Ordered assembly of the V(D)J synaptic complex ensures accurate recombination

Abstract

Recombination of gene segments at the immunoglobulin and T-cell receptor loci requires that the RAG1 and RAG2 proteins bring together DNA signal sequences (RSSs) with 12- and 23-bp spacers into a synaptic complex and cleave the DNA. A RAG1/2 multimer that can cleave both signals is shown to assemble on an isolated RSS, and the complementary RSS enters this complex as naked DNA. When RAG1/2 is allowed to bind 12 and 23 RSSs separately prior to their mixing, synaptic complex assembly and cleavage activity are greatly reduced, indicating that only a complex initially assembled on a single RSS leads to productive cleavage. RAG1/2 complexes assembled on 12 RSSs will only incorporate 23 partners, while complexes assembled on 23 RSSs show a 5- to 6-fold preference for 12 partners. Thus, initial assembly on a 12 RSS most accurately reflects the strict 12/23 coupled cleavage observed in the cell. Additional cellular factors such as chromatin may ensure that RAG1/2 first assembles on a 12 RSS, and then a free 23 RSS enters to activate cleavage.

Fig. 1. Steps in RSS cleavage by RAG1/2. In Figures 1–6, 12 RSSs are depicted as open triangles and 23 RSSs are depicted as filled triangles, with both triangles pointing away from the coding flank. (A) RAG1/2 binds to the intact RSS and introduces a nick between the signal and coding DNA. The 3′ hydroxyl (OH) in the nicked species attacks the opposite strand in a direct transesterification reaction to cleave the DNA, leaving a 3′ OH on the blunt-ended RSS. RAG1/2 can also carry out transesterification on pre-nicked substrates. (B) RAG1/2, with the help of HMG1 or HMG2, assembles a synaptic complex including a pair of RSSs (canonically, one 12 RSS and one 23 RSS). The synaptic complex is competent to carry out coupled cleavage of both partners in Mg²⁺. This cartoon is not meant to reflect the stoichiometry of RAG1 and RAG2 protomers present in the synaptic complex.

Fig. 2. Cleavage and RAG1/2 binding under two different assembly conditions. (A) Cleavage reactions (10 µl) were assembled in Ca²⁺ in one of two ways. (1) RAG1/2 at the concentration indicated in (B), HMG1 and pre-nicked 12 RSS substrate (the position of the label is indicated by an asterisk; the OH has been omitted) were first combined (5 min, 37°C), followed by the addition of pre-nicked 23 RSS partner (5 min, 37°C), and finally Mg²⁺ (15 min, 37°C). (2) Pre-nicked 12 RSS substrate and 23 RSS partner were each individually incubated with HMG1 and RAG1/2 at the concentration indicated in (B) (5 min, 37°C), these mixes were then combined (5 min, 37°C), and finally Mg²⁺ was added (15 min, 37°C). (B) Reaction products were separated on denaturing polyacrylamide gels as described in Materials and methods. Positions of pre-nicked substrate and hairpin (HP) cleavage products are shown to the right of the gel. Substrate (%) converted to HP product is indicated. (C) Binding reactions using intact RSS substrates were assembled as described in (A), omitting the addition of MgCl₂ and the final 15 min incubation. Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described in Materials and methods. The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated.

Fig. 3. Cleavage under various assembly conditions in the presence of specific competitor. (A) Assembly of cleavage reactions in Ca²⁺ was staged as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to pre-nicked 12 RSS substrate (the position of the label is indicated by an asterisk). Competitor was intact 12 RSS, and intact 23 RSS acted as partner. Positions of pre-nicked substrate and hairpin (HP) cleavage products are shown to the right of the gel. Substrate (%) converted to product is indicated. (B) Reactions were staged as per (A), but pre-nicked 23 RSS was used as substrate, intact 23 RSS was used as competitor, and intact 12 RSS acted as partner.

Fig. 4. Cleavage of intact substrate in the presence of specific competitor without pre-incubation in Ca²⁺. Cleavage reactions were assembled as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk). Reactions did not include Ca²⁺. Some reactions included specific competitor (intact 12 RSS, lanes 4–6; intact 23 RSS, lanes 10–12) and partner (intact 23 RSS, lanes 3–5; intact 12 RSS, lanes 9–11). Positions of substrates as well as nicked and hairpin (HP) products are shown on either side of the gel. Substrate (%) converted to HP product is indicated.

Fig. 5. Gel mobility-shift assay for detection of synaptic complex assembly. (A) Binding reactions were assembled in Ca²⁺ and analyzed as described in Materials and methods. Intact 12 RSS (squares) or 23 RSS (circles) substrate (2 nM) was combined with RAG1/2 at the concentrations indicated and HMG1 (5 min, 25°C). Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described. Substrate (%) bound in the single end complex was determined. (B) Intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk) was incubated with RAG1/2 (110 nM) and HMG1 (5 min, 37°C), followed by the addition of 23 or 12 RSS partner, respectively, at the concentrations indicated (5 min, 37°C). Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described. The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated.

Fig. 6. Gel mobility-shift assay for detection of synaptic complex assembly in the presence of specific competitor. (A) Binding reactions were assembled as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk). Some reactions included specific competitor (intact 12 RSS, lanes 4–6; intact 23 RSS, lanes 10–12) and partner (intact 23 RSS, lanes 3–5; intact 12 RSS, lanes 9–11). The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated. (B) Binding reactions were assembled as in (A) with labeled 23 RSS substrate present in the first stage (5 min, 37°C) and 12 RSS (squares) or 23 RSS (circles) partner, at the concentrations indicated added in the second (5 min, 37°C). Complexes were separated and quantified as described. Substrate (%) bound in the synaptic complex is indicated.

Fig. 7. Model for assembly of the synaptic complex. RAG1/2 in solution may include all protein components necessary for binding to two RSSs (1a and 3a), or a bivalent complex could assemble after binding of a monovalent complex to a single RSS (not shown). Heptamer and nonamer binding domains may be contributed by different protomers within the complex (Swanson, 2001). In the bivalent complex, the heptamer and nonamer binding domains within one RSS binding site are optimally arranged to bind a 12 RSS (1a, white binding site), while these domains are farther apart in the second RSS binding site, which can only bind to a 23 RSS (3a, white binding site). These conformations may interchange rapidly in solution (2). Initial binding to a 12 RSS locks that binding site because of the fixed length of the 12 bp spacer (1b); the second site must then be occupied by a 23 RSS (1c). Initial binding to a 23 RSS (3b) does not lock the complex because of the relative flexibility of the 23 bp spacer (3c). The second RSS to enter the complex can be either a 12 (3d) or a 23 (3e). HMG proteins are not shown in this model; they would presumably bind within the 23 bp spacer and increase its bend. Heptamer (7), nonamer (9) and spacer regions are indicated; RSSs are depicted as entering the complex in opposite orientations (DNA arrows).