A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Initiation of V(D)J recombination involves the synapsis andcleavage of a 12/23 pair of recombination signal sequences byRAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factorsof the HMG box family, such as HMG-1, are known to assist inthese processes. After cleavage, the RAG proteins remain boundto the cut signal ends and, at least in vitro, support the integrationof these ends into unrelated target DNA via a transposition-likemechanism. To investigate whether the protein complex supportingsynapsis, cleavage, and transposition of V(D)J recombinationsignals utilized the same complement of RAG and HMG proteins,I compared the RAG protein stoichiometries and activities ofdiscrete protein-DNA complexes assembled on intact, prenicked,or precleaved recombination signal sequence (RSS) substratesin the absence and presence of HMG-1. In the absence of HMG-1,I found that two discrete RAG-1/RAG-2 complexes are detectedby mobility shift assay on all RSS substrates tested. Both containdimeric RAG-1 and either one or two RAG-2 subunits. The additionof HMG-1 supershifts both complexes without altering the RAGprotein stoichiometry. I find that 12/23-regulated recombinationsignal synapsis and cleavage are only supported in a protein-DNAcomplex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly,the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1is dispensable for its activity.

INTRODUCTION

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Introduction

The antigen binding regions of immunogobulin and T-cell receptorsare encoded in arrays of noncontiguous coding segments, calledvariable (V), diversity (D), and joining (J), that are assembledduring lymphocyte development to generate the variable-regionexon of the mature antigen receptor gene (5). The assembly process,termed V(D)J recombination, involves a series of site-specificDNA rearrangements mediated by recombination signal sequences(RSSs) abutting the coding segments. Each RSS contains conservedheptamer and nonamer elements, separated by nominally conservedspacer DNA that is either 12 or 23 bp long (12-RSS and 23-RSS,respectively). The RSS facilitates the generation of functionalantigen receptors via the "12/23 rule," a restriction that limitsrecombination to a pair of coding segments whose flanking RSSsbear spacer DNAs of different lengths.

The products of recombination-activating genes 1 and 2, RAG-1and RAG-2 (35, 42), initiate V(D)J recombination by introducinga DNA double-strand break (DSB) at a 12/23 pair of RSSs (atthe heptamer-coding segment border). The cleavage reaction generatestwo distinct DNA intermediates: blunt, 5'-phosphorylated signalends (SE) and coding ends terminating in covalently sealed DNAhairpin structures (38, 39, 43). DSB intermediates are generatedin two biochemical steps: first-strand nicking at the 5' endof the heptamer, followed by a direct transesterification reactionin which the 3'-OH exposed in the nicking step attacks the phosphodiesteron the antiparallel DNA strand (26, 52). The RAG proteins alsomediate other reactions in vitro, including a disintegrationreaction similar to those catalyzed by retroviral integrases(28), the nicking of DNA hairpins (6, 45), the cleavage of 3'flap structures (41), and the insertion of SE into double-strandedDNA via transposase-like strand transfer reactions (1, 19).

The RAG-RSS complex relevant to physiological V(D)J recombinationis expected to contain two RSSs (one 12-RSS and one 23-RSS),which are brought together by the RAG proteins into a 12/23paired signal complex (PC). In the context of the PC, also calleda synaptic complex, DSBs are introduced at both RSSs in a coordinatedor coupled cleavage reaction. Synaptic complex formation andcoupled cleavage adhering to the 12/23 rule have been recapitulatedin vitro (13, 40). Both are facilitated by certain nonspecificDNA-bending proteins of the HMG box family (e.g., HMG-1 andHMG-2), and both exhibit a divalent metal ion dependence. Ca²⁺is sufficient to promote PC formation but not DNA cleavage.Coupled cleavage of 12/23 RSS pairs requires Mg²⁺; Mn²⁺ decouplesthe reaction, allowing cleavage to occur at an isolated RSS.

Both cleavage steps are minimally supported in vitro in a protein-DNAcomplex, termed a stable or single complex (SC), containinga RAG-1 dimer and one or two subunits of RAG-2 bound to an isolatedRSS (4, 46, 48). HMG-1 and HMG-2 can be stably incorporatedinto the SC (30, 47, 51). The addition of HMG-1/HMG-2 does notsignificantly affect the cleavage of isolated 12-RSS substratesby the RAG proteins but stimulates both nicking and hairpinformation (the latter preferentially) on 23-RSS substrates (51),even in the absence of synapsis (47).

In contrast to the SC, whose RAG protein stoichiometry has beencharacterized, the number of RAG-1 and RAG-2 protomers presentin the PC remains unclear. In one model of the PC, RAG-1 andRAG-2 are bound as a mixed tetramer containing two moleculesof each protein, based on the following lines of evidence: (i)RAG-1 alone forms a stable dimer in solution (4) and retainsthis configuration when bound to DNA (37), regardless of whetherRAG-2 is present (48); (ii) RAG-1 and RAG-2 associate as a tetramerin solution (in a 1:1 ratio) (4); and (iii) two active sitesare present in each dimer of RAG-1, one contributed from eachRAG-1 subunit (22, 46). However, recent studies provide indirectevidence suggesting that the PC contains a larger number ofRAG-1 protomers, possibly a tetramer (22, 31). Discriminatingbetween these models necessarily requires a direct approachthat compares the RAG stoichiometries within the SC and PC sideby side. A related issue, as yet unresolved, is whether signalend complexes (SEC) that support transposition require the samecomplement of RAG and HMG proteins as the PC for their assemblyand activity.

Evidence has implicated both synaptic-complex assembly and thehairpin step of V(D)J cleavage as critical control points inthe enforcement of the 12/23 rule (17, 53). However, the relativecontribution of each factor, as well as the role of the HMGprotein in these processes, remains uncertain, as no study hasdirectly compared the catalytic activities of preformed pairedRSS complexes.

To address these issues, I compared the stoichiometries andcatalytic activities of the SC, PC, and SEC directly by usinga combination of mobility shift and in-gel enzyme assays. Ifind that two distinct SC complexes are assembled on intact,prenicked, and precleaved RSS substrates (SC1 and SC2). BothSC complexes contain a stable RAG-1 dimer. One species containsa single RAG-2 monomer (SC1), whereas a slower-migrating species(SC2) contains two RAG-2 subunits. Both complexes are supershiftedby HMG-1 (forming HSC1 and HSC2). The PC (and SEC) assembledwith an RSS pair contains the same complement of RAG proteinsas HSC2. The 12/23 rule is found to be imposed both during PC(and SEC) assembly and during catalysis of strand cleavage (orstrand transfer). Interestingly, both the SC2 and SEC speciesassembled from precleaved substrates support transposition.Thus, HMG-1 is required for 12/23-regulated PC assembly andRSS substrate cleavage but is dispensable for RAG-mediated transposition.

MATERIALS AND METHODS

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Materials and Methods

DNA constructs. Eukaryotic expression constructs encoding core RAG-1 and RAG-2fused at the amino terminus to one or two copies of maltosebinding protein (MBP) have either been previously describedor were assembled from previously published constructs by conventionaltechniques (M2R2) (47, 48). The prokaryotic expression constructpET11d-hHMG-1 has been described elsewhere (15). Fusion proteinsused in this study are depicted in Fig. 1.

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FIG. 1. RAG proteins and RSS substrates used in this study. (A) Schematic diagrams of RAG-1, RAG-2, and HMG-1 fusion proteins (encoded residues are in parentheses) designated at the left. MBP, myc (M), and polyhistidine (H) sequences are also indicated. (B) Single and double MBP-RAG fusion proteins were coexpressed in the indicated combinations and purified by amylose affinity chromatography. Wild-type and active-site mutant (D600A) forms of RAG-1 (WT-MR1 and MT-MR1, respectively) were coexpressed with RAG-2 and similarly prepared. Protein samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by silver staining. The positions of the RAG-1 and RAG-2 fusion proteins are shown at the left. (C) Schematic diagrams of radiolabeled intact (12-RSS, 23-RSS), prenicked (12N-RSS, 23N-RSS), and precleaved (12-SE, 23-SE) RSS substrates, along with the position of the ³²P end label (_*P). Heptamer and nonamer sequences are shaded. The lengths of the flanking and spacer regions are also shown.

Protein expression and purification. Single and double MBP-RAG expression constructs (wild type ormutant) were cotransfected with pRSVT in 293 cells by calciumphosphate precipitation (49). Typically, 14 10-cm-diameter tissueculture dishes were transfected with 20 µg of expressionconstruct(s) (20 µg for individually expressed proteinsor 10 µg of both RAG-1 and RAG-2 constructs for coexpressedRAGs)/plate and 1 µg of pRSVT/plate and harvested at 48h posttransfection. Fusion proteins were purified by amyloseaffinity chromatography as previously described (48, 49). Polyhistidine-taggedhuman HMG-1 was expressed in Escherichia coli and purified bya previously published procedure (47).

Oligonucleotide binding assays. Intact and prenicked 12- and 23-RSS substrates were assembledand purified as described previously (46). Precleaved substratescontaining only an RSS SE were similarly prepared by annealingcold 5'-phosphorylated DG10 (12-SE) or DG4 (23-SE) to theirrespective complements labeled at the 5' end with ³²P (26).Diagrams of the RSS substrates used in this study are shownin Fig. 1. Electrophoretic mobility shift assays (EMSA) wereperformed under the same conditions described previously (49),except that DAR 81/82 was omitted as a competitor. Briefly,binding reaction mixtures (10 µl) containing individuallyexpressed ( $~$ 30 ng) or coexpressed RAG proteins ( $~$ 100 ng) and HMG-1(5 µg/ml; when indicated in Fig. 2, 4, and 5) were preincubatedwith radiolabeled substrate DNA ( $~$ 0.02 pmol) in the presenceof 5 mM Ca²⁺ for 1 min at 25°C. Subsequently, either 25-fold-(SE substrates) or 50-fold (intact and prenicked)-excess coldpartner DNA (either intact, prenicked, or precleaved, as appropriate)was added, as determined by titration experiments (data notshown). Samples were incubated for an additional 10 min at 25°Cand then fractionated at 4°C on native 4% gels. Protein-DNAcomplexes were visualized from dried gels by autoradiographywith a Molecular Dynamics Storm 860 PhosphorImager.

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FIG. 2. RAG synaptic complexes contain a RAG-1/RAG-2 tetramer core. EMSAs were performed by using ³²P-end-labeled intact (A), prenicked (B), or precleaved (C) substrates containing either a canonical 12-RSS (left) or 23-RSS (right). Individually expressed (i) or coexpressed (c) single and/or double MBP-RAG proteins, defined in Fig. 1A, were incubated with radiolabeled substrate (Fig. 1C) in the absence or presence of HMG-1 and/or cold partner DNA as indicated above the gel. In some cases (e.g., lanes 12, 16, 29, and 33), samples contained an equal mixture (m) of the two coexpressed RAG protein preparations identified in the previous two lanes (e.g., lane 12 contains a mixture of coexpressed RAG proteins found in lanes 10 and 11). Protein-DNA complexes were fractionated by EMSA and visualized by autoradiography with a PhosphorImager. The positions of complexes containing dimeric RAG-1 alone (R1), dimeric RAG-1 and monomeric RAG-2 (SC1), and a RAG-1/RAG-2 tetramer (SC2) are shown at left. Complexes supershifted by the incorporation of HMG-1 are also indicated (HSC1 and HSC2). The positions of paired RSS complexes containing HMG-1 and a RAG-1/RAG-2 tetramer bound to intact (PC), prenicked (PC), or precleaved (SEC) substrates are noted at the right. Whether four, five, or six MBP moieties are distributed among the RAG subunits in these complexes is also indicated (M4, M5, and M6, respectively).

In-gel cleavage and transposition assays. Preformed RAG complexes assembled on intact and prenicked substrateswere assayed for cleavage activity by an in-gel cleavage assaydescribed previously (46). Briefly, preparative-scale bindingreaction mixtures (50 µl) were assembled in the presenceof Ca²⁺ and fractionated by EMSA as described above. After disassembly,the gel was submerged in buffer containing 5 mM MgCl₂ and incubatedat 37°C for 1 h to initiate DNA cleavage within fractionatedprotein-DNA complexes. Following electrophoretic transfer ofthe DNA to DEAE-cellulose, reaction products attributed to complexesof interest (as visualized by autoradiography) were recovered,normalized, and fractionated by denaturing gel electrophoresis.A similar approach was used to determine transposition activityof RAG complexes assembled on SE substrates, except that sampleswere fractionated on native gels containing the target plasmidpcDNA1 (4.0 kb; Invitrogen), which was added to the acrylamidemixture before pouring the gel (2.5 µg/ml). After EMSA,the gel was incubated in transposition buffer (25 mM MOPS [morpholinepropanesulfonicacid]-KOH [pH 7.0], 60 mM potassium glutamate, 10 mM CaCl₂,10% glycerol) at 37°C for 1 h. Reaction products were recoveredas described above and fractionated on a linear 4 to 20% gradientnative polyacrylamide gel. PhosphorImager scans of dried gelswere quantified with ImageQuant software.

RESULTS

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Results

Paired RSS complexes contain a RAG-1/RAG-2 tetramer core. As part of an ongoing effort to understand the molecular processesunderlying V(D)J recombination, I have worked to characterizediscrete protein-DNA complexes assembled on single RSS substratescontaining various combinations of individually purified RAG-1,RAG-2, and HMG-1 (or HMG-2) (46-49). To extend these results,I wished to biochemically characterize RAG synaptic complexescontaining a pair of RSS substrates in order to clarify theirrelationship to lower-order RAG-RSS complexes described previously(either lacking or containing HMG-1). For this study, formsof core RAG-1 and RAG-2 proteins, fused at the amino terminusto either one or two copies of MBP, were expressed individuallyor coexpressed in 293 cells and purified by amylose affinitychromatography (Fig. 1). A hexahistidine-tagged form of humanHMG-1 was overexpressed in bacteria and purified from clarifiedlysates by Ni²⁺-chelation chromatography.

I first compared the requirements for assembly of single andpaired RSS complexes by an EMSA, focusing particularly on therole of HMG-1 in protein-DNA complex formation. The DNA substratesused in these experiments contained a canonical 12- or 23-RSSthat was either intact, prenicked, or precleaved (containingonly an SE) (Fig. 1C and 2). Under these conditions, HMG-1 bindsall three substrates tested in the absence of the RAG proteins(data not shown). RAG-2 shows no binding activity (Fig. 2, lanes1 and 18), but RAG-1 alone binds all three substrates to lowlevels (R1; Fig. 2, lanes 2 and 19). When individually expressedRAG-1 and RAG-2 are mixed and analyzed, a protein-DNA complexof retarded mobility, termed SC1, is clearly observed in reactionscontaining intact or prenicked DNA substrates (Fig. 2A and B,lanes 3 and 20) but is only faintly visible in reactions containingprecleaved substrates (Fig. 2C, lanes 3 and 20). A minor, slower-migratingspecies is also detected (at higher exposure) in samples containingintact or prenicked substrates (SC2). It is important to noteat this point that SC1 and SC2 are designated based on theirrelative electrophoretic mobilities and their appearance inreactions containing only a single type of RSS substrate, ratherthan their intrinsic activities, which as shown below may bealtered by the addition of cold partner DNA.

When both RAG-1 and RAG-2 are coexpressed, the abundances ofSC1 and SC2 in all samples are increased (Fig. 2, lanes 4 and21). Thus, coexpressed RAG proteins are used in the remainingsamples. For intact substrates, the abundances of both the SC1and SC2 complexes are reduced upon addition of cold intact partnerDNA; the levels of reduction are comparable irrespective ofwhether the partner DNA contains a 12- or 23-RSS (Fig. 2A, lanes5 and 6 and 22 and 23). In contrast, when cold prenicked orprecleaved partner DNA is added to their respective samples,the formation of the SC1 complex is diminished, while SC2 complexformation is stimulated in a 12/23-dependent fashion, suggestingthe presence of two RSS substrates in this complex (Fig. 2B and C;compare lanes 5 and 6 and 22 and 23). In all three cases,however, the mobility of the complex is not significantly alteredby addition of cold partner DNA.

When binding reactions containing coexpressed RAG proteins aresupplemented with HMG-1 in the absence of partner DNA, boththe SC1 and SC2 complexes are supershifted (HSC1 and HSC2; Fig.2, lanes 7 and 24). For intact substrates, the addition of HMG-1and cold partner DNA stimulates the 12/23-regulated formationof a complex that comigrates with HSC2. The preference for theappropriate RSS partner over the inappropriate partner is aboutthree- to fourfold. This species is considered to contain anRSS pair and is therefore designated PC (or SEC) at this pointfor consistency with previously published reports describingconditions for the detection of paired RSS complexes by EMSA(17). In contrast, the level of HSC1 complex formation is reduced(Fig. 2A, compare lanes 8 and 9 and 25 and 26). For prenickedand precleaved substrates, the addition of cold partner DNAhas the same effect as in the absence of HMG-1 but the abundanceof the PC (and SEC) is slightly greater than that of counterpartslacking HMG-1 (Fig. 2B and C, lanes 9 and 10 and 25 and 26).

I next wished to examine the stoichiometry of the RAG proteinsin the context of the PC (or SEC) for all of the DNA substrateslisted above. To determine RAG protein stoichiometry, I coexpressedforms of RAG-1 and RAG-2 fused to one or two copies of MBP (thusaltering their molecular weights) and analyzed protein-DNA complexformation by EMSA (48). The detection of one complex (or more)whose electrophoretic mobility is intermediate to those observedin binding reactions containing only one form of the RAG proteinindicates heterodimer (or multimer) formation. As expected,the PC (or SEC) obtained from samples containing either MR1or M2R1m coexpressed with MR2 displayed distinct mobilities,reflecting the difference in their relative molecular weights(PC [or SEC-]-M4 and PC [or SEC-]-M6, respectively; Fig. 2,compare lanes 10 and 11 and 27 and 28). When these two RAG proteinpreparations were mixed in the binding reaction, no speciesof intermediate mobility were detected by EMSA (Fig. 2, lanes12 and 29). However, samples containing both forms of RAG-1coexpressed with RAG-2 showed the presence of a single predominantspecies of intermediate mobility (PC [or SEC-]-M5; Fig. 2, lanes13 and 30), consistent with a RAG-1 dimer configuration in thePC. The PCs assembled from reactions containing MR1 coexpressedwith either MR2 or M2R2 also displayed distinct electrophoreticmobilities (Fig. 2, compare lanes 14 and 15 and 31 and 32).Interestingly, when both RAG protein preparations were mixed,an intermediate species was clearly evident (Fig. 2, lanes 16and 33). Similar results were also obtained when MR1 was coexpressedwith both forms of RAG-2 (Fig. 2, lanes 17 and 34). These datasuggest that two RAG-1 and two RAG-2 subunits are present inthe PC (and SEC) and that, unlike RAG-1, the RAG-2 subunitsfreely reassort in solution. Consistent with the conclusionthat the PC (and SEC) contains a RAG-1/RAG-2 tetramer core,the PCs (and SECs) assembled from M2R1m or M2R2 comigrate (Fig.2, compare lanes 11 and 15 and 28 and 32), as predicted if sixMBP moieties are distributed among four RAG protomers. In contrast,the minor HSC1 species detected in the same two samples do notcomigrate, an outcome expected in principle if HSC1 containsa RAG-1 dimer and monomeric RAG-2. Indeed, this stoichiometrywas verified for HSC1 by using combinations of single and doubleMBP-RAG proteins in the absence of partner DNA (data not shown).By the same approach, SC2 was also found to possess the sameRAG stoichiometry as the PC (and SEC) (Fig. 3). Thus, both SC1and HSC1 contain a stable RAG-1 dimer and monomeric RAG-2, andSC2, HSC2, and PC (and SEC) contain a stable RAG-1 dimer andtwo subunits of RAG-2 capable of reassorting in solution.

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FIG. 3. The RAG protein stoichiometries of the SC2 and PC (or SEC) species are identical and differ from that of the SC1 species by the addition of a single RAG-2 subunit. Combinations of single and/or double MBP-RAG proteins were incubated with ³²P-end-labeled prenicked 12-RSS substrate in the absence of HMG-1 with or without cold prenicked partner 23-RSS as shown above the gel (see the legend to Fig. 2 for more detail, including definitions of "i," "c," and"m"). Comparable results were obtained with intact and precleaved substrates as well.

The PC supports 12/23-dependent cleavage of intact and prenicked substrates. I next evaluated the catalytic activity of the complexes observedby EMSA. To assess RSS cleavage activity, I used an in-gel cleavageassay to compare catalytic activities of multiple, discreteprotein-DNA complexes in a single native polyacrylamide gel(46). In the first experiment, wild-type or active-site mutant(D600A) RAG-1 (14, 21, 23) coexpressed with RAG-2 was incubatedunder binding conditions (in the presence of Ca²⁺) with intact12- or 23-RSS substrates in the absence or presence of HMG-1and/or cold partner DNA in combinations comparable to thoseshown in Fig. 2. Samples were fractionated by EMSA, and thegel was soaked in buffer containing Mg²⁺ for 1 h at 37°Cto initiate DNA cleavage within the protein-DNA complexes. Autoradiographsof the DNA following electrophoretic transfer to DEAE-celluloseshow that wild-type and mutant RAG-1s assemble protein-DNA complexesto comparable levels (Fig. 4A). DNA derived from the SC1, SC2,HSC1, HSC2, and PC species was recovered, normalized, and fractionatedby denaturing gel electrophoresis (Fig. 4B). To account forslight variations in the actual amount of DNA loaded on thegel (despite attempted normalization), the cleavage productswere quantified and expressed as percentages of total DNA presenton the gel.

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FIG. 4. 12/23-regulated RAG-mediated hairpin formation requires HMG-1. (A) Wild-type or active-site mutant (D600A) MR1 coexpressed with MR2 (WT-MR1/MR2 or MT-MR1/MR2, respectively) was incubated with intact 12-RSS (left) or 23-RSS (right) substrates with or without HMG-1 and/or cold partner DNA (12-RSS or 23-RSS) under binding conditions, as indicated above the gel, and subjected to an in-gel cleavage assay (see Materials and Methods). The autoradiographs of electrophoretically transferred DNA shown here were used to isolate reaction products from complexes of interest. The positions of SC1, HSC1, SC2, and HSC2/PC (comigrating) species, defined for Fig. 2, are shown at the left. (B) Recovered reaction products from panel A were normalized and fractionated by denaturing gel electrophoresis. Positions of nicked and hairpin products are shown at the left and right. The hairpin species shown on gels of 12- and 23-RSS substrates have previously been shown to comigrate. The percentages of nicked (%N) and hairpin (%HP) products in each lane are quantified below the gel and account for slight variations in the amount of DNA actually loaded. (C) In-gel cleavage experiments using prenicked 12-RSS (left) or 23-RSS (right) substrates were performed as described above and presented in the same order. Autoradiographs of DNA transferred from the gel resemble those shown in panel A and reveal a distribution of protein-DNA complexes similar to that observed in comparable lanes depicted in Fig. 2B (data not shown). Quantitative analysis of hairpin formation is shown below the gel. Note that all reaction products shown on both sides of panels B and C are derived from a single native gel subjected to the in-gel cleavage reaction. The abundance and distribution of the cleavage products observed are representative of independent experiments.

Consistent with previous results (46), significant nicking wasdetected in 12-RSS substrates recovered from wild-type SC1 formedin the absence of cold partner DNA, as well as low levels ofthe hairpin product (Fig. 4B, lane 1). The abundance of nicks,but not hairpin products, is slightly enhanced in the SC2 speciesobtained from the same lane (Fig. 4B, lane 2). The additionof intact 12- or 23-RSS partner DNA, while reducing complexformation, appears to modestly stimulate nicking independentof RSS composition (Fig. 4B, lanes 3 to 6). Supplementing HMG-1in the absence of cold partner DNA supershifts the SC1 and SC2species (HSC1 and HSC2, respectively) but does not appreciablyalter the cleavage activity of either complex (Fig. 4B, lanes7 and 8). Addition of cold 12-RSS partner DNA, while impairingHSC1 and PC formation, does not dramatically change the cleavageactivity of either complex (Fig. 4B, lanes 9 and 10). However,while the addition of cold 23-RSS partner DNA has relativelylittle effect on HSC1 activity, PC cleavage activity is stimulatedapproximately fourfold relative to that observed in the PC formedin the absence of cold 12-RSS partner DNA (Fig. 4B, lanes 11and 12; compare lane 10 to lane 12). Importantly, the PC formedby using a RAG-1 D600A mutant is essentially inactive, evenif cold partner DNA is added (Fig. 4B, lane 13 to 15).

An in-gel cleavage assay using an intact 23-RSS substrate yieldedresults generally consistent with the data described above.However, the 23-RSS SC1 and SC2 species assembled in the absenceof partner DNA generate lower levels of nicked products, havea propensity to nick aberrantly, and produce virtually no hairpinproduct compared to their 12-RSS SC counterparts (Fig. 4B, lanes16 and 17). The addition of cold partner DNA enhances nickingactivity appreciably (two- to threefold); nicking is preferentiallystimulated in the SC2 species when 12-RSS partner DNA is added(Fig. 4B, lanes 18 to 21). The HSC1 species formed in the presenceof HMG-1 exhibits enhanced nicking activity with reduced levelsof aberrant nicking and supports hairpin formation to detectablelevels (Fig. 4B, lane 22). The HSC2 species obtained from thesame lane possesses the same activity as HSC1 with respect tonicking and hairpin formation (Fig. 4B, lane 23). However, whencold 12-RSS partner DNA is added, hairpin formation is not significantlyaltered in HSC1 but is preferentially stimulated in the PC obtainedfrom the same lane (Fig. 4B, lanes 24 and 25). Such stimulationis not observed when cold 23-RSS partner DNA is added (Fig.4B, lanes 26 and 27). As observed with the 12-RSS substrate,no cleavage activity is detected in control PCs containing aRAG-1 active-site mutant, regardless of whether cold partnerRSS is present (Fig. 4B, lanes 28 to 30).

To determine whether the nicking step was rate limiting forthe 12/23-regulated stimulation of hairpin formation observedin PCs assembled on intact RSS substrates, I next performeda similar experiment using a prenicked, rather than intact,RSS substrate (Fig. 4C). The results are largely consistentwith those shown in Fig. 4B, except that a slight 12/23-dependentstimulation (approximately twofold) of hairpin formation inthe 23-RSS SC2 complex is detectable, and the addition of coldprenicked 23-RSS partner DNA simulates hairpin formation inthe PC, albeit to a lesser extent than prenicked 12-RSS partnerDNA. Taken together, these results show that a PC containinga RAG-1/RAG-2 tetramer and HMG-1 supports 12/23-regulated synapsisand cleavage of RSS substrates in the presence of Mg²⁺.

HMG-1 promotes synapsis of SE substrates but is dispensable for RAG-mediated transposition. To assess whether the SEC described in Fig. 2 supports transpositionof SE, I used an in-gel assay similar to the one described above,except that plasmid DNA was added to the acrylamide solutionused to pour the native gel for the EMSA. The plasmid DNA inthis assay serves as the target of the strand transfer reactioncatalyzed by the RAG proteins. In preliminary experiments, theplasmid pcDNA1 was shown to be large enough so that the protein-DNAcomplexes migrated faster than the plasmid DNA in a native 4%gel and small enough to allow sufficient electrophoretic transferto DEAE-cellulose following the EMSA (data not shown). Wild-typeor active-site mutant (D600A) RAG-1 coexpressed with RAG-2 wasincubated under binding conditions (in the presence of Ca²⁺)with radiolabeled precleaved substrate in the absence or presenceof HMG-1 and/or cold partner DNA in combinations comparableto those for Fig. 4. Protein-DNA complexes were separated byan EMSA, and the gel was soaked in buffer containing 10 mM Ca²⁺for 1 h at 37°C, conditions known to support transpositionin vitro (19). Autoradiographs of the DNA following electrophoretictransfer to DEAE-cellulose show that the wild-type and mutantforms of RAG-1 support protein-DNA complex formation to comparablelevels (and to levels similar to the data shown in Fig. 2C),with the exception that the RAG-1 active-site mutant appearsto assemble SC2 complexes more efficiently than wild-type RAG-1(Fig. 5A). The basis for this phenomenon is unknown but is currentlyunder investigation. The DNA recovered from the SC1, HSC1, SC2,HSC2, and SEC species was normalized and fractionated on a native4 to 20% linear-gradient gel along with radiolabeled, linearizedpcDNA1 and free precleaved substrate DNA (Fig. 5B, lanes 1 and2). Double-ended insertion results in a DNA DSB of pcDNA1, withradiolabeled substrate DNA covalently linked to both ends ofthe linearized plasmid (19).

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FIG. 5. HMG-1 is dispensable for RAG-mediated transposition in a preformed SEC. (A) Precleaved 12-RSS (left) or 23-RSS (right) substrates were incubated with wild-type or active-site mutant (D600A) MR1 coexpressed with MR2 (WT-MR1/MR2 or MT-MR1/MR2, respectively) with or without HMG-1 and/or cold partner DNA (12-SE or 23-SE) under binding conditions, as indicated above the gel, and then subjected to an in-gel transposition assay (see Materials and Methods). The autoradiographs of the DEAE-cellulose paper to which the DNA was transferred are shown. The positions of SC1, HSC1, SC2, HSC2/SEC (comigrating) species, defined in Fig. 2, are shown at the left. (B) Reaction products were isolated from complexes of interest by using the autoradiographs in panel A and analyzed on a native linear 4 to 20% gradient gel. The position of pcDNA1, linearized by double-ended insertion, is indicated at the left. Linearized 5'-end-labeled pcDNA1 (lanes 1 and 17) and precleaved substrate (lanes 2 and 18; indicated at the left) serve as markers. The percentages of integrated substrate DNA are quantified below the gel (%TP).

Unexpectedly, a wild-type SC2 species assembled on a precleaved12-RSS formed in the absence of cold partner DNA supports transposition,as a radiolabeled product that comigrates with linearized, radiolabeledpcDNA1 is clearly visible (Fig. 5B, lane 4). A smaller amountof this product is also observed in DNA recovered from the SC1species present the same lane of the gel (Fig. 5B, lane 3).However, as this may be due to a low level of cross-contaminationwith SC2, whether the SC1 species truly supports some degreeof transposition is unclear. As an important control, no double-endedinsertion product is recovered from SC1 or SC2 complexes assembledby using a RAG-1 active-site mutant (Fig. 5B, lane 5 and 6),demonstrating that the radiolabeled substrate is covalentlylinked to the plasmid rather than associated via RAG-DNA interactions.Interestingly, HMG-1 suppresses transposition activity in HSC1and HSC2 species formed in the absence of cold partner DNA (Fig.5B, lanes 7 and 8). When cold 23-SE partner DNA is added inthe absence of HMG-1, transposition activity in SC2 is reducedabout twofold relative to the activity exhibited by its counterpartformed in the absence of partner DNA and HMG-1 (Fig. 5B, comparelane 11 to lane 4). This outcome is consistent with the possibilitythat two radiolabeled 12-SE substrates are present in SC2, oneof which is exchanged for a cold 23-SE when this RSS partneris present. When both HMG-1 and cold 12-SE partner DNA are present,the SEC possesses no more activity than HSC2 (Fig. 5B, comparelane 13 to lane 8). However, when cold 23-SE partner DNA ispresent, transposition activity is stimulated to a level comparableto that for the 12/23 SC2 species (Fig. 5B, compare lane 15to lane 11). Importantly, all complexes formed by using a RAG-1active-site mutant failed to support transposition (Fig. 5B,lanes 4, 5, 9, 10, 12, 14, and 16).

Interestingly, the SC2 assembled on a radiolabeled 23-SE substratein the absence of HMG-1 and cold partner DNA exhibits littletransposition activity, but, in the presence of cold 12-SE partnerDNA, transposition is stimulated $~$ 40-fold (Fig. 5B, compare lanes20 and 27). In contrast to complexes assembled on precleaved12-RSS substrates, HMG-1 did not suppress transposition activitywhen incorporated into the SEC formed in the absence of coldpartner DNA (Fig. 5B, lane 24), nor did it greatly enhance activity(relative to the SC2 complex) when cold 12-SE partner DNA waspresent (Fig. 5B, compare lanes 27 and 29). However, when bothHMG-1 and cold 23-SE partner DNA were present, the activityof the SEC dropped dramatically, to a level similar to thatfor HSC2 assembled in the absence of cold partner DNA (Fig.5B, compare lane 31 to lane 13). Based on these data, I concludethat a RAG-1/RAG-2 tetramer supports transposition. In the absenceof HMG-1, the RAG-1/RAG-2 tetramer supports both 12/12 and 12/23transposition to similar levels; both reactions are intrinsicallymore favorable than 23/23 transposition. HMG-1 appears to activelyinhibit 12/12 transposition, thereby making 12/23 transpositionthe most favorable. However, transposition activities in preformed12/23 SC2 and SEC species are essentially identical, suggestingthat HMG-1 is dispensable for RAG-mediated transposition.

DISCUSSION

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Discussion

A RAG-1/RAG-2 tetramer core supports 12/23-regulated synapsis, cleavage, and transposition of V(D)J recombination signals. All site-specific recombination reactions require the orchestratedsynapsis of recombination sites mediated (in every example todate) by a multisubunit protein complex. V(D)J recombinationprovides an important example of this process, in which twolymphoid cell-specific proteins, RAG-1 and RAG-2, collaboratewith ubiquitous nonspecific DNA-bending factors (e.g., HMG-1and -2) to facilitate the assembly of a nucleoprotein complexthat supports the initiation of V(D)J rearrangement. As partof an ongoing effort to understand how RAG-1 and RAG-2, withassistance by HMG-1, restrict binding and cleavage to a 12/23pair of recombination signals, I investigated the compositionand assembly of RAG synaptic complexes with model V(D)J recombinationsignals. In this study, I examined the requirements for synapsis,cleavage, and transposition of RSS substrates by using a combinationof mobility shift and in-gel enzyme assays. The results presentedhere extend previous work by demonstrating that (i) a RAG-1/RAG-2tetramer supports 12/23-regulated synapsis, cleavage, and transpositionof V(D)J recombination signals; (ii) the synaptic complex isassembled by association of a stable RAG-1 dimer with two RAG-2subunits that freely reassort in solution; (iii) HMG-1 stimulatesboth PC and SEC formation, yet, once the complexes are formed,HMG-1 is required only for 12/23-dependent cleavage and is dispensablefor RAG-mediated transposition activity; and (iv) the 12/23rule is clearly imposed, both at the level of RSS binding andat the level of hairpin formation.

Implications for synaptic complex assembly. The data presented here suggest that, in the absence of HMG-1,the RAG proteins together assemble at least two distinct protein-DNAcomplexes with isolated recombination signals, termed SC1 andSC2. SC1 contains a stable RAG-1 dimer and monomeric RAG-2,consistent with data published previously (48), and SC2 containsan additional subunit of RAG-2. This result is in agreementwith a report by Sadofsky and colleagues concluding that RAG-1and RAG-2 form a tetramer in solution and retain this configurationwhen bound to DNA (4). However, in that study, the authors didnot examine whether the RAG-1/RAG-2 tetramer they observed supported12/23-regulated synapsis and cleavage of V(D)J recombinationsignals. In accord with previous studies (30, 47, 51), the SC1and SC2 species are supershifted by the incorporation of HMG-1(forming HSC1 and HSC2, respectively). Data shown here suggestthe RAG protein stoichiometry is not altered by the presenceof HMG-1. Although HMG-1 stimulates 23-RSS cleavage within anHSC1 species containing monomeric RAG-2, as noted previously(47), 12/23-regulated synapsis and cleavage of intact RSS substratesare only supported when the RAG-1/RAG-2 tetramer and HMG-1 occurin the context of a PC.

The conclusion reached here that the SC2 and PC species bothcontain a RAG-1/RAG-2 tetramer contrasts with studies publishedrecently by two other laboratories (22, 31). In a study by Oettingerand coworkers (31), RAG protein stoichiometry in SC and PC speciesassembled on intact RSS substrates was analyzed by using anapproach similar to that presented here. Consistent with myresults, the SC2 and PC were found to contain a pair of RAG-2subunits and to possess the same complement of RAG-1 protomers,which do not reassort upon mixing. However, in contrast to datapresented here, the authors could not could unambiguously determineRAG-1 stoichiometry in the SC or PC by using two different-sizeforms of RAG-1 coexpressed in bacteria, possibly due to limitationsof that expression system. Instead, the relative content ofRAG-1 and RAG-2 within the SC and PC species was evaluated indirectly.The authors showed by EMSA that the mobilities of the SC2 andthe PC assembled with MBP-RAG-1 and core RAG-2 (expressed inHeLa cells) were slightly retarded relative to those of comparablecomplexes assembled with core RAG-1 and MBP-RAG-2 (expressedin insect cells), leading to the conclusion that RAG-1 is inexcess over RAG-2 in both complexes. However, this interpretationmay be confounded by differences in the sources and compositionsof the RAG proteins used in the experiments, which could contributeto subtle changes in the mobilities of the complexes. Two factorsto consider include differences in posttranslational modificationof RAG-2 expressed in HeLa versus insect cells and differencesin the composition and length of the adaptor used to fuse MBPto RAG-1 and RAG-2, as these expression constructs were preparedby different cloning strategies (21, 26). In this study, allRAG proteins were prepared from mammalian cells expressed byusing similar plasmid constructs.

In another study by Roth and colleagues (22), a RAG-1 heterodimercontaining an active-site mutation on one subunit was foundto cleave a 12/23 plasmid substrate at both RSSs about fourfoldless effectively than a wild-type RAG-1 heterodimer. These dataled the authors to conclude, based on probability arguments,that the synaptic complex likely contains a pair of RAG-1 dimers.However, since the synaptic complex was not directly observedin this assay, one cannot determine from the data whether thecleavage events observed occurred within the context of a singlecomplex or instead represented the combined activities of morethan one complex. Because the RAG-1 dimer was present in largemolar excess ( $~$ 100-fold) over the DNA substrate in these experiments,the cleavage events observed may reflect nearly simultaneousbinding and cleavage at each RSS by separate RAG-1 heterodimersthat do not physically interact, a possibility made more significantby the fact that cleavage can occur in vitro in the absenceof synapsis (47). The probability of double cleavage in thisscenario is the same as that in the two-dimer model, but thedifference is that the two RAG-1 dimers do not associate inthe same protein-DNA complex. If the PC were to contain a RAG-2dimer (about which there is agreement between the study of Mundyet al. [31] and the data presented here) and a RAG-1 tetramer,then the PC assembled here from MR1/M2R2 would be unlikely tocomigrate with the PC assembled from M2R1m/MR2, as the lattercomplex would contain an additional two MBP modules (representing $~$ 80 kDa of protein). The fact that all RAG-2 homo- and heterodimericSC2 and PC species analyzed here comigrate with comparable speciescontaining RAG-1 on the same gel argues strongly for the presenceof a RAG-1 dimer in these complexes. The MBP moieties are unlikelyto influence the RAG protein stoichiometry, as dimeric configurationsfor RAG-1 and RAG-2 have been established by using other approaches(4, 31, 37).

I considered the possibility that a pair of RAG tetramer complexes(one HSC2 containing a labeled RSS and another HSC2 containingan unlabeled partner RSS), rather than a single RAG tetramercomplex (bound to a pair of RSS substrates), supports synapsisand cleavage through transient protein-protein interactionswithin the gel matrix. However, this possibility seems unlikelyfor three reasons. First, comparable HSC1 and SC2 species donot exhibit 12/23-regulated cleavage, which might reasonablybe expected in this scenario since these complexes contain complementsof protein similar to the PC. Second, diffusion of the protein-DNAcomplexes within the gel matrix would likely be impeded by theirlarge size ( $>=$ 500 kDa), impairing their ability to assemble anappropriate paired RSS complex. Third, the binding of two RSSsby a single RAG-1/RAG-2 tetramer is more plausible, given thefollowing observations: (i) PC formation is stimulated in a12/23-regulated fashion (which would not be expected if transienthigher-order RAG complexes were involved in RSS synapsis), (ii)the nonamer binding domains on both RAG-1 subunits are intactand able to bind DNA (46), and (iii) RAG synaptic complexesevaluated for DNA stoichiometry by mobility shift assays demonstratethe presence of two RSSs within the PC (20).

How is the synaptic complex thought to be assembled? Evidencepresented here and elsewhere suggests the following sequenceof events (Fig. 6). First, RAG-1 and RAG-2 associate in solution,forming a multimeric complex (3, 4, 27, 48). Next, the preassociatedRAG-1/RAG-2 complex binds a single RSS (4, 18, 48). Finally,an appropriate partner RSS is integrated into a PC (17), likelythrough a direct-capture mechanism (31). Evidence presentedhere suggests that the RAG-1/RAG-2 tetramer and HMG-1 togethermediate synapsis. However, the precise order of events thatleads to the integration of HMG-1 and the second RAG-2 subunitinto the PC remains unclear. Once the PC is assembled, the RAGproteins initiate DNA cleavage, retaining their tetramer configurationthrough both the nicking and transesterification steps of V(D)Jrecombination. The postsynaptic complex persisting after DNAcleavage is expected to retain the same RAG protein stoichiometry,as a RAG-1/RAG-2 tetramer complex supports SE synapsis. Althoughthe SEC described here supports transposition, the physiologicalsignificance of this activity remains uncertain. The model proposedfor the composition and assembly of the PC has several attractivefeatures, including (i) its consistency with earlier observationsthat a RAG-1 dimer contains two active sites (46), assemblesa 1:1 mixed-tetramer complex with RAG-2 in solution (4), andmediates both heptamer and nonamer contact in the presence ofRAG-2 (49); (ii) its ability to accommodate variations in theaccessibility of antigen receptor loci (5); and (iii) its sensitivityto intracellular RAG-2 levels, which are regulated in the cellcycle by a phosphorylation-dependent degradation mechanism (25).

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FIG. 6. A sequential-binding model of synaptic complex assembly, adapted from Sadofsky (40). In solution, RAG-1 (shaded ovals) exists as a dimer and associates with one or two RAG-2 subunits (hatched and open circles). A RAG complex minimally containing a RAG-1 dimer and monomeric RAG-2 associates with one RSS (a 12-RSS is shown here for simplicity) and then captures an appropriate RSS partner (31). RAG-1 interacts with both the nonamer (long hatched box) and the heptamer (short hatched box), and RAG-2 is localized at the heptamer-coding junction (12, 48, 49). The second RAG-2 subunit may associate with the complex either before or after acquisition of the second RSS (in this case, a 23-RSS). The HMG protein (open pentagon) selectively promotes binding to the 23-RSS through interactions with RAG-1 (2, 47, 51), although the number of protomers involved and the timing of their association with the RAG complex are unclear. Although not directly tested, a trans mode of RAG-1 association is depicted, consistent with the RAG-1 active-site organization observed in SC1 (46). Studies of the minimal intersignal distance required to support coupled cleavage and recombination suggest the heptamer-to-heptamer orientation of the RSSs shown here (11, 44). The RAG tetramer stoichiometry would be retained through both cleavage steps of V(D)J recombination and in the postcleavage complex as well (not shown). For additional details, see the text.

The RAG-1/RAG-2 tetramer configuration proposed here also hasprecedents in studies of transposases. Bacteriophage Mu transposition,for example, is mediated by a protein-DNA complex, termed atranspososome, containing two Mu end DNA segments bound to aMuA tetramer (24). All four subunits interact specifically withDNA, occupying two of three potential binding sites at eachMu end (29), yet only two of four potential active sites areutilized for the chemical steps involved in Mu transposition(32, 54). However, aside from a superficial resemblance in theoverall number of protomers in the synaptic complex, V(D)J recombinationbears few other similarities to Mu transposition, especiallyrelative to Tn5 (and Tn10) transposition (36), with which itshares more features. These include (i) the generation of DNAhairpin intermediates (7), (ii) the presence of a single enzymebinding site at each DNA end undergoing cleavage (36), and (iii)the structural resemblance between RAG-1 and the Tn5 transposasewith respect to the spacing and organization of critical active-sitecarboxylate residues (the DDE motif) (33, 46). The similaritybetween RAG-1 and the Tn5 transposase is shown here to extendto the dimeric configuration of both proteins within synapticcomplexes (8, 10). However, an important distinction betweenthe two recombination systems is that only one protein, thetransposase, is necessary to catalyze all reactions underlyingTn5 transposition, whereas RAG-1 requires RAG-2 for V(D)J recombinaseactivity. Despite this distinction, the protein complexes mediatingsynapsis in both cases apparently contain two potential activesites, both of which are used to catalyze sequential strandcleavage and strand transfer reactions.

Distinct roles for HMG-1 in synapsis, cleavage, and transposition. HMG-1 (or HMG-2) is known to play a critical role in promotingRAG synaptic complex assembly and activity (13, 40). One wayHMG-1 assists in this process is by facilitating RAG bindingto and bending of the 23-RSS via interactions with the RAG-1homeodomain and the RSS nonamer (2, 47). Two lines of evidencepresented here suggest that HMG-1 additionally promotes associationwith the coding sequence to help organize a "competent" synapticcomplex capable of selectively cleaving 12/23 RSS pairs. First,12/23-regulated synapsis of intact RSS substrates requires thepresence of HMG-1, but HMG-1 is not stringently required forsynapsis of prenicked or precleaved substrates, indicating thatthe intact coding end itself acts as a barrier to synaptic complexassembly. HMG-1 may help the RAG proteins overcome this barrier,possibly by bending the DNA and/or stabilizing conformationalchanges or other structural intermediates integral to synapsis(9, 50). Second, 12/23-regulated cleavage of prenicked RSS substrates(transesterification) requires HMG-1, despite the less stringentrequirement for HMG-1 in the assembly of paired complexes usingthese substrates. Therefore, in addition to promoting the synapsisof intact substrates, HMG-1 may assist the RAGs during the transesterificationstep by stabilizing a cleavage-competent complex capable ofsupporting hairpin formation in the context of a PC.

There remains some controversy over where the 12/23 rule isimposed, with both synapsis and transesterification being implicatedas key control points in the enforcement of the 12/23 rule (17,53). The data presented here provide direct evidence for a middleground, as speculated in a recent review (13), in which the12/23 rule is enforced both at the level of RSS binding (atleast three- to fourfold) and at the level of hairpin formation(approximately fourfold). Although both factors individuallyare relatively modest under the conditions tested here, theycould combine to provide about a 15-fold preference for 12/23substrates, about half the $>=$ 30-fold preference observed in vivo(16). The three- to fourfold preference for 12/23 synapsis foundhere likely underestimates the RAGs' true selectivity for 12/23RSS pairs in vivo, as high local concentrations of chromosomalDNA would act as an effective nonspecific competitor for RAGbinding in vivo. Moreover, chromosomal accessibility of antigenreceptor loci is known to be tightly regulated and may imposetemporal ordering to RAG-RSS association (5).

Like 12/23-regulated cleavage of intact and prenicked substrates,transposition in vitro also occurs in the context of a complexcontaining a RAG-1/RAG-2 tetramer core and exhibits dependenceon the 12/23 rule. Remarkably, while HMG-1 is required for synapsisof intact substrates and promotes 12/23-regulated cleavage ofintact and prenicked substrates, HMG-1 is dispensable for transpositionactivity once the SEC is formed, as the SC2 and SEC speciesassembled with a 12/23 pair of RSSs have the same transpositionactivity. This observation is consistent with recent evidenceindicating that HMG-1 is not required for capture of targetDNA (34) and that SEC formation occurs at low (but detectable)levels in the absence of the HMG protein (14). Interestingly,incorporation of HMG-1 is found to suppress 12/12 transposition,which otherwise occurs quite readily under these conditions,but has little effect on 23/23 transposition, which is not efficientin any case. Thus, in addition to being required for synapsisand cleavage of RSS substrates, HMG-1 may have an unexpectedrole in suppressing 12/12 transposition.

ACKNOWLEDGMENTS

I appreciate the technical assistance of Dustin Volkmer andLei Wang.

This work was supported by a grant to P.C.S. from the AmericanCancer Society (RSG-01-020-01-CCE) and the Health Future Foundation.

FOOTNOTES

* Mailing address: Department of Medical Microbiology and Immunology, Creighton University, School of Medicine, 2500 California Pl., Omaha, NE 68178. Phone: (402) 280-2716. Fax: (402) 280-1875. E-mail: pswanson{at}creighton.edu.

REFERENCES

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