short target DNA duplications flanking the transposed fragment, consistent with canonical transposition. In vitro transposition assay with purified RAG1/RAG2 proteins and defined DNA substrates; product characterization by sequencing Nature High 9723614 9727489
1997 After DNA cleavage at recombination signals, RAG1 and RAG2 form a stable post-cleavage synaptic complex with signal-end DNA that also incorporates HMG-1/HMG-2 and components of DNA-dependent protein kinase (DNA-PK). In vitro V(D)J cleavage system with nuclease sensitivity assays, gel mobility shift assays, and immunoprecipitation experiments Cell High 9094713
2000 Two aspartic acid residues in RAG1, D600 and D708, constitute a single catalytic active site structurally related to the active sites of transposases/integrases and are responsible for all catalytic (DNA cleavage) functions of the RAG protein complex. Computational secondary structure prediction followed by site-directed mutagenesis and V(D)J recombination activity assays Molecular Cell High 10678172
2015 Crystal structure of the mouse RAG1-RAG2 complex at 3.2 Å resolution shows a 230 kDa Y-shaped RAG1-RAG2 heterotetramer, with the amino-terminal domains of two RAG1 chains forming an intertwined stalk and each RAG1-RAG2 heterodimer composing one arm, with the active site in the middle and RAG2 at the tip. The architecture is similar to hairpin-forming transposases Hermes and Tn5. X-ray crystallography at 3.2 Å resolution Nature High 25707801
1998 RAG1 exhibits only weak (3–5-fold) preference for RSS over random DNA when alone; RAG2 does not bind DNA by itself, but together RAG1 and RAG2 form a more stable and specific RAG1-RAG2-DNA complex that is active in V(D)J cleavage. The nonamer is protected with contacts in the minor groove, and the heptamer is rendered more accessible, indicating DNA distortion near the coding/signal border. Gel retardation (EMSA), footprinting with 1,10-phenanthroline-copper and dimethyl sulfate protection assays Molecular and Cellular Biology High 9671477
1999 RAG1 and RAG2 possess intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip, implicating them in initiating coding-end processing and P-nucleotide generation during V(D)J recombination. In vitro nuclease activity assay with synthetic and cleavage-generated hairpin substrates in Mn2+ and Mg2+ conditions Molecular and Cellular Biology High 10330156
1998 RAG1 and RAG2 can reverse the cleavage reaction by joining an RSS to a broken coding sequence end, producing hybrid joints, demonstrating a strand-transfer (rejoining) activity of the RAG proteins. In vitro joining assay with RAG proteins and defined DNA substrates; product characterization Science High 9535663
2003 The N-terminal region of RAG1 (containing the RING finger domain) functions as an E3 ubiquitin ligase, mediating ubiquitylation of a test substrate and formation of polyubiquitin chains in vitro, independent of RAG1's recombinase catalytic core. In vitro ubiquitylation assay with purified RAG1 N-terminal domain fragments Genes & Development High 12629039
2003 RAG1 undergoes auto-ubiquitylation in cells; in vitro, the RING finger domain mediates its own ubiquitylation at a conserved lysine residue, preferentially using ubiquitin-conjugating enzyme UbcH3/CDC34, and requires an intact RING finger motif. In vitro ubiquitylation assay with purified RAG1 RING finger domain; cell-based ubiquitylation assay; RING finger mutant analysis PNAS High 14671314
1995 RAG1 and RAG2 form a stable complex in primary thymocytes and in adherent cells. Most cells localize RAG proteins at the nuclear periphery, but when overexpressed in fibroblasts, RAG1 localizes to the nucleolus. Nucleolar localization is mediated by basic amino acid-containing domains that also serve as NLS and RNA-binding sequences. RAG1-interacting proteins SRP1 and Rch1 bind directly to RAG1 NLS and mediate nuclear/nucleolar translocation. Co-immunoprecipitation from thymocytes; subcellular fractionation; immunofluorescence localization; deletion/domain analysis in transfected cells Immunity High 8777717
2009 Crystal structure of the RAG1 nonamer binding domain (NBD) bound to its nonamer DNA recognition motif reveals a tightly interwoven dimer that simultaneously binds and synapses two nonamer DNA elements, with each NBD contacting both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex. X-ray crystallography; fluorescence resonance energy transfer; biochemical binding and synapsis assays Nature Structural & Molecular Biology High 19396172
2000 RAG1/RAG2 can resolve transposition intermediates by two pathways: (1) hairpin formation on target DNA adjacent to transposed RSS ends, consistent with a mechanism leading to chromosomal translocations, and (2) disintegration (removal of transposed donor DNA from the intermediate). At physiological magnesium concentrations, disintegration is favored, which may explain why RAG-mediated transposition is rare in cells. In vitro transposition/disintegration assay with purified RAG proteins and defined branched DNA substrates at varying Mg2+ concentrations Cell High 10892649
2003 RAG-mediated transposition is suppressed by physiological concentrations of GTP and by the full-length RAG2 protein, both acting by blocking non-covalent capture of target DNA. Ca2+ ions can stimulate transposition even in the presence of full-length RAG2 and GTP. In vitro transposition assay with defined concentrations of GTP, Ca2+, and full-length vs. core RAG2 protein The EMBO Journal High 12682024
2010 In vivo, RAG1 and RAG2 bind in a highly focal manner to a small region of active chromatin encompassing Ig kappa and TCR alpha J gene segments (recombination centers) in a developmental stage- and lineage-specific manner. RAG1 binding was detected only at RSS-containing regions, while RAG2 binds at thousands of H3K4me3-containing sites genome-wide. Each RAG protein is independently capable of specific binding within recombination centers, and RAG1 can bind in the absence of RAG2. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in primary lymphoid cells Cell High 20398922
2010 Enhancers control RAG1 binding globally at Jα or Dβ/Jβ gene segments, promoters and transcription direct RAG1 binding locally at the Tcra and Tcrb loci, and RAG1 can be targeted to chromatin in the absence of RAG2, providing direct confirmation of the accessibility model of V(D)J recombination regulation. Chromatin immunoprecipitation (ChIP) using mutant Tcra and Tcrb alleles lacking enhancers or promoters in primary lymphocytes Journal of Experimental Medicine High 21115692
2010 The C-terminal regions of RAG1 (aa 1009-1040) and RAG2 (aa 388-520, including the PHD domain) collaborate to inhibit the hairpinning stage of DNA cleavage (autoinhibition). The RAG2 C-terminal region stabilizes the RAG1/2 heterotetramer but destabilizes the RAG-DNA pre-cleavage complex. Binding of the RAG2 PHD domain to histone H3K4me3 peptide reverses this inhibition and restores hairpinning activity. In vitro DNA cleavage assays with truncated/full-length purified RAG proteins; histone peptide binding assays PNAS High 21149691
2009 Electron microscopy of the post-cleavage signal-end complex (SEC) revealed an anchor-shaped particle containing two protomers each of RAG1 and RAG2. The N-termini of RAG1 and RAG2 are positioned at opposing ends, and the DNA beyond the RSS nonamer emerges from the same face of the complex near RAG1 N-termini. Stoichiometric and molecular mass analysis; negative-staining electron microscopy of isolated RAG1/2-DNA complexes Molecular Cell High 19647518
1999 Core RAG1 (aa 384-1008) exists as a dimer in solution and as the minimal DNA-binding species at an RSS. RAG1 recognizes both conserved nonamer and heptamer sequences of the RSS. Core RAG1 contains two zinc ions. HMG2 is stably incorporated into the recombinant RAG1/RSS complex and increases the affinity of RAG1 for the RSS even in the absence of RAG2. Electrophoretic mobility shift assay (EMSA); competition binding assays; zinc analysis; in vitro cleavage assay with recombinant proteins Nucleic Acids Research High 10390537
2007 RAG1 residue W956 is required for both the nicking and hairpin stages of DNA cleavage, consistent with a base-flip mechanism where W956 stacks on a flipped base during hairpin formation, analogous to W298 in the Tn5 transposase. Abasic DNA substrates rescue the W956A RAG1 mutant defect, supporting a base-flip model. In vitro V(D)J cleavage assays with site-directed RAG1 mutants and abasic DNA substrates PNAS High 17307873
2004 The isolated central domain of RAG1 has inherent single-stranded DNA cleavage activity that does not require but is enhanced by RAG2; the C-terminal domain of RAG1 inhibits this catalytic activity, but the inhibition is suppressed on double-stranded DNA substrates, suggesting autoregulation of RAG1 DNA cleavage activity. In vitro ssDNA and dsDNA cleavage assays with isolated RAG1 domain fragments (central domain and C-terminal domain separately and in combination) Molecular and Cellular Biology High 15254250
2008 Full-length RAG1 (but not core RAG1) physically associates with the NHEJ factors Ku70 and Ku80, providing a biochemical link between the cleavage and joining phases of V(D)J recombination. Co-immunoprecipitation and pulldown assays comparing full-length vs. core RAG1 Nucleic Acids Research Medium 18281312
2011 The cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP, is assembled into RAG protein-DNA complexes, and supports in vitro ubiquitylation activity insensitive to RAG1 RING domain mutations. B lineage-specific VprBP disruption arrests B-cell development and impairs V(D)J rearrangements, with D-JH coding joints showing longer junctional insertions and higher mutation frequencies. Co-immunoprecipitation; in vitro ubiquitylation assay; conditional knockout mouse model with immunophenotyping and V(D)J junction sequencing The EMBO Journal High 22157821
2015 RAG1 ubiquitylates histone H3; a RAG1 RING-finger C325Y mutation abrogates mono-ubiquitylation of H3, blocks V(D)J recombination at the cleavage step, and causes un-ubiquitylated H3 to retain RAG1 at chromatin via interaction with RAG1 N-terminal amino acids 1-218. Histone H3 ubiquitylation by RAG1 thus triggers RAG1 release, allowing transition to the cleavage phase. Knockin mouse model (C325Y); in vitro ubiquitylation assay; chromatin immunoprecipitation; Co-IP/pulldown domain mapping Cell Research High 25572281
2015 The RAG1-RAG2 interaction has an affinity of KD ~0.4 μM; the interaction requires a predicted α-helix near the RAG1 C terminus (aa 997-1008) and a region from aa 479-559, with Asp-546 and Glu-547 being particularly critical. Structural modeling suggests these residues lie near the RAG1 active site, raising the possibility that RAG2 binding alters the RAG1 active site conformation. Biolayer interferometry; pulldown assays; site-directed mutagenesis; structural modeling based on Hermes transposase structure Journal of Biological Chemistry High 25745109
2009 RAG1 exhibits a high-affinity non-sequence-specific DNA binding mode that masks RSS recognition under physiological conditions. Addition of RAG2 suppresses this non-specific RAG1-DNA association, resulting in a large differential in binding affinity for RSS versus non-RSS sites, establishing a major mechanism by which RAG2 enables effective RSS recognition. Fluorescence anisotropy and EMSA comparing RAG1 alone vs. RAG1+RAG2 binding to RSS and non-RSS substrates Journal of Molecular Biology High 19232525
2009 Atomic force microscopy of pre-cleavage RAG synaptic complexes reveals that the complex contains approximately twice the protein content as a single RAG-RSS complex, consistent with a pair of RAG heterotetramers. The RSSs in the synaptic complex are predominantly in a side-by-side configuration without DNA strand crossover, and synapsis is mediated by RAG protein-protein interactions. Atomic force microscopy (AFM) of RAG1/RAG2-DNA complexes; mass measurement Journal of Biological Chemistry Medium 19502597
2013 HMGB1 is recruited cooperatively to RAG1-DNA complexes: HMGB1 alone has only a weak interaction with RAG1 in the absence of DNA, but the presence of DNA synergistically increases HMGB1 binding to RAG1, without strict RSS sequence specificity. This cooperative binding explains the stable integration of HMGB1 in the V(D)J recombinase complex. Fluorescence anisotropy with Alexa488-labeled HMGB1; pulldown assays; comparison of RAG1-HMGB1 interaction with and without DNA Nucleic Acids Research High 23325855
2009 RAG1 protein undergoes major conformational changes upon binding the RSS: intrinsic fluorophores shift from hydrophobic to solvent-exposed environments, and circular dichroism reveals structural changes. The RSS-induced conformational change may influence RAG1's interaction with RAG2 and synaptic complex formation. Protein intrinsic fluorescence spectroscopy; circular dichroism; acrylamide quenching; frequency domain fluorescence anisotropy decay Journal of Biological Chemistry Medium 12488446
2020 RAG1 associates with numerous nucleolar proteins in a manner dependent on amino acids 216-383, and a motif within this region is required for nucleolar localization. Disruption of nucleoli or mutation of the RAG1 nucleolar localization motif increases V(D)J recombination activity, while removal of amino acids 1-215 (required for nucleolar egress) reduces recombination, indicating that nucleolar sequestration of RAG1 is a negative regulatory mechanism. Proximity-dependent biotin identification (BioID) interactome analysis; fluorescence microscopy; V(D)J recombination activity assays in transformed and primary pre-B cell lines PNAS High 32047031
2018 VprBP (DCAF1) restrains RAG1 protein levels post-transcriptionally and independently of Dicer: loss of VprBP stabilizes RAG1 protein by protecting it from degradation that requires both 20S proteasome and cullin-RING E3 ligase activity. RAG1 stabilization through small molecule inhibition of cullin-RING E3 ligase promotes V(D)J recombination in pre-B cells. Conditional VprBP knockout B cells; western blot for RAG1 protein levels; proteasome and cullin-RING ligase inhibitor experiments; V(D)J recombination assay Journal of Immunology High 29925675
2016 RAG1 genome-wide binding is driven by two distinct modes of chromatin interaction: (1) an H3K4me3-focused, promoter-associated mode dependent on the RAG2 PHD finger, and (2) an H3K27Ac-focused, enhancer-associated mode dependent on the non-core regions of RAG1. Sequence-specific DNA binding contributes minimally to RAG1 targeting outside antigen receptor loci. Deep RAG1 ChIP-seq analysis combined with chromatin feature correlation; comparison of core vs. full-length RAG1 binding patterns Nucleic Acids Research High 27436288
2009 In vivo evidence from a RAG1-S723C knockin mouse shows that this mutation is proficient for DNA cleavage but exhibits defects in post-cleavage complex formation and end joining, resulting in impaired lymphocyte development, decreased V(D)J rearrangements, and aberrant DNA double-strand breaks at rearranging loci. The mutation predisposes to thymic lymphomas with chromosomal translocations in a p53 mutant background. Knockin mouse model (RAG1-S723C); immunophenotyping; V(D)J rearrangement analysis; genomic DSB detection; tumor analysis Blood High 19126872
2014 RUNX1 protein binds to the Dδ2-23RSS in the human TCR-δ locus, interacts directly with RAG1, and enhances RAG1 deposition at this site, thereby imposing the use of two Dδ gene segments in human TCR-δ chains and dictating ordered TCR-δ gene assembly. ChIP assays; co-immunoprecipitation of RUNX1 and RAG1; functional V(D)J recombination assays with RSS mutants Journal of Experimental Medicine Medium 25135298
2016 Ancestral RAG1 proteins (Transib transposase and sea urchin RAG1-like) have a latent ability to initiate V(D)J recombination when co-expressed with RAG2, and Transib transposase transposition is stimulated by RAG2. RAG1 alone can perform low levels of V(D)J recombination in the absence of RAG2, losing the requirement for the 12/23 rule, implicating RAG2 in the origins of the 12/23 restriction. Cell-based V(D)J recombination assays with ancestral RAG1-like proteins ± RAG2; in vitro transposition assay with Transib transposase ± RAG2 Genes & Development High 27056670
2021 Human RAG1 aggregates in the nucleus in the absence of RAG2, exhibiting extremely low V(D)J recombination activity. RAG2 interacts with RAG1 to disrupt RAG1 aggregates and thereby activate robust V(D)J recombination. Mouse and zebrafish RAG2 cannot disrupt human RAG1 aggregates as efficiently as human RAG2, indicating species-specific coevolution. Fluorescence imaging of RAG1 aggregation in cells; V(D)J recombination activity assays; cross-species RAG2 complementation experiments Cell Reports Medium 34644584
2006 RAG proteins can bind and cleave a cryptic RSS (cRSS) located within an IgVH gene segment with sequence specificity; however, cleavage at the cRSS bypasses formation of the DNA hairpin intermediate and instead produces nicks on both strands, suggesting an alternative RAG cleavage mechanism depending on substrate sequence. EMSA binding assays; in vitro cleavage assays with canonical RSS vs. cRSS substrates; strand-specific nick analysis Journal of Biological Chemistry Medium 16531612
2016 DNA damage activates the ATM kinase which causes loss of FOXO1 binding to the Erag enhancer and FOXO1 cleavage, resulting in rapid downregulation of RAG1/2 mRNA and protein. RAG1/2-induced DNA breaks in pre-B cells also downmodulate RAG1/2 expression via this ATM-FOXO1 pathway, establishing a negative feedback regulatory mechanism. Genotoxic stress treatment (ionizing radiation, etoposide, bleomycin) of primary pre-B, pro-B, and pro-T cells; qRT-PCR; western blot; ChIP for FOXO1 at Erag; ATM inhibitor experiments; V(D)J recombination assay Journal of Immunology High 27559048
2021 miR-29c directly targets and downregulates RAG1 expression in a B cell stage-specific manner; CRISPR-Cas9 editing of the miR-29c binding site in RAG1 confirms direct interaction. Modulation of miR-29c levels alters V(D)J recombination efficiency in pre-B cells; miR-29c null mice show reduced mature B cells. CRISPR-Cas9 genome editing of miR-29c target site in RAG1; miR-29c overexpression/inhibition in pre-B cells; V(D)J recombination assay; miR-29c knockout mice immunophenotyping Cell Reports High 34260911

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1990 RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science (New York, N.Y.) 1175 2360047
1998 Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 560 9723614
1998 DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 414 9727489
2005 RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS biology 337 15898832
2002 Target-selected inactivation of the zebrafish rag1 gene. Science (New York, N.Y.) 306 12098699
1997 RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 244 9094713
1991 Thymocyte expression of RAG-1 and RAG-2: termination by T cell receptor cross-linking. Science (New York, N.Y.) 237 1831564
2010 The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 234 20398922
1996 Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science (New York, N.Y.) 223 8953043
1993 Expression and V(D)J recombination activity of mutated RAG-1 proteins. Nucleic acids research 168 8284210
1997 Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish. Immunogenetics 149 9089097
2015 Crystal structure of the V(D)J recombinase RAG1-RAG2. Nature 130 25707801
2006 An ancient evolutionary origin of the Rag1/2 gene locus. Proceedings of the National Academy of Sciences of the United States of America 125 16505374
2000 Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Molecular cell 124 10678172
1999 Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2. Science (New York, N.Y.) 116 10446057
1998 Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences. Molecular and cellular biology 113 9671477
1994 Regulation of RAG-1 and CD69 expression in the thymus during positive and negative selection. European journal of immunology 105 8020549
2015 Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon. Biology direct 92 25928409
1995 Localization, interaction, and RNA binding properties of the V(D)J recombination-activating proteins RAG1 and RAG2. Immunity 92 8777717
1999 DNA hairpin opening mediated by the RAG1 and RAG2 proteins. Molecular and cellular biology 89 10330156
1998 Rejoining of DNA by the RAG1 and RAG2 proteins. Science (New York, N.Y.) 85 9535663
2000 Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity. The Journal of experimental medicine 82 11085752
2004 Mutations in genes required for T-cell development: IL7R, CD45, IL2RG, JAK3, RAG1, RAG2, ARTEMIS, and ADA and severe combined immunodeficiency: HuGE review. Genetics in medicine : official journal of the American College of Medical Genetics 81 14726805
2012 Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 76 23150522
2009 Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis. Nature structural & molecular biology 76 19396172
2014 RAG1/2 knockout pigs with severe combined immunodeficiency. Journal of immunology (Baltimore, Md. : 1950) 74 24973446
1993 Characterization and expression of recombination activating genes (RAG-1 and RAG-2) in Xenopus laevis. Journal of immunology (Baltimore, Md. : 1950) 74 8376769
2011 Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer. Leukemia 71 21617701
2005 Evolution of RAG-1 in polyploid clawed frogs. Molecular biology and evolution 68 15703243
1996 RAG1, RAG2 and pre-T cell receptor alpha chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation. European journal of immunology 68 8977312
2003 The RAG1 N-terminal domain is an E3 ubiquitin ligase. Genes & development 67 12629039
2010 Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination. The Journal of experimental medicine 64 21115692
1995 The recombination activation gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis. Immunogenetics 61 7642230
1999 A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2. Nucleic acids research 59 10390537
2009 Characterization of rag1 mutant zebrafish leukocytes. BMC immunology 58 19192305
2003 Regulation of RAG1/RAG2-mediated transposition by GTP and the C-terminal region of RAG2. The EMBO journal 57 12682024
2003 Autoubiquitylation of the V(D)J recombinase protein RAG1. Proceedings of the National Academy of Sciences of the United States of America 57 14671314
2000 RAG1/2-mediated resolution of transposition intermediates: two pathways and possible consequences. Cell 57 10892649
2001 RAG1 and RAG2 expression by B cell subsets from human tonsil and peripheral blood. Journal of immunology (Baltimore, Md. : 1950) 56 11123315
1995 RAG1 and RAG2 expression in human intestinal epithelium: evidence of extrathymic T cell differentiation. European journal of immunology 53 7774617
2013 Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome. The Journal of allergy and clinical immunology 51 24332219
2010 Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proceedings of the National Academy of Sciences of the United States of America 49 21149691
1996 Regions of RAG1 protein critical for V(D)J recombination. European journal of immunology 49 8625984
2020 Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nature cell biology 46 31907413
2009 The roles of the RAG1 and RAG2 "non-core" regions in V(D)J recombination and lymphocyte development. Archivum immunologiae et therapiae experimentalis 46 19333736
1999 Omenn syndrome: a disorder of Rag1 and Rag2 genes. Journal of clinical immunology 46 10226883
2008 Evidence for Ku70/Ku80 association with full-length RAG1. Nucleic acids research 45 18281312
2009 Asynchronous RAG-1 expression during B lymphopoiesis. Journal of immunology (Baltimore, Md. : 1950) 43 20007571
2001 Effect of HIV integrase inhibitors on the RAG1/2 recombinase. Proceedings of the National Academy of Sciences of the United States of America 43 11756686
2009 Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation. Blood 41 19126872
1997 Identification and characterization of the murine Rag1 promoter. Molecular immunology 41 9464529
2017 Sexual and Vertical Transmission of Zika Virus in anti-interferon receptor-treated Rag1-deficient mice. Scientific reports 40 28775298
2009 Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex. Molecular cell 40 19647518
2004 Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1. Immunological reviews 39 15242397
2018 Hypomorphic Rag1 mutations alter the preimmune repertoire at early stages of lymphoid development. Blood 37 29743177
2011 VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity. The EMBO journal 35 22157821
1992 Activation of V(D)J recombination by RAG1 and RAG2. Trends in genetics : TIG 35 1492366
1997 Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions. Journal of immunology (Baltimore, Md. : 1950) 34 9379036
2016 Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination. Genes & development 33 27056670
2015 RAG1 deficiency may present clinically as selective IgA deficiency. Journal of clinical immunology 33 25739914
2014 Integrated genetic approaches identify the molecular mechanisms of Sox4 in early B-cell development: intricate roles for RAG1/2 and CK1ε. Blood 33 24786772
2019 Rag1 immunodeficiency-induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress. Aging cell 32 31348603
2018 Rag1-null Dahl SS rats reveal that adaptive immune mechanisms exacerbate high protein-induced hypertension and renal injury. American journal of physiology. Regulatory, integrative and comparative physiology 32 29537860
2013 Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA. Nucleic acids research 32 23325855
2013 An amphioxus RAG1-like DNA fragment encodes a functional central domain of vertebrate core RAG1. Proceedings of the National Academy of Sciences of the United States of America 32 24368847
2017 Naïve B cells reduce fungal dissemination in Cryptococcus neoformans infected Rag1-/- mice. Virulence 30 28837391
2021 MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development. Cell reports 27 34260911
2020 Nucleolar localization of RAG1 modulates V(D)J recombination activity. Proceedings of the National Academy of Sciences of the United States of America 27 32047031
2016 The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling. Journal of immunology (Baltimore, Md. : 1950) 27 27559048
2009 Molecular mechanism underlying RAG1/RAG2 synaptic complex formation. The Journal of biological chemistry 27 19502597
2008 AID and RAG1 do not contribute to lymphomagenesis in Emu c-myc transgenic mice. Oncogene 26 18408759
2018 Generation of Immunodeficient Rats With Rag1 and Il2rg Gene Deletions and Human Tissue Grafting Models. Transplantation 25 29688994
2020 T and B Lymphocyte Deficiency in Rag1-/- Mice Reduces Retinal Ganglion Cell Loss in Experimental Glaucoma. Investigative ophthalmology & visual science 24 33320171
2017 Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks. Journal of immunology (Baltimore, Md. : 1950) 24 28213501
2014 Atypical severe combined immunodeficiency caused by a novel homozygous mutation in Rag1 gene in a girl who presented with pyoderma gangrenosum: a case report and literature review. Journal of clinical immunology 24 25104208
2009 A non-sequence-specific DNA binding mode of RAG1 is inhibited by RAG2. Journal of molecular biology 24 19232525
2015 NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity. Blood 23 26153519
2007 Biochemical and folding defects in a RAG1 variant associated with Omenn syndrome. Journal of immunology (Baltimore, Md. : 1950) 23 18056378
2006 Omenn syndrome--review of several phenotypes of Omenn syndrome and RAG1/RAG2 mutations in Japan. Allergology international : official journal of the Japanese Society of Allergology 23 17075247
2002 RAG1-DNA binding in V(D)J recombination. Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy. The Journal of biological chemistry 23 12488446
2001 RAG1 and RAG2 in V(D)J recombination and transposition. Immunologic research 23 11417858
2021 Natural Killer Cells Are Present in Rag1-/- Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke. Translational stroke research 22 34105078
2016 RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2. Nucleic acids research 22 27436288
1995 Lymphocyte development and function in T-cell receptor and RAG-1 mutant mice. International reviews of immunology 22 7494108
2021 RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination. Cell reports 21 34644584
2015 RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination. Cell research 21 25572281
2003 Cloning of shark RAG2 and characterization of the RAG1/RAG2 gene locus. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 21 12551847
2006 DNA cleavage of a cryptic recombination signal sequence by RAG1 and RAG2. Implications for partial V(H) gene replacement. The Journal of biological chemistry 20 16531612
2015 Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2. The Journal of biological chemistry 19 25745109
2014 RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement. The Journal of experimental medicine 19 25135298
2007 Requirements for DNA hairpin formation by RAG1/2. Proceedings of the National Academy of Sciences of the United States of America 19 17307873
2005 Novel RAG1 mutation in a case of severe combined immunodeficiency. Pediatrics 19 16061569
1992 Restricted expression of recombination activating gene (RAG-1) in mouse lymphoid tissues. Cell biochemistry and function 19 1628381
1996 Chromatin structure and transcriptional regulation of human RAG-1 gene. Blood 18 8916942
2022 Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells. Frontiers in immunology 17 36032098
2019 RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia. Journal of Cancer 17 31333801
2005 Identification of a third evolutionarily conserved gene within the RAG locus and its RAG1-dependent and -independent regulation. European journal of immunology 17 15971274
2018 VprBP (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation. Journal of immunology (Baltimore, Md. : 1950) 16 29925675
2017 RAG1/2 induces genomic insertions by mobilizing DNA into RAG1/2-independent breaks. The Journal of experimental medicine 16 28179379
2004 DNA cleavage activity of the V(D)J recombination protein RAG1 is autoregulated. Molecular and cellular biology 16 15254250

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