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Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Recombination is initiated by two lymphoid-specific factors, recombination-activating genes 1 and 2 (RAG1 and RAG2) (4, 5). These genes are located 8 kb apart within the same genomic locus and were first identified by their capacity to activate rearrangement of an artificial substrate in a fibroblast cell line. Functional disruption of either gene by either homologous recombination in mice (6, 7) or by amino acid substitutions or truncations in humans with severe combined immunodeficiency or Omenn syndrome (8, 9, 10) has confirmed the fundamental role of RAGs in V(D)J recombination.
Numerous aspects of the recombination reaction are dependent upon the interplay between RAG1 and RAG2 during the rearrangement process. Although RAG1 has recently been shown to contain the catalytic residues of the recombinase and can alone bind the RSS in part through a helix-turn-helix motif homologous to the DNA binding domain of the Hin recombinase (11, 12, 13, 14, 15), such a complex cannot mediate DNA nicking (16, 17, 18). Upon recruitment of RAG2, contacts between the recombinase and the RSS are modified (13, 14, 19, 20), resulting in stabilization of the recombinase-RSS complex (21, 22) and bending of the RSS (23), with significant distortion of the heptamer coding flank border (19, 20). The interaction of RAG2 with RAG1 likely triggers a conformational change in RAG1 that activates its hydrolytic machinery and targets nicking at the border of the RSS and the coding flank. The free hydroxyl group of the nicked strand is then used in a transesterification reaction by the RAG1/RAG2 complex to form a covalently sealed hairpin (24). The hydrolytic mechanism of the RAG1/RAG2 recombinase is again employed for resolving the hairpin intermediate (25, 26) and for processing numerous 3' overhang structures (27). The minimal regions of both proteins that are sufficient for mediating recombination in vivo are referred to as the active cores and span residues 384-1008 in RAG1 (28) and residues 1–383 in RAG2 (24, 29).
Substantial evidence suggests that most steps of V(D)J recombination occur with the 12 and 23 RSSs juxtaposed in a tightly regulated synaptic complex (30, 31, 32, 33, 34, 35). Formation and maintenance of this complex in which double strand breaks are generated requires both RAG1 and RAG2. Following cleavage, both RAGs are integral components of the postcleavage complex (33, 36). In addition, in a reaction paralleling the mechanism used by transposases, the postcleavage complex of RAG1 and RAG2 can transpose cut signal ends into unrelated DNA via a nucleophilic attack (37, 38).
The multifaceted capacities of the RAG1/RAG2 complex for directing DNA recognition before alternating hydrolysis, transesterification, and hydrolysis reactions within a regulated synaptic complex suggest a dynamic interface between RAG1 and RAG2. This interaction has previously been explored in a number of ways. The RAG proteins have been observed to colocalize in the periphery of the nucleus of thymocytes by indirect immunofluorescence and have been coprecipitated from thymocytes as well as from various cell lines transiently overexpressing the RAG proteins (39, 40). In addition, reconstitution experiments using purified RAG1 and RAG2 have demonstrated a direct interaction between the two proteins in the absence of DNA (22, 41). Mapping of the domains of interaction between RAG1 and RAG2 has been undertaken only in part, with aa 504-1008 of RAG1 shown to complex with RAG2 (aa 1–491) (40). RAG2, which has recently been suggested by sequence analysis to form a ß-propeller-like structure composed of six kelch repeat motifs (42, 43), has not been further subjected to deletional mapping.
In this study, we report a detailed analysis of the dominant domains of interaction between the active cores of RAG1 and RAG2. We show that the predicted sixth kelch motif of RAG2 is largely responsible for mediating interaction with RAG1 and that mutation of amino acid W317 within this region abolishes complex formation between RAG1 and RAG2 with subsequent deleterious effects on RSS recognition and cleavage. Moreover, we show that zinc finger B (ZFB) at the C terminus of RAG1 is involved in recruitment of RAG2. In all, these data complement and extend our view of the importance of RAG1-RAG2 interactions for the activation of V(D)J recombination and provide a view of RAG2 as a multimodular adapter protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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GST fusion constructs were generated by subcloning
BamHI/NotI-digested PCR products of RAG1 and RAG2
into the eukaryotic expression vector pEBG, which is under the
transcriptional regulation of the elongation factor 2 promoter
(39). Amino acid substitutions within RAG2 were generated
in pBluescript using the Bio-Rad (Richmond, CA) Phagemid kit and were
transferred as KpnI/EarI fragments into pEBG2
C
(11). All constructs were sequenced.
RAG1 and RAG2 interaction assays
Recombinant proteins were overexpressed in the human embryonal kidney fibroblast cell line HEK293T by calcium phosphate transfection (39). Cells were harvested 48 h posttransfection in PBS and subsequently were lysed in IP lysis buffer (25 mM Tris-HCl (pH 8.0); 250 mM NaCl; 1 mM MgCl2; 0.5% Nonidet P-40; 5% glycerol; and 2 µg/ml of the aprotinin, leupeptin, and pepstatin protease inhibitors). Extracts were spun down for 10 min at 4°C, and the supernatants were incubated with 20 µl of pre-equilibrated GST beads for 2 h at 4°C with rocking. Beads were washed five times in IP-lysis buffer, resuspended in SDS gel-loading buffer (50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), and boiled for 10 min at 95°C. Proteins were then resolved by SDS/PAGE and transferred to nitrocellulose membranes that were blocked by a 30-min incubation in TBST-1% milk. Membranes were incubated with the primary mAb (anti-HA (1:200 dilution) and/or anti-GST (1:1000 dilution)) for 2 h in TBST-1% milk before three washes. Membranes were then incubated with secondary Ab (conjugated with either alkaline phosphatase or HRP) for 30 min. Protein bands were visualized by incubation either with the alkaline phospatase substrate (Promega, Madison, WI) or with luminol (enhanced chemiluminescence system; Amersham, Arlington Heights, IL).
Protein expression and purification
GST fusions of RAG1 and RAG2 were overexpressed in HEK293T cells and purified as previously described (11). Proteins were dialyzed in cleavage buffer (25 mM Tris-HCl (pH 8.0), 150 mM KCl, 2 mM DTT, and 20% glycerol), quantified by Coomassie blue staining after SDS/PAGE, and stored at -80°C in single-use aliquots.
Oligonucleotide DNA cleavage substrates
The 12-RSS upper-strand oligonucleotide (22) was 5'
end-labeled with 
-32P ATP using T4
polynucleotide kinase (NEB, Beverly, MA) as described by the
manufacturer. The unlabeled lower-strand was then annealed by
incubation at 75°C for 2 min before slow cooling to room temperature.
The unincorporated nucleotides were removed by passage through a
Sepharose spin column (G-50 or G-25; Boehringer Mannheim, Indianapolis,
IN), and the double-stranded, radiolabeled substrates were then ethanol
precipitated and resuspended at a concentration of 0.05 pmol/µl.
In vitro cleavage reactions
Cleavage reactions were performed essentially as described (22, 24). A total of 100 ng of RAG1 and RAG2 were incubated for 90 min at 37°C with 0.05 pmols of 32P-labeled 12 RSS in 12.5 mM MOPS-KOH (pH 7.0), 5 mM Tris-HCl (pH 7.0), 30 mM KCl, 60 mM KOAc, 1.4 mM DTT, 0.5 µM nonspecific single-stranded DNA, 0.05 µg/µl BSA, 10% DMSO, 4% glycerol, and 0.5 mM MgCl2 or MnCl2 in a final volume of 20 µl. Reactions were stopped by the addition of 0.1% SDS and denaturing gel-loading buffer (98% deionized formamide, 10 mM EDTA (pH 8.0), 0.025% xylene cyanol FF, and 0.025% bromophenol blue) and were resolved on 16% polyacrylamide/6 M urea denaturing gels.
EMSAs
Conditions were essentially as previously described (21, 22). A total of 50 ng of RAG1 and RAG2 were incubated with 0.05 pmols of 32P-labeled 12 RSS at 30°C for 10 min in 25 mM MOPS-KOH (pH 7.0), 5 mM Tris-HCl (pH 7.0), 30 mM KCl, 120 mM KOAc, 2.4 mM DTT, 1 µM nonspecific single-stranded DNA, 0.1 µg/µl BSA, 20% DMSO, 4% glycerol, and 1 mM MgCl2 in a final volume of 10 µl. The complexes were then cross-linked by addition of 1 µl glutaraldehyde (final concentration, 0.1% v/v) and incubated for 10 min at 30°C. Complexes were resolved on 4% native polyacrylamide gels.
In vivo recombination assays
In vivo recombination assays were performed in HEK293T cells
essentially as previously described (9, 23). HEK293T (or
HEK293, NIH3T3) cells were cotransfected with the deletional
recombination substrate pJH289 (5 µg) (44) and 6 µg of
the expression vectors for GST-RAG1N330 and the various GST-RAG2
mutants. Cells were harvested 48 h posttransfection, and DNA was
isolated as described (5) and analyzed for recombination
frequency by PCR analysis (20 cycles of 94°C for 30 s, 65°C
for 60 s, and 74°C for 60 s). The linear range of the PCR
assay was determined by serial dilutions of the rescued recombined
plasmid. A total of 0.2% of the recovered plasmid was used in all
assays. Oligonucleotides detect the recombined products by annealing to
the joined signal ends and to the CAT gene present in pJH289 (oligos
RA5 and RA14, respectively) (45, 46). As a loading
control, a 154-bp fragment of the CAT gene was amplified (oligos RA1
and RA14) (23) under identical conditions (data not
shown). Amplified products were visualized by autoradiography after
electrophoresis on a 10% polyacrylamide gel.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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To identify the region of RAG2 required for coprecipitation of
RAG1, a panel of RAG2 deletion mutants was constructed as GST
N-terminal fusions (Fig. 1
A).
The GST tag not only facilitates detection and purification of the
recombinant proteins but also provides increased solubility and/or
stability while leaving recombination activity of episomal substrates
essentially unaltered (39). The recombinant forms of
GST-RAG2 were transiently overexpressed in the human kidney fibroblast
cell line HEK293T, together with an N-terminal HA-tagged form of the
RAG1 active core (HA-RAG1N330–1040). GST-RAG2 proteins were
purified from the cell extracts on glutathione-agarose beads, and
coprecipitation of RAG1 was evaluated by Western blot analysis.
Equivalent levels of RAG1 expression in each assay were confirmed by
blotting total cellular extracts (data not shown). Both the full-length
(aa 1–527) and active core (aa 1–388) forms of RAG2 were able to
efficiently coprecipitate RAG1 (Fig. 1B, lanes 1
and 2), whereas GST alone did not associate with RAG1
(lane 7). Deletion of aa 314–388 entirely abolished
precipitation of RAG1 (lane 3) and, accordingly,
further deletions of the RAG2 core were also unable to associate with
RAG1 (lanes 4 and 5). These findings
suggest that aa 314–388 are involved in mediating interaction with
RAG1, and indeed a peptide spanning this region successfully
precipitated RAG1, albeit at somewhat reduced levels (lane
6). To exclude the possibility that the RAG1-RAG2 association was
mediated through nonspecific binding of contaminating genomic DNA, the
above interactions were analyzed in the presence of ethidium bromide,
which disrupts protein interactions mediated through DNA
(47). In fact, RAG1/RAG2/RSS complexes can be entirely
disrupted with 100 µg/ml ethidium bromide as determined by mobility
shift analysis (data not shown). Because the presence of 400 µg/ml
ethidium bromide did not alter the level of association between aa
314–388 of RAG2 and RAG1 (data not shown), we conclude that the
interaction of RAG2 with RAG1 is not mediated through nonspecific DNA
binding.
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N330–1040) (Fig. 1
C, lanes 2 and
5) repeats 4 205–253(205–253) and 5 254–305(254–305) were unable to
efficiently precipitate RAG1 (Fig. 1C, lanes 3
and 4). The current data suggest that the predicted sixth
kelch repeat of RAG2 provides a dominant interface of interaction with
RAG1. However, the effectiveness of such an experiment is dependent
upon the selection of appropriate borders for each of the repeats.
Hence, we cannot entirely exclude the possibility that RAG2 may in part
use one of the other repeats for making contacts with RAG1.
To further define the minimum RAG2 domain required for coprecipitation
of RAG1, a series of GST fusion peptides spanning the interaction site
was constructed. Only the peptides encompassing aa 314–388 and
314–371 (Fig. 1D, lanes 2 and 3) were
able to coprecipitate RAG1, indicating that the minimum domain of
interaction is between aa 314 and 371. Recently, sequence analysis has
revealed that RAG2 possesses a six-fold symmetrical structure with each
repeat composed of a kelch motif (42, 43). Intriguingly,
aa 314–371 coincide with the sixth kelch motif. Crystallographic
analysis has revealed that the 
50-aa kelch motif acquires a fold
consisting of four ß strands, which is reminiscent of the superbarrel
fold observed in the proteins of the sialidase family (48, 49). Kelch motifs have been observed in numerous proteins,
including galactose oxidase from Dactylium dendroides
(48), the 
- and ß-scruin proteins from
Limulus polyhemus (50), the kel1p
protein involved in cell fusion and morphology in Saccharomyces
cerevisiae (51), the kelch protein of
Drosophila that is involved in cytoplasmic transport from
nurse cells to oocytes (52), and the mouse
IAP-promoted placental (MIPP) protein (53). Our
finding that a single kelch motif can mediate protein-protein
interactions between RAG2 and RAG1 support the model that RAG2 is
indeed composed of six discrete repeats formed from four antiparallel
ß strands. Predominant contact with RAG1 through the C-terminal kelch
motif suggests that the N-terminal five repeats may be available to
establish interactions with other proteins involved in the V(D)J
recombination reaction. Because kelch associates with actin in the ring
canals in Drosophila egg chambers (54) and -
and ß-scruin are actin-bundling proteins (55), it is
exciting to speculate that RAG2 may localize the recombinase to the
nuclear periphery by binding structural components of the nuclear
matrix through one of its kelch motifs. A capacity to form discrete
contacts through individual blades of a putative ß-propeller would
imply that RAG2 may function as a multimodular adapter protein involved
in coordinating macromolecular assemblies during the recombination
process.
In previous studies, mutational and deletional analyses have
demonstrated that aa 1–383 of RAG2 are essential for
RAG1/RAG2-mediated recombination of episomal substrates in transiently
transfected cells (29, 56). Because aa 372–388 are
dispensable for interaction between RAG2 and RAG1, we next examined
whether they are also nonessential for RAG1/RAG2-mediated RSS binding
and cleavage. A GST fusion of the RAG2 active core spanning aa 1–371
(RAG2C371), as expected, was able to efficiently precipitate RAG1
(Table I). Accordingly, along with RAG1,
RAG2C371 displayed comparable activity to RAG2C388 for complex
formation on the 12 RSS (Fig. 2A, lanes 3 and
7), for 12 RSS cleavage (Fig. 2B, lanes
3, 7, 13, and 17), and for in
vivo recombination of an exogenous substrate (Table I). Thus, we
conclude that the minimal active core of RAG2 required for both in
vitro and in vivo activity spans aa 1–371. Deletion of the region
corresponding to the predicted sixth kelch motif (RAG2C313), which
abolishes formation of the RAG1/RAG2 complex, abolished entirely both
12 RSS binding (Fig. 2A, lane 8) and cleavage
activity (Fig. 2B, lanes 8 and 18),
thus demonstrating the importance of this region for establishing a
productive DNA binding and cleavage complex. However, although the
putative sixth kelch domain alone could coprecipitate RAG1, it could
not activate the DNA recognition (Fig. 2A, lane
9) and hydrolytic mechanisms (Fig. 2B, lanes
9 and 19) of the recombinase, indicating that the first
five kelch motifs are also critical for the activity of the RAG1/RAG2
complex. In all, the data demonstrate that although the sixth kelch
repeat is capable of interacting with RAG1, it is not sufficient for
activating the catalytic capacity of RAG1 to initiate the first steps
of the recombination reaction.
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To identify amino acid residues within the proposed sixth kelch
motif of RAG2 that are potentially critical for mediating interaction
with RAG1, we compared the sequences of all known RAG2 molecules from
various species. Interestingly, limited sequence conservation is
observed in this region (Fig. 3A). W317 and F318 are among
the most conserved residues. We explored the role of these two residues
in RAG1/RAG2 complex formation by introducing the conservative amino
acid substitutions WF317/8YY. After coexpression in 293T cells,
RAG2C(WF317/8YY) was unable to coprecipitate RAG1 (Fig. 3B, lane 4). Single amino acid mutations
RAG2CW317Y and RAG2CF318Y revealed that W317 is essential for
coprecipitation of RAG1, whereas mutation F318Y did not interfere with
RAG1 interaction (Fig. 3B, lanes 2 and
3). Functional analysis of W317Y demonstrated that
disruption of RAG1 interaction concurrently abolished 12 RSS binding
(Fig. 2A, lane 4) and cleavage (Fig. 2B, lanes 4 and 14) as well as the
capacity for in vivo recombination (Table I). On the other hand, F318Y,
which was capable of interacting with RAG1, also permitted complex
formation on the 12 RSS (Fig. 2B, lane 5),
efficient 12 RSS cleavage (Fig. 2B, lanes 5 and
15), and in vivo recombination (Table I).
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RAG1 ZFB is involved in RAG1-RAG2 DNA-independent interaction
Having established a predominant RAG2 domain involved in the interaction with RAG1, we sought to further define the regions of RAG1 required for coprecipitation of RAG2. McMahan et al. (40) have previously identified a large region of RAG1 spanning aa 504-1008 that is able to precipitate RAG2 after overexpression in COS cells. This region has been shown to contain a single C2H2 zinc binding domain (ZFB, aa 727–750), which possesses weak and apparently nonspecific DNA binding capacity (59). The N terminus of RAG1, which is dispensible for in vivo recombination, contains two zinc binding domains (a ring finger and a zinc finger A (ZFA)), which together form a highly specific dimerization interface between RAG1 molecules (59, 60). Due to the ability of the N-terminal zinc binding domains to coordinate protein interactions, we tested the role of ZFB in mediating RAG1 and RAG2 interaction.
A series of GST-fused RAG1 deletion mutants was generated (Fig. 4A) and coexpressed with an
HA-tagged form of the RAG2 active core (HARAG2C388) in HEK293T
cells. Complexes were purified on glutathione beads, and
coprecipitation of RAG2 was detected by Western blot analysis. In
agreement with McMahan et al. (40), the C terminus of RAG1
(aa 500-1040) was able to coprecipitate RAG2 (Fig. 4B,
lane 5). Deletion of aa 758-1040 (aa 500–758), which left
ZFB intact, had no effect on the interaction with RAG2 (Fig. 4B, lane 7, and C, lane 3),
whereas a further deletion of aa 726-1040, which completely removes
ZFB, abolished the interaction (Fig. 4B, lane 6).
Moreover, a peptide encompassing ZFB (aa 692–758) was still able to
coprecipitate RAG2 (Fig. 4C, lane 5), whereas a
peptide spanning the N-terminal
C2H2 zinc-binding motif ZFA
(aa 330–389) and GST alone failed to interact with RAG2 (Fig. 4C, lanes 4 and 6). Because the
interaction between ZFB and RAG2 was unaltered by the introduction of
400 µg/ml ethidium bromide (Fig. 4C, lane 12),
we conclude that the recruitment of RAG2 by ZFB of RAG1 is not mediated
through nonspecific DNA binding. Equal levels of RAG2 expression in all
of the assays presented in Fig. 4 were evaluated by blotting crude
extracts (data not shown). Although ZFB represents a predominant
interface of interaction, our data do not permit us to exclude the
possibility that RAG1 and RAG2 interactions are established through
other domains present in RAG1. In addition, attempts to determine
whether the sixth kelch motif of RAG2 directly interacts with ZFB of
RAG1 were hampered by the poor solubility of both short peptides when
expressed as HA-tagged forms (data not shown).
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In this study we present minimal domains in RAG1 and RAG2 required for efficient complex formation. Whether these domains are central for all the steps of RSS recognition, synapsis, and cleavage or whether different interfaces of interaction are utilized for each step of the reaction remains to be addressed. Nonetheless, the deletional mapping of the interaction domains between RAG1 and RAG2 presented in this study provides an important first step toward an understanding of the intricate contacts required to form a RAG1/RAG2 complex that is active for RSS binding and cleavage.
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Acknowledgments |
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Footnotes |
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2 Current address: Hellenic Pasteur Institute, Laboratory of Molecular Genetics, 127 Vas. Sofias Avenue, 11521 Athens, Greece
3 Eugenia Spanopoulou died on September 2, 1998.
4 Address correspondence and reprint requests to Dr. Sandro Santagata, Mount Sinai School of Medicine, Ruttenberg Cancer Center, Box 1130, 1425 Madison Avenue, New York, NY 10029.
5 Abbreviations used in this paper: RSS, recombination signal sequence; RAG, recombination-activating gene; ZFB, zinc finger B; ZFA, zinc finger A.
Received for publication November 5, 1999. Accepted for publication March 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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