Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination

Nagawa, Fumikiyo; Kodama, Motomune; Nishihara, Tadashi; Ishiguro, Kei‐ichiro; Sakano, Hitoshi

doi:10.1128/mcb.22.20.7217-7225.2002

Cited by 21 publications

(33 citation statements)

References 44 publications

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“…Ordered assembly usually begins with RAG1/2 binding to the 12RSS, followed by capture of free 23RSS (3,4). Changes in the sensitivity of a 12RSS to chemical modification occur upon RAG1/2 binding, consistent with some unwinding at the heptamer-coding border (5,6). Double-strand breaks at the coding-RSS border are produced by a nick 5Ј of the heptamer; nucleophilic attack on the opposing strand by the 3Ј hydroxyl group at the nick then creates a hairpin by a transesterification reaction (7).…”

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confidence: 99%

Requirements for DNA hairpin formation by RAG1/2

Grundy

Hesse²,

Gellert³

2007

Proc. Natl. Acad. Sci. U.S.A.

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The rearrangement of antigen receptor genes is initiated by doublestrand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some ''cut-and-paste'' transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.immunoglobulin gene ͉ recombination ͉ transposase I n many vertebrates, the vast antigen-binding repertoire of immunoglobulins and T cell receptors is created by combinatorial V(D)J recombination using an array of variable (V), diversity (D), and joining (J) gene segments in B and T cell genomic DNA (1). A complex of the RAG1 and RAG2 gene products is responsible for initiating DNA cleavage and probably for mediating the recruitment of nonhomologous end-joining factors to process and join the broken V, (D), and J segments.The sites of DNA cleavage are directed by recombination signal sequences (RSSs) that flank each coding segment. The two types of RSSs (denoted 12RSS and 23RSS) consist of conserved heptamer and nonamer motifs separated by a spacer of 12 or 23 nonconserved base pairs. Normally, DNA cleavage occurs when the RAG complex recognizes and pairs a 12RSS and a 23RSS, thus ensuring the correct recombination of a V to a J or of a V to a D and of a D to a J segment (termed the ''12/23 rule'') (2). Ordered assembly usually begins with RAG1/2 binding to the 12RSS, followed by capture of free 23RSS (3, 4). Changes in the sensitivity of a 12RSS to chemical modification occur upon RAG1/2 binding, consistent with some unwinding at the heptamer-coding border (5, 6). Double-strand breaks at the coding-RSS border are produced by a nick 5Ј of the heptamer; nucleophilic attack on the opposing strand by the 3Ј hydroxyl group at the nick then creates a hairpin by a transesterification reaction (7). The resulting double-strand break consists of a hairpin on the coding flank and a blunt-ended signal sequence. In the presence of Mg 2ϩ , the complete reaction takes place only in the presence of an RSS pair (coupled cleavage) (8, 9). The mechanism of DNA cleavage has been characterized in vitro, mainly with truncated RAG proteins that retain activity but are mor...

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confidence: 99%

Requirements for DNA hairpin formation by RAG1/2

Grundy

Hesse²,

Gellert³

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…DNA Substrates-Oligonucleotides were synthesized and purified as described (43)(44)(45). Sequences used were as follows * This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Yamada Science Foundation.…”

Section: Methodsmentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. The 32 P-labeled, biotinylated, and 3Ј-dideoxyoligonucleotides were prepared as described (43)(44)(45). The nicked RSS was prepared by annealing three oligonucleotides: an RSS bottom strand, a 5Ј-phosphorylated top strand of the signal end DNA, and a coding top strand (with a 3Ј-dideoxy or 3Ј-OH end).…”

Section: Methodsmentioning

confidence: 99%

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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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“…The resulting 3Ј-hydroxyl group (3Ј-OH) then attacks the bottom strand to form a hairpin structure at the coding end (CE) and a blunt end at the signal end (SE). After the cleavage of RSSs, the SEs stably stay with the RAG proteins in vitro (2,18,21,36). Physical association of the CEs with the SE complex has been shown to occur in vitro (18,56).…”

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In Vitro Processing of the 3′-Overhanging DNA in the Postcleavage Complex Involved in V(D)J Joining

Nishihara

Nagawa

Nishizumi

et al. 2004

Molecular and Cellular Biology

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The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3-processing activity is observed not only with the core RAG2 but also with the full-length protein.V(D)J joining plays key roles in activating and diversifying the antigen receptor genes. In the initial phase of V(D)J joining, the protein products of recombination-activating genes (RAG1 and RAG2) (39, 48) recognize the recombination signal sequences (RSS), each consisting of a conserved 7-mer (CACAGTG) and a conserved 9-mer (ACAAAAACC), separated by a spacer of constant length of either 12 or 23 bp (6,17,35,44,45,46,52,53). For the coordinate cleavage of RSSs, synaptic complex formation of the 12-and 23-RSSs is required (8,18,59). RSS DNA is cleaved by RAG proteins in two successive steps, nicking and hairpin formation (31, 58). A nick is first introduced at the coding and 7-mer border on the top strand. The resulting 3Ј-hydroxyl group (3Ј-OH) then attacks the bottom strand to form a hairpin structure at the coding end (CE) and a blunt end at the signal end (SE). After the cleavage of RSSs, the SEs stably stay with the RAG proteins in vitro (2,18,21,36). Physical association of the CEs with the SE complex has been shown to occur in vitro (18,56). This association in the presence of other repair proteins appears to be necessary for the CE processing in vivo (18,20,41,56,60).In the joining phase of V(D)J recombination, CEs are processed and ligated to form a coding joint. Several factors are required in DNA end joining, including the Ku heterodimer (Ku70/80), the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), Artemis, XRCC4, and DNA ligase IV (4,11,14,15,27,28,34,38,42,43,54,61). It has been shown that the Artemis/DNA-PKcs complex opens the hairpin a few nucleotides from the tip, generating the 3Ј-overhanging structure (30, 50). After the hairpin opening, the CEs are modified by nucleotide deletions and additions for junctional diversification (50). For nucleotide additions, terminal deoxynucleotidyltransferase (TdT) has been reported to be responsible for the non-germ line encoded nucleotides (N) (13, 24). In contrast, the exact mechanism for the nucleotide deletions remains largely unknown. The RAG proteins are known to mediate the strand transfer of the SE to the hairpin CE or to the doublestranded (ds) DNA, resulting in the aberrant jo...

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Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination

Cited by 21 publications

References 44 publications

Requirements for DNA hairpin formation by RAG1/2

Requirements for DNA hairpin formation by RAG1/2

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

In Vitro Processing of the 3′-Overhanging DNA in the Postcleavage Complex Involved in V(D)J Joining

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