INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

V(D)J recombination assembles separate antigen receptor gene segments into an exon encoding the antigen binding domain of immunoglobulin and T-cell receptor proteins. The V(D)J recombinase consists of two proteins, RAG-1 and RAG-2, which bind to recognition sequences (RSS) located adjacent to the V, D, and J coding segments. Recombination is initiated by the RAG proteins, which introduce a nick precisely between the RSS and the coding segment, creating a free 3' OH which is then used as a nucleophile to attack the opposite strand, resulting in a blunt, 5'-phosphorylated signal end and a hairpin coding end (17, 22).

Although nicking can occur at a single RSS, efficient hairpin formation normally requires a pair of different RSS, one with a 12- and one with a 23-nucleotide spacer (known, respectively, as the 12-RSS and the 23-RSS), with the two being assembled into a synaptic complex (9, 30). This restriction, known as the 12/23 rule, prevents immunologically irrelevant recombination events from scrambling the immune receptor loci. The 12/23 rule can, however, be bypassed under certain conditions, such as when Mn²⁺ is substituted for the physiological divalent cation, Mg²⁺ (30).

In order to understand the mechanism of cleavage and the molecular basis of the 12/23 rule, it is necessary to delineate the organization of the nucleoprotein complexes that carry out cleavage at a single RSS and at a 12/23 RSS pair. Recent work has shown that a single molecule of DNA containing an RSS is bound by a RAG-1 dimer, with one or two monomers of RAG-2, and that this nucleoprotein complex is competent for cleavage (3, 21, 29). This single RSS complex is quite stable; it is resistant to incubation with a 250-fold excess of specific competitor at 37°C for at least 1 h (8). Furthermore, purified RAG-1 dimers are stable in solution, even in the absence of DNA, remaining associated after overnight incubation at 4°C (21). Additional evidence for the stability of RAG-1 dimers is provided by the observation that heterodimer formation requires coexpression of two different RAG-1 molecules: mixing of individually purified RAG-1 homodimers did not result in detectable heterodimer formation (29).

Although the stoichiometry of RAG-1 molecules bound to a single RSS has been established, our understanding of the anatomy of a functional RAG-RSS complex remains incomplete. A question of particular mechanistic importance is whether the two cleavage steps, nicking and hairpin formation, utilize one or both active sites present in the RAG-1 dimer bound at a single RSS. Precedents exist for both possibilities. The Tn7 transposase utilizes two distinct active sites, in two different transposase proteins, to catalyze cleavage of the top and bottom strands (24). In contrast, the transposases of bacteriophage Mu, Tn10, and Tn5 each use a single active site to carry out cleavage and strand transfer (4, 11, 18, 19, 32).

To address these questions, we have taken advantage of catalytically deficient RAG-1 mutants. We recently identified three catalytic amino acids (D600, D708, and E962) in RAG-1 that are thought to be involved in coordinating a divalent metal ion that is essential for cleavage (14). There is evidence that two of these amino acids, D708 and D600, directly contact the metal ion (7, 12, 14). Importantly, mutant proteins bearing a single-amino-acid substitution at any of these three positions exhibit essentially wild-type RSS binding and 12/23 synaptic complex formation but are completely unable to carry out either nicking or hairpin formation (7, 12, 14). Because metal ions are required for DNA binding (2, 23), it is likely that these catalytically deficient mutants can bind metal ions but fail to position them properly for catalysis. Moreover, pairwise combinations of these mutant proteins do not restore enzymatic activity when assayed either in vivo or in vitro, demonstrating that these three catalytic amino acids are not contributed by different monomers of RAG-1 (unpublished observations). We have used purified mutant heterodimers of RAG-1 that contain one wild-type monomer and one catalytically deficient monomer to determine that a single RAG-1 monomer performs both nicking and hairpin formation at a single RSS. Furthermore, analysis of cleavage of paired 12/23 complexes suggests that the synaptic complex contains two RAG-1 dimers.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Protein purification. Baculovirus transfer vectors encoding the mutant RAG proteins or their wild-type counterparts were made by subcloning into the pFastBac transfer vector (GIBCO/BRL). Sf9 cells were coinfected with one recombinant baculovirus encoding a RAG-1 molecule with an N-terminal maltose binding protein (MBP) fusion and three copies of a C-terminal Myc tag (MBP-RAG-1[384-1008]-Myc3, ~130 kDa, referred to as mR1) and a second recombinant baculovirus encoding a RAG-1 molecule lacking the N-terminal MBP fusion but containing a C-terminal polyhistidine and three copies of the Myc tag (RAG-1[384-1008]-His9-Myc3, ~85 kDa, referred to as R1h) (2, 13, 17). Typically, 500-ml volumes of Sf9 cells at a density of 2 × 10⁶ cells/ml were infected with high-titer virus stocks. Cells were harvested ~60 h postinfection and lysed in 30 ml of lysis buffer (20 mM Tris-Cl, pH 7.9, at 4°C; 0.5 M NaCl; 20% glycerol; 2 mM -mercaptoethanol) plus 10 mM imidazole by dounce homogenization (20 strokes, tight pestle). The resulting lysate was centrifuged at 100,000 × g for 30 min at 4°C. The supernatant was loaded onto a 0.5-ml metal chelating Sepharose column (Pharmacia) charged with NiSO₄. The column was washed with 10 ml of lysis buffer containing 60 mM imidazole and eluted with 5 ml of lysis buffer containing 250 mM imidazole. The eluate was diluted with 8 ml of amylose A (25 mM sodium phosphate, pH 7.2; 0.5 M NaCl; 10% glycerol; 1 mM dithiothreitol [DTT]) and loaded onto a 0.5-ml amylose resin column. The column was washed with 10 ml of amylose A, and the protein was eluted with amylose A plus 10 mM maltose. Fractions containing the RAG proteins were dialyzed against 1,000 volumes of storage buffer (25 mM K-HEPES, pH 7.5; 150 mM potassium glutamate; 20% glycerol; 2 mM DTT) for 3 h at 4°C. The protein was aliquotted, flash-frozen in liquid nitrogen, and stored at 80°C. Glutathione S-transferase (GST)-tagged RAG-2(1-383) was purified as described previously (25, 26).

Electrophoretic mobility shift assays. Purified RAG-1 and RAG-2 proteins (100 ng of each, as measured by Coomassie blue-stained gels) were incubated with 25 fmol of the annealed oligonucleotide substrate DAR39/40 (17) in 10 µl of reaction buffer (37.8 mM HEPES-KOH, pH 7.5; 51 mM potassium glutamate; 10% glycerol; 3 mM DTT; 2.5 pmol of the nonspecific competitor oligonucleotide FM117 [17]; 1 mM MgCl₂; 60 µg of bovine serum albumin [BSA] per ml; 0.006% NP-40; 20% dimethyl sulfoxide). The incubations were at 30°C for 30 min, and they were cross-linked by the addition of glutaraldehyde (to 0.1%), with an additional incubation for 10 min at 37°C, as described previously (2, 8). DNA binding was analyzed by nondenaturing electrophoresis through a 4% polyacrylamide gel run in 1× Tris-borate-EDTA (TBE) (80 mA · h at 4°C). Dried gels were visualized by autoradiography and/or with a phosphorimager.

Oligonucleotide cleavage assays. Oligonucleotide cleavage assays were performed as described previously (13). RAG-1 and RAG-2 (100 ng of each; approximately 400 fmol of RAG-1 dimer) were incubated with 250 fmol of annealed DAR39/40 (17) (DAR39 was 5' ³²P end labeled) in a 10-µl reaction mixture (40 mM HEPES-KOH, pH 7.5; 60 mM potassium glutamate; 5; 60 mM potassium glutamate; 10% glycerol; 3 mM DTT; 1 mM MnCl₂; 60 µg of BSA per ml; 0.006% NP-40) at 37°C for 45 min unless otherwise stated. The reactions were stopped by adding an equal volume of a solution containing 94% formamide, 20 mM EDTA, and 0.05% bromophenol blue. The reaction products were separated by electrophoresis through a 10% acrylamide gel containing 30% formamide, 0.67× TBE, 7 M urea, and 12.5 mM HEPES-KOH, pH 7.5, for 2 h at 75 W. Wet gels were visualized by autoradiography or with a phosphorimager.

Plasmid cleavage assays. RAG-1 and RAG-2 were incubated with 20 ng of pJH290 for 3 h at 30°C (50 mM HEPES-KOH, pH 8.0; 11 mM KCl; 4 mM NaCl; 15 mM KGlu; 5 mM MgCl₂; 4% glycerol; 1 mM DTT; 100 ng of BSA per µl; 20 ng of HMG-1 per µl). The cleavage reactions were terminated by the addition of 100 µl of stop buffer (100 mM Tris-Cl, pH 8.0; 0.2% sodium dodecyl sulfate; 0.35 mg of proteinase K per ml; 10 mM EDTA) and incubated at 55°C for 1 h. The deproteinized cleavage products were phenol-chloroform extracted, ethyl alcohol precipitated, resuspended in 20 µl of Tris-EDTA, and digested with PvuII (0.1 U) for 30 min at 37°C. Half of the digested reaction products were then separated by electrophoresis through a 4.5% acrylamide gel, transferred to a solid support, and hybridized with a randomly primed 693-bp PvuII fragment from pJH290 that is complementary to all cleavage products.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Expression and purification of heterodimeric RAG-1 protein. To purify heterodimers containing one wild-type monomer and one catalytically deficient monomer (D708N, D600N, or E962Q), we coinfected insect cells with recombinant baculoviruses encoding two different versions of RAG-1, one with an N-terminal MBP fusion (referred to as mR1) and one with a C-terminal polyhistidine tag (referred to as R1h). Three different dimers can be formed within a cell infected by both viruses, as diagrammed in Fig. 1A: an mR1-mR1 homodimer, an mR1-R1h heterodimer, and an R1h-R1h homodimer. Assuming random association, these three species should be formed in a 1:2:1 ratio. Heterodimers (mR1-R1h) were purified by sequential affinity chromatography using an Ni²⁺ chelating resin (which binds the polyhistidine tag) followed by an amylose A resin (which binds the MBP tag). This sequential chromatography scheme purifies heterodimers away from contaminating homodimers (purified heterodimer preparations contain less than 5% homodimer [see below]).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Heterodimeric RAG-1 protein preparations containing equal amounts of the two RAG-1 monomers. (A) Three possible combinations (i, ii, and iii, respectively) of RAG-1 dimers resulting from random assortment during coinfection. Form ii is the only dimer retained after sequential affinity chromatography. (B) Representative Western blot analysis of heterodimeric RAG-1 protein preparations using the anti-c-Myc antibody (clone 9E10). The quantitation of band intensity was carried out by fluorimager analysis. The average molar ratios (fluorimager units for mR1 divided by fluorimager units for R1h for both amounts loaded [1x and 2x]) were calculated from three independent protein preparations of each heterodimer.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Electrophoretic mobility shift analysis of homodimers and heterodimers. Electrophoretic mobility shift analysis of a 12-RSS-containing oligonucleotide substrate was performed with either homodimeric or heterodimeric RAG-1 and GST-tagged RAG-2. All reaction mixtures contain GST-tagged RAG-2, but only RAG-1 is listed above the lanes for simplicity. Dialysis buffer used for RAG-1 purification was used for no RAG-1 control. The heterodimers shift the probe to an intermediate size relative to the large and small homodimers. In panel B, samples were run for a longer time.

One functional RAG-1 active site is sufficient for both nicking and hairpin formation in Mn²⁺. The catalytically competent RAG-12-RSS complex (also called the 12-SC) contains a dimer of RAG-1, one 12-RSS, and either one or two RAG-2 molecules (3, 21, 29). How many functional active sites within the RAG-1 dimer bound to a single RSS are necessary for nicking and hairpin formation? The simplest possibility is either that (i) one RAG-1 monomer nicks and the other monomer is responsible for hairpin formation or that (ii) a single monomer's active site performs both catalytic steps. These two models can be tested using the purified mutant heterodimers which contain one wild-type RAG-1 monomer and one mutant RAG-1 monomer that is incapable of either nicking or hairpin formation. Model 1 predicts that a standard substrate would be nicked, without hairpin formation (since nicking must precede hairpin formation); use of a prenicked substrate would allow hairpin formation. According to model 2, both nicking and hairpin formation should be observed with the use of standard substrates.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   One functional RAG-1 monomer is capable of nicking and hairpin formation at a single RSS. Abbreviations: U, uncut substrate; HP, hairpin product; N, nicked intermediate. (A) Standard cleavage reactions using a radiolabeled 12-RSS-containing oligonucleotide substrate and Mn2+ were performed. (B) Cleavage was allowed to proceed for the times noted. (C) RAG-1 and RAG-2 proteins were diluted with dialysis buffer prior to incubation, and cleavage was allowed to proceed for 45 min. (D) Schematic of substrate and cleavage products. *, radiolabel.

View larger version (47K): [in this window] [in a new window]   FIG. 4.   Mutant heterodimer cleavage activity is competitor resistant. Abbreviations: U, uncut substrate; HP, hairpin product; N, nicked intermediate; PN, prenicked substrate. (A) This assay was staged, using two incubations. In the first incubation, RAGs and substrate were incubated in Ca2+ to allow complex formation in either the presence or the absence of specific cold competitor. In the second incubation, Mn2+ was added to allow catalysis in either the presence or the absence of specific cold competitor. (B) Cleavage in the presence of Mg2+ or Mn2+ was tested on an uncut substrate or a prenicked substrate.

One RAG-1 active site is also sufficient for both cleavage steps at a single RSS in the presence of Mg²⁺. The experiments described above were performed in the presence of Mn²⁺, which is known to relax the specificity of many nucleases (31), including the RAG proteins (17). Furthermore, Mn²⁺ nonspecifically rescues catalytic activities of several mutant RAG proteins (but not the DDE mutants) by unknown mechanisms (14). Therefore, it was important to examine single-RSS cleavage in the presence of Mg²⁺ (hairpin formation was inefficient but detectable under these conditions). As we observed in experiments with Mn²⁺, both mutant heterodimer preparations were capable of nicking and hairpin formation in the presence of Mg²⁺, yielding levels of these products that were approximately twofold lower than those observed with wild-type heterodimers (Fig. 4B, lanes 1 to 3). The mutant heterodimers were also capable of catalyzing hairpin formation using a prenicked substrate, again approximately 50% less efficiently than the wild type both in the presence of Mg²⁺ (Fig. 4B, lanes 5 through 7) and in the presence of Mn²⁺ (Fig. 4B, lanes 9 through 11). Thus, a single functional RAG-1 active site in the RAG-RSS complex was sufficient for nicking and hairpin formation in the presence of both Mn²⁺ and Mg²⁺. The ratios of nick formation to hairpin formation for wild-type and mutant heterodimers were closely matched at early time points. In some experiments, after prolonged incubation the mutant heterodimers showed somewhat less hairpin formation than wild-type heterodimers. The mechanistic basis for this behavior is unclear. It is important to note, however, that cleavage at a single RSS is not necessarily a physiologic reaction (analysis of cleavage at a 12/23 RSS pair is presented below).

Cleavage of a 12/23 synaptic complex requires two RAG-1 dimers. Under physiological conditions, hairpin formation is substantially stimulated by the formation of a synaptic complex in which the RAG proteins are bound to both a 12- and a 23-RSS (6, 9, 30). The stoichiometry of this complex with respect to RAG-1 has not been established. Two models have been proposed for the formation of these complexes (3, 27, 29). A RAG complex containing a RAG-1 dimer associated with a single RSS could simply capture a second RSS, leading to a synaptic complex containing only one dimer of RAG-1. Alternatively, the synaptic complex could form by collision of two RAG-RSS complexes, leading to a complex containing two RAG-1 dimers. Examples of both dimeric (19) and tetrameric (18, 32) configurations are provided by bacterial transposases. In both cases, only one active monomer at each end of the transposable element is necessary for all catalytic activities (4, 11, 18, 19, 32).

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Two dimers of RAG-1 exist in a synaptic complex. Abbreviations: U, uncut substrate; SC, cleavage at one RSS; DC, cleavage at both RSS; CE, coding end. (A) Cleavage was allowed to proceed for the times noted; (B) RAG-1 and RAG-2 proteins were diluted with dialysis buffer prior to incubation and cleavage was allowed to proceed for 30 min.