<b>The 12/23 rule is enforced at the cleavage step of V(D)J recombination <i>in vivo</i></b>

Steen, Sharri Bockheim; Gomelsky, Larissa; Roth, David B.

doi:10.1046/j.1365-2443.1996.d01-259.x

Cited by 78 publications

(38 citation statements)

References 38 publications

(73 reference statements)

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“…When lentiviruses carrying either a 12- or a 23-RSS were co-transduced into REH cells, so stimulation of RAG cleavage requires synapsis in trans , the amount of intact 12- and 23-RSS substrate was reduced to 70% and 74%, respectively, relative to samples where no partner is present (Figure 5A, left). This is consistent with the level of cutting previously published for cutting in trans using vectors with individual RSSs (Steen et al., 1996). When a SJ-carrying lentivirus is transduced with either a 12- or 23-RSS, in each case cutting occurs at the RSS, while negligible cleavage of the SJ is detected (Figure 5A).…”

Section: Resultssupporting

confidence: 92%

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

et al. 2019

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Summary V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo . Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction “cut-and-run” and suggest that it could be a significant cause of lymphocyte genome instability.

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Section: Resultssupporting

confidence: 92%

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

et al. 2019

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“…According to the current model, the RAG complex first binds to one RSS to form a signal complex (SC), which can recruit a partner RSS to form the paired complex (PC), also called the synaptic complex, in which double-strand breaks are generated between the coding gene segments and the RSSs8–10. While nicking can occur in both the SC and PC, hairpin formation is largely restricted to the PC and is sensitive to 12/23 synapsis8,11–14. In the second phase of V(D)J recombination, the ends are processed and joined by proteins of the non-homologous end joining pathway15.…”

Section: Introductionmentioning

confidence: 99%

Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis

Yin

Bailey

Innis

et al. 2009

Nat Struct Mol Biol

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The products of recombination activating genes (RAG) 1 and 2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA-binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer-binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1/2.

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“…Recombination occurs only between gene segments where one segment is flanked by a 12-RSS and the other is flanked by a 23-RSS, a restriction known as the 12/23 rule. While the mechanisms underlying the 12/23 rule are poorly understood, the rule is enforced during the synapsis and hairpin formation steps (5,6,11,12). Gel shift experiments performed by Jones and Gellert (6) showed that the preference for 12/23 synapsis was stronger if the RAG proteins were incubated first with the 12-RSS than if they first saw the 23-RSS.…”

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confidence: 99%

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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The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo

Cited by 78 publications

References 38 publications

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability

Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis

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

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