RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule

Nishihara, Tadashi; Nagawa, Fumikiyo; Imai, Takeshi; Sakano, Hitoshi

doi:10.1074/jbc.m709890200

Cited by 12 publications

(15 citation statements)

References 63 publications

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“…5). RAG binding to the RSS has been shown to perturb DNA structure near the site of cleavage (41), and data from RSS substrates containing mismatches in the heptamer supported the idea that such DNA distortion facilitates nicking (42), as has been shown for hairpin formation (43)(44)(45)(46)(47). Based on this, we propose that the J␤2.5 coding flank inhibits a DNA distortion necessary for nicking at the coding flank-heptamer boundary and that this is overcome upon synapsis with 3=D␤1.…”

Section: Discussionmentioning

confidence: 58%

Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the “Beyond 12/23” Rule

Banerjee

Schatz

2014

Molecular and Cellular Biology

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bAt the Tcrb locus, V␤-to-J␤ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the "beyond 12/23" rule (B12/23 rule). Previous work showed that V␤ recombination signal sequences (RSSs) do not recombine with J␤ RSSs because J␤ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled J␤ RSSs recombine with D␤ RSSs? We report here that the nicking of some J␤ RSSs can be substantially stimulated by synapsis with a 3=D␤1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12-or 23-bp spacers (12-or 23-RSSs, respectively).T he first step in the assembly and diversification of antigen receptor genes is V(D)J recombination, a site-specific recombination reaction that joins variable (V), diversity (D), and joining (J) coding segments at immunoglobulin and T-cell receptor (TCR) loci. During B-and T-cell development, the recombination-activating gene 1 and 2 proteins (RAG1/2) initiate V(D)J recombination (1, 2) at sites specified by recombination signal sequences (RSSs), which flank each V, D, and J gene segment.The biochemistry of V(D)J recombination can be divided into two phases: cleavage and joining (3, 4). The cleavage phase is catalyzed by a recombinase complex comprised of RAG1/2 (collectively, RAG) and the ubiquitous DNA binding/bending protein HMGB1 or the closely related HMGB2 protein (5). The first step in the cleavage phase is the binding of the recombinase complex to one RSS, forming a signal complex (SC). After SC formation, synapsis occurs, in which a partner RSS is captured to form the paired complex (PC) (6, 7). RAG catalyzes the nicking of DNA at the heptamer-RSS boundary, giving rise to a free hydroxyl group. Double-strand breaks are catalyzed in the PC via a transesterification reaction whereby the free hydroxyl created by nicking directly attacks the opposite strand (8). These four ends created by cleavage are resolved in the joining phase by the proteins of the nonhomologous end-joining (NHEJ) repair pathway to form a precise signal joint containing the RSSs and an imprecise coding joint containing the antigen receptor gene segments (reviewed in reference 9).Each RSS is comprised of three sequence elements: a relatively well-conserved heptamer (consensus CACAGTG) and nonamer (consensus ACAAAAACC) and a less well-conserved spacer of either 12 or 23 bp (12-RS...

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Section: Discussionmentioning

confidence: 58%

Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the “Beyond 12/23” Rule

Banerjee

Schatz

2014

Molecular and Cellular Biology

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“…However, the sensitivity of TϪ1 was greatly increased in the presence of Ca 2ϩ which supports stable complex formation, but not catalysis. The sensitivity of TϪ1 indicates a significant distortion of the DNA following the first nick, as has been suggested by others on the basis of similar evidence (2,12,50,59,68). If the distortion at TϪ1 equates to V(D)J base flipping, then its location in the coding flank mirrors the reversed hairpin polarity of the hairpin compared to Tn5.…”

Section: Resultsmentioning

confidence: 66%

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks

Bischerour

Roth

et al. 2009

Molecular and Cellular Biology

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Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position ؊1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.Antibody and T-cell receptor (TCR) diversity is generated by V(D)J recombination initiated by the RAG proteins, RAG1 and RAG2. The recombination signal sequences (RSSs), where recombination takes place, have a distinctive arrangement resembling transposon ends. The relationship between V(D)J recombination and transposition was established beyond doubt by the discovery of RAG-mediated transposition and by the identification of a triad of conserved active-site residues. This evidence placed RAG1 firmly within the family of transposases and retroviral integrases that have a characteristic DDE triad of amino acid residues that coordinate catalytic metal ions in the active site (1,26,30,35,39,46). Later, the Transib family of transposons was identified as the likely ancestral group of RAG1 (33).In V(D)J recombination, the RAG proteins excise the DNA between a pair of RSSs. This fragment is the equivalent of an excised transposon, and it takes no further part in the canonical V(D)J recombination reaction. Instead, the variable regions of the genes encoding antibodies and TCR are created by the imprecise rejoining of the flanking DNA, referred to as the "coding flank." A key feature of the cleaved coding flanks is that they have covalently closed hairpin ends. The asymmetric resolution of these hairpins contributes to the diversification of the coding sequences during rejoining. The hairpins themselves arise as a consequence of the molecular mechanism RAG-mediated RSS cleavage.The crystal structure for the catalytic core of the human immunodeficiency virus type 1 integrase protein revealed a structural fold shared in common with RNase H and the Holliday junction resolving enzyme RuvC (22). RNase H and RuvC monomers each perform a simple nicking reaction that requires a single phosphoryl transfer ...

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“…One predicted bend lies near the nonamer-spacer junction, the same location where 23SC footprinting data suggest HMGB1 makes contact with the phosphate backbone (8,10). A second predicted bend is located near the heptamer-coding flank junction, where it has been proposed that HMGB1/2 interacts with distorted DNA structures thought to be created by RAG before hairpin formation (3,8,46,56). A third bend is predicted to occur near the heptamer-spacer junction, a region in which RAG-DNA backbone interactions and photocrosslinking to HMGB1 have been detected in the 23SC (8,10,11).…”

Section: Discussionmentioning

confidence: 99%

RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

Ciubotaru

Trexler

Spiridon

et al. 2013

Nucleic Acids Research

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During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.

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RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule

Cited by 12 publications

References 63 publications

Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the “Beyond 12/23” Rule

Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the “Beyond 12/23” Rule

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks

RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

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