Initiation of V(D)J recombination in vitro obeying the 12/23 rule

Eastman, Quinn; Leu, Thomas; Schatz, David G.

doi:10.1038/380085a0

Cited by 217 publications

(198 citation statements)

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“…Synapsis plays an important role in regulating the cleavage step in several other site-directed recombination systems (Landy 1989;Chen et al 1992;Kim & Landy 1992;Mizuuchi 1992;Mizuuchi et al 1992;Grindley 1993;Haniford & Kleckner 1994). Both our data and experiments performed in cell-free systems in the presence of Mg 2+ support such a model, since abundant signal ends are not observed unless a 12/23 RSS pair is present in the substrate molecule (Eastman et al 1996;van Gent et al 1996). Several lines of evidence provide additional support for a synapsis model.…”

Section: A Synapsis Model For V(d)j Recombinationsupporting

confidence: 69%

“…In the presence of Mn 2+ , single RSS cleavage events are observed in 12/23 substrates and in substrates containing a single RSS or a 12/12 RSS pair (Eastman et al 1996;van Gent et al 1996). However, when Mg 2+ is used as the divalent metal ion, cleavage occurs in a temporally coordinated fashion at both RSS and is largely dependent upon the presence of a 12/23 RSS pair (Eastman et al 1996;van Gent et al 1996). Together with our data from living cells, these results indicate that the cleavage step of V(D)J recombination is regulated in vivo by the 12/23 rule.…”

Section: The 12/23 Rule Is Enforced At or Prior To The Cleavage Stepmentioning

confidence: 99%

“…Other studies using nuclear extracts from lymphoid cells supplemented with truncated RAG-1 protein found that substrates containing a single RSS or a 12/12 RSS pair are cleaved as efficiently as substrates containing a 12/23 RSS pair in the presence of Mn 2+ . However, subsequent studies have shown that altered reaction conditions (Mg 2+ in place of Mn 2+ ) promote 12/23-regulated cleavage in cell-free systems (Eastman et al 1996;van Gent et al 1996). Neither the indirect data from in vivo experiments nor the results from cell-free systems establish the extent to which cleavage requires a 12/23 RSS pair under physiological conditions.…”

Section: Introductionmentioning

confidence: 98%

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

1996

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Background: V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both.

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Section: A Synapsis Model For V(d)j Recombinationsupporting

confidence: 69%

Section: The 12/23 Rule Is Enforced At or Prior To The Cleavage Stepmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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

1996

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“…Thus, the products of this reaction are a blunt end and a hairpinned end. The recombinases, RAG1 and RAG2, require a divalent cation, Mg 2+ or Mn 2+ , to catalyse these steps van Gent et al 1996b;Eastman et al 1996). As in the HIV-1 integrase reaction, the cleavage step can use nucleophiles other than water, such as ethylene glycol (van Gent et al 1996a).…”

Section: Other Related Reactionsmentioning

confidence: 99%

Polynucleotidyl transfer reactions in site‐specific DNA recombination

1997

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Site-specific DNA rearrangement reactions are widespread among organisms. They are used, for example, by vertebrates to boost immune response diversity, and in turn by parasitic organisms to evade the host immune system by surface antigen switching. Parasitic genetic elements ubiquitous to most organisms invade new host genomic sites by a variety of types of site-specific recombination. Polynucleotidyl transfer reactions are central to these DNA recombination reactions. The recombinase of each reaction system that 'catalyses' such chemical reactions at specific DNA sites are apparently designed to accomplish unique DNA geometrical specificity, or delicate control over the extent or direction of the reaction, with the sacrifice of protein turnover. Here we discuss our current understanding of several issues that relate to the polynucleotidyl transfer steps in several of the better studied site-specific recombination reactions.

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“…1B). Under the relaxed condition using Mn 2ϩ (27,31,47), instead of Mg 2ϩ , the DH3u cleaved RSS DNA as efficiently as the wild-type RAG1 (Fig. S1).…”

Section: Rag1 Mutants For Synapsis-dependent Rss Cleavage-tomentioning

confidence: 96%

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

Nishihara¹,

Nagawa

Imai

et al. 2008

Journal of Biological Chemistry

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In V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, recombinationactivating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule.V(D)J recombination plays key roles in activating and diversifying the antigen receptor genes in mammals (1). In the initial phase of the recombination, the protein products of recombination-activating genes, RAG1 and RAG2 (2, 3), recognize and cleave the recombination signal sequences (RSSs), 3 each consisting of conserved 7-mer (CACAGTG) and 9-mer (ACAAAAACC) motifs, separated by a spacer of either 12 or 23 bp (4 -13). V(D)J recombination takes place only between two RSSs with different spacer lengths, one containing a 12-bp spacer and the other containing a 23-bp spacer (14). This is the 12/23 rule for V(D)J recombination.It has been proposed that V(D)J joining is a reversal of an ancient accidental insertion of a transposable element into a primordial V gene, later exploited by the vertebrate immune system during evolution (6). Consistent with this hypothesis, the postcleavage complex of the RAG proteins is known to possess transposition activity, both in vitro and in vivo (15-21). Furthermore, computational analyses of the fly and mosquito genomes revealed a transposon named Transib, which codes for a RAG1-like transposase (22). In sea urchin Strongylocentrotus purpuratus, a pair of genes (SpRag1L and SpRag2L) resembling RAG1 and RAG2 were found in tandem in the genome (23, 24). Many RAG1-like pseudogenes are also present in the sea urchin genome (22, 24 -26). Although the physiological roles and biochemical properties are yet to be studied for the SpRag1/2L proteins, they may represent the evolutionary intermediates of the vertebrate RAG1 and RAG2.During V(D)J recombination in mice, the RAG proteins cleave RSS DNA in two successive steps, nicking and hairpin formation (27,28). A nick is first introduced at the coding/7-mer border on the top strand, and the resulting 3Ј-hydroxyl group (3Ј-OH) attacks the bottom strand to generate a hairpin structure at the coding end. Although nicking can occur without the partner RSS, the subsequent hairpin formation requires the synaptic formation between 12-and 23-RSSs (29 -40). Previous works suggested that the RAG1-RAG2 complex is formed pre...

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Initiation of V(D)J recombination in vitro obeying the 12/23 rule

Cited by 217 publications

References 18 publications

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

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

Polynucleotidyl transfer reactions in site‐specific DNA recombination

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

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