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Originally published In Press as doi:10.1074/jbc.M403706200 on April 28, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29821-29831, July 9, 2004
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Ku70/Ku80 and DNA-dependent Protein Kinase Catalytic Subunit Modulate RAG-mediated Cleavage

IMPLICATIONS FOR THE ENFORCEMENT OF THE 12/23 RULE*

Dennis J. Sawchuk,abc Jorge Mansilla-Soto,cd Claudio Alarcon,d Netai C. Singha,d Hanno Langen,e Marco E. Bianchi,f Susan P. Lees-Miller,g Michel C. Nussenzweig,ahi and Patricia Cortesdj

From the aLaboratory of Molecular Immunology and hHoward Hughes Medical Institute, The Rockefeller University, New York, New York 10021, dImmunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, eGenetics, F. Hoffmann-La Roche, Ltd., CH-4070 Basel, Switzerland, fUniversità Vita Salute San Raffaele, via Olgettina 58, 20132, Milano, Italy, and the gDepartment of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, April 2, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
V(D)J recombination allows for rearrangement of antigen receptor gene variable (V), diversity (D), and joining (J) segments, resulting in a diverse array of antigen receptors. The reaction occurs in B and T cells and requires the recombinase activity of the lymphoid-specific RAG1 and RAG2 (1, 2). RAG1/2 are directed to the correct sites of recombination by recombination signal sequences (RSSs),1 each consisting of a conserved nonamer and heptamer element, separated by a less conserved spacer of 12 (12RSS) or 23 (23RSS) nucleotides (3).

The recombination reaction can be divided into two steps. First, RAGs bind and cleave at the heptamer/coding borders of one 12RSS and one 23RSS, generating hairpin coding ends and blunt 5'-phosphorylated signal ends (4). Second, DNA breaks are repaired, with hairpin coding ends being opened, processed, and ligated to form an imprecise coding joint, and signal ends are precisely ligated to form a signal joint (4).

In vivo and in vitro experiments have shown that the processing-repair step involves the participation of lymphoid-specific terminal deoxynucleotidyl transferases (TdTS and TdTL) (5), the ubiquitous nonhomologous end-joining (NHEJ) factors, including Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, DNA ligase IV, Artemis (for a review, see Refs. 6 and 7), and DNA polymerase µ (8). How all of these factors are specifically recruited and coordinately assembled during V(D)J recombination is largely unknown (for further discussion see Ref. 9). However, it has been proposed that they are recruited through interactions of Ku70/80 with RAG-generated ends (10-12).

Recombination must be correctly targeted, both spatially and temporally. One critical level of regulation is that a 12RSS and 23RSS pairing is highly preferred for recombination. This preference, termed the 12/23 rule, operates at the cleavage step of the reaction in vivo and in vitro (13-16). RAG1/2 were first shown to establish the 12/23 rule in vitro (17), with a 3-4-fold preference for a 12/23 substrate. We and others have shown that high mobility group protein 1 or 2 (HMGB1 or HMGB2) stimulates RAG-mediated cleavage (18, 19). HMGB1 was later shown to enforce the 12/23 rule in vitro (13, 15, 20, 21). This conclusion was reached using either a trans-RSS cleavage assay (13, 21) or cleavage assays in which the cis-RSS substrate contained a gap (20) or a nick (15) between the RSSs.

Using a cis-RSS cleavage assay containing RAGs, we reported that additional ubiquitous factors present in whole cell extract (WCE) enhance the 12/23 rule (19). We have fractionated and characterized this extract and here demonstrate that DNA-PKcs and Ku70/Ku80 play a role in 12/23-regulated cleavage. Since the 12/23 rule is set at the cleavage step, this finding implicates DNA-PKcs and Ku70/Ku80 as components of the precleavage complex upstream of DNA repair.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
The mammalian expression vectors for GST-core RAG1 and GST-core RAG2 have been described (34). Plasmids pEF-FT-RAG1 and pEFHT-RAG2 encode thioredoxin (Trx) fusions to core RAG1 (residues 380-1040) or core RAG2 (residues 1-383), tagged at the N terminus with a FLAG (RAG1) or an HA (RAG2) epitope. Subcloning details can be obtained upon request.

Recombination substrates pJH290 12/23 (35), 12/12, and 23/23 (19) were PCR-amplified using RA2 and RA14 primers to yield a 560-bp amplicon containing the RSSs. The PCR fragment was then cloned into PCR2.1 vector using TA cloning (Invitrogen). The subcloned deletion substrates are identified as pDS12/23, pDS12/12, and pDS23/23.

Cell Culture and Transfection
293T cell culture and transfection were as described (19, 36).

Purification of Recombinant Proteins
RAGs—GST-RAG proteins were copurified, as described (19). FLAG-Trx-RAG1·HA-Trx-RAG2 complex was purified by incubating the cell lysate with anti-FLAG M2-agarose affinity gel (Sigma); after subsequent washes, the fusion proteins were eluted at 4 °C in elution buffer consisting of 25 mM Tris-HCl (pH 8.0, 4 °C), 200 mM NaCl, 20% glycerol, and 200 µg/ml FLAG peptide (Sigma). Protein preparations were kept at -80 °C. Concentrations of GST-RAG1 and GST-RAG2 were determined to be 100 and 200 ng/µl, respectively. For FLAG-Trx-RAG and HA-Trx-RAG2, the concentrations were 100 and 50 ng/µl, respectively.

HMGB1—Cells from a 1-liter bacterial (BL21) culture were resuspended in 30 ml of lysis buffer (20 mM Tris·HCl, pH 7.5, 200 mM NaCl, 1.0% Nonidet P-40, and protease inhibitors) and then successively treated with lysozyme (50 µg/ml) and DNase I (200 µg/ml and 5 mM MgCl2) for 1-2 h at 4 °C. The lysed mixture was centrifuged at 16,000 rpm for 30 min to collect the supernatant. The supernatant was passed through a 2-ml Ni2+-nitrilotriacetic acid column, washed extensively with the wash buffer (50 mM NaH2PO4, pH 8.0, 0.5 M NaCl, 5 mM{beta}-mercaptoethanol, 0.5% Tween 20, 10 mM imidazole, and 10% glycerol), and then the protein was eluted with 350 mM imidazole in wash buffer. The sample was dialyzed in buffer B (50 mM NaH2PO4, pH 8.0, 0.1 M NaCl, 5 mM {beta}-mercaptoethanol, and 10% glycerol) and loaded on a 20-ml DEAE column. Elution was made with a 0.1-1.0 M KCl gradient, and the fractions containing HMGB1 (it was eluted near 220 mM KCl) was pooled together and dialyzed against buffer B. The dialyzed sample was loaded on a 5-ml MonoQ column, and the protein was eluted with a 0.1-0.5 M KCl gradient. His6-HMGB1 was eluted between 200 and 250 mM KCl. The fractions containing HMGB1 were pooled together and dialyzed against 25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2 mM dithiothreitol, and 10% glycerol.

Large Scale Culture of 293T Cells and Whole Cell Extract Preparation
293T cells were grown in spinner culture in Joklik's minimal essential medium (Sigma), and 100 liters of suspension cells were grown and harvested (National Cell Culture Center, Minneapolis, MN). Whole cell extracts were prepared as described (16, 19).

Purification of the Cleavage-enhancing and Cleavage-inhibitory Activities
Column fractions were analyzed by two cleavage assays: one assaying only for cleavage enhancing activity, to locate active peaks, and the other utilizing the positive peak in combination with the S300 activity (regulated cleavage assay). The 293T WCE was loaded onto a High Q column equilibrated with BC100 (20 mM Tris-Cl pH 7.9, 0.2 mM EDTA, 20% glycerol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). The flow-through (possessing the 12/23 enforcing activity) was loaded onto a High S column equilibrated with BH100 (20 mM HEPES, pH 7.9, 1 mM MgCl2, 0.1 mM EDTA, 20% glycerol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). The column was step-eluted at 300, 600, and 1000 mM KCl. The regulatory activity separated into cleavage-enhancing and cleavage-inhibitory activities at this point. For cleavage-enhancing activity, see the Supplementary Material. For cleavage-inhibitory activity, the 300 mM elution from the High S column was further fractionated into several columns, but the inhibitory activity was lost.

Purification of Ku70, Ku80, and DNA-PKcs
Using human placenta or HeLa cells as a source, Ku70/Ku80 heterodimer and DNA-PKcs were separately purified as previously described (37).

Preparation of DNA Cleavage Substrate
End-labeled linear double stranded cis-cleavage substrates were obtained by EcoRI digestion of the pDS substrates, liberating a DNA fragment containing the respective RSS pair. 250 ng of the cleavage substrate was then radioactively end-labeled with exo- Klenow enzyme and [{alpha}-32P]dATP and [{alpha}-32P]TTP. The labeled substrates were gel-purified, ethanol-precipitated, and resuspended in 5 mM Tris-Cl (pH 7.9). Body-labeled linear double-stranded cis-cleavage substrates were obtained by PCR using the pJH290 DNAs, RA2, and RA14 primers and [{alpha}-32P]dCTP. Radioactive PCR products were first purified by column (Qiagen) and further purified by gel extraction; DNA was quantified by agarose gel-ethidium bromide staining.

Cleavage Assay
Assays Containing Linear DNA—Cleavage reaction conditions were as previously described (16, 19), except that 5 mM MgAc2 was used in the assay (50 µl). For protein purification experiments, assays contained combinations of 100 ng of GST-RAG1 (19 nM), 200 ng of GSTRAG2 (58 nM), column pools containing the cleavage enhancing and cleavage inhibitory activities, and 1.5 µg of HMGB1 (1.2 µM) when required. Reconstitution experiments using GST-RAGs contained the above proteins plus 10-100 ng of Ku70/Ku80 (1.3-13 nM) and 10-100 ng of DNA-PKcs (0.4-4 nM). Reconstitution experiments using Trx-RAGs contained 100 ng of FLAG-Trx-RAG1 (23 nM), 50 ng of HA-Trx-RAG2 (18 nM), 50-500 ng of HMGB1 (40-400 nM), 100-400 ng of Ku70/Ku80 (13-52 nM), and 100-400 ng of DNA-PKcs (4-16 nM). After sample processing, gel electrophoresis, and gel drying, DNA cleavage products were visualized using a Storm PhosphorImager (Amersham Biosciences) and quantified using ImageQuant software (Amersham Biosciences). For body-labeled linear substrates, signal end (SE) product was used as a direct parameter to quantify cleavage activity. To properly compare between different linear DNA substrates, the levels of SE product were expressed as a percentage of the radioactivity of its corresponding DNA substrate. -Fold preference of 12/23 substrate was determined by comparing the percentages of SE products between 12/23 and 12/12 or 23/23 substrate-containing reactions.

Assays Containing Plasmid DNA—Cleavage reactions were carried out with 1 µg (6.7 nM) of pDS plasmids (12/23, 12/12, or 23/23) and the same amount of proteins as above, except that 400 ng of FLAG-Trx-RAG1 (92 nM) and 200 ng of HA-Trx-RAG2 (72 nM) were used. After a 1.5-h incubation, samples were processed exactly as described above, except that precipitated DNA was resuspended in 0.1x TE buffer and digested with the single cutting enzyme NcoI (10 units) for 3 h. After digestion, DNA was fractionated on a native 1.1% agarose gel, transferred to nylon membranes, UV-cross-linked, and probed with a radiolabeled probe obtained by PCR using the RA2 and RA14 primers and pJH290 12/23 as template. Data were then visualized and quantified as described. SE product was used as a direct parameter to compare between different substrates.

Immunodepletions
The monoclonal antibody 162 (NeoMarkers), recognizing the Ku70/Ku80 native heterodimer, or an isotype-matched control anti-keratin monoclonal antibody LHK6B (NeoMarkers) was cross-linked to Protein A-Sepharose. 200 µl of S300 regulatory fraction was incubated with 10 µl of cross-linked anti-Ku or anti-keratin beads for 1 h at 4 °C. Beads were pelleted by centrifugation, and supernatants were transferred to a second microcentrifuge tube and incubated again with 10 µl of anti-Ku or anti-keratin beads. This process was repeated four times.

Western Blotting
Protein samples were resolved on a 4-20% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. Chicken anti-HMGB2 antibody was used for HMGB proteins. For Ku70/Ku80 Western blots, monoclonals N3H10 and 111 were used (Neomarkers). The antibody 18-2 (Neomarkers) was used to detect DNA-PKcs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a 12/23 Rule Enforcing Activity—Our previous work demonstrated that RAG1 and RAG2 complemented with 293T WCE show strict 12/23 regulation in vitro (19). To facilitate the characterization of the regulatory activity found in the 293T WCE, we used cis-cleavage substrates (19) equally labeled at both ends (Fig. 1A). Cleavage at both RSSs yields 90- and 180-base pair radiolabeled DNA fragments from the 12/23, 12/12, and 23/23 substrates; single RSS cleavage events produce fragments of ~380 or 470 base pairs (Fig. 1A). The 12/23 rule predicts that only coupled cleavage products (cleavage at both RSSs) from the 12/23 substrate should be formed.



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  		FIG. 1.
cis-Cleavage assay contrasting effects of HMGB1, unfractionated, and fractionated 293T WCE. A, schematic of cis-cleavage substrates. 32P incorporation is indicated by the multiple asterisks on the 5'- and 3'-side of each substrate. 12RSSs and 23RSSs are indicated by open and filled triangles, respectively. B, 293T WCE confers 12/23 specificity. The major band at the top of each panel is the intact cleavage substrate. Single RSS cleavages, yielding 12 or 23 signal ends, are shown at the left of each panel. Hairpin coding ends are also denoted schematically to the left of each panel. Cryptic RSS cleavage is indicated by the large asterisks. C, two chromatographic fractions from the 293T WCE combined confer 12/23 RAG cleavage specificity. DNA substrates were incubated with RAG1/2, fractionated 293T WCE, 300 mM KCl, and 600 mM KCl High S column fractions. The positions of cleavage substrate and products are the same as described for B.

 
Copurified GST-core-RAG1 and GST-core-RAG2 (RAG1/2) were able to cleave all substrates (Fig. 1B, lanes 1 and 2). Purified recombinant HMGB1 added to the reaction preferentially enhanced RAG-mediated cleavage of substrates possessing a 23RSS (Fig. 1B, lanes 5 and 6). Since the combination of RAG1/2 and HMGB1 still produced high levels of cleavage of 12/12 and 23/23 substrates, this protein combination was evidently not sufficient to enforce the 12/23 rule (Fig. 1B, lanes 5 and 6). In contrast, the addition of 293T WCE to the reaction gave only coupled cleavage products from the 12/23 substrate and suppressed cleavage of 12/12 and 23/23 substrates in this system (Fig. 1B, lanes 3 and 4). Thus, 293T WCE possesses an activity that promotes 12/23-regulated cleavage, as previously described (19).

To identify the factors involved, we generated WCE from a 100-liter 293T cell culture. We then fractionated the WCE using a High Q column as a negative chromatography step. The flow-through from this column was applied to a High S column at 100 mM KCl and step-eluted at 300, 600, and 1000 mM KCl. This resolved two regulatory activities (Fig. 1C, lanes 2 and 3). The 300 mM High S fraction (S300) was inhibitory, decreasing RAG-mediated cleavage of all substrates (Fig. 1C, lane 2). In contrast, the 600 mM High S fraction (S600) had a cleavage-enhancing activity similar to that of HMGB1 (compare Fig. 1B, lanes 5 and 6, with Fig. 1C, lane 3). 12/23-regulated cleavage was reconstituted only when the S300 and S600 fractions were combined, producing a marked 12/23 RSS preference for cleavage (Fig. 1C, lane 4). Thus, inhibitory and enhancing components of the regulatory activity found in the WCE could be separated by High S column chromatography.

Purification and Characterization of the Cleavage-enhancing and -inhibitory Activities—We purified the cleavage-enhancing activity present in the S600 pool by fractionation as depicted in Supplementary Fig. 1. Mass spectrometry identified the protein as human HMGB2 (data not shown). This protein reacted with anti-HMGB2 antibody on Western blots but migrated faster than native HMGB1 or HMGB2 from WCE (Supplementary Fig. 1C). Its faster electrophoretic mobility and binding characteristics on DNA-cellulose suggested that the form of HMGB2 purified is lacking its acidic carboxyl tail (22). Removal of HMGB acidic tail allows for much higher DNA binding and bending characteristics (23). Therefore, cleavage assays were performed to directly compare the HMGB species found in the DNA-cellulose fraction with full-length HMGB1 (Supplementary Fig. 1D). In the presence of the S300 fraction, HMGB1 conferred 12/23 specificity to the RAGs, yet 1.5 µg of HMGB1 were required to compare with the activity found in 10 ng of the DNA-cellulose cleavage enhancing fraction (Supplementary Fig. 1D). We conclude that the cleavage-enhancing activity found in the purified S600 fraction is due to the presence of this fast migrating form of HMGB2 and that full-length HMGB1 could replace the purified activity.

We initially attempted to purify the inhibitory activity found in the S300 fraction by column chromatography, but after two subsequent columns the activity was lost (data not shown). Further attempts to purify the regulatory activity failed. As an alternative strategy to identify and characterize the activity present in the S300 fraction, we assayed for proteins known to participate in the DNA repair stage of V(D)J recombination. Western blot analysis demonstrated that the S300 fraction contained Ku70/Ku80, DNA-PKcs, and XRCC4 proteins (Fig. 2A and data not shown). Inhibitory activities of Ku70/Ku80 toward transcription factors have been described (24). To determine whether Ku could be responsible for the cleavage-inhibitory activity of the S300 fraction, we immunodepleted Ku70/Ku80 from the S300 fraction using a monoclonal antibody that exclusively recognizes the native heterodimer (Fig. 2B). Two sequential depletions left only residual Ku proteins in the supernatant, which could not be further depleted (Fig. 2B). An isotype control depletion, using anti-keratin antibodies, retained all Ku70/Ku80 heterodimer in the supernatant (Fig. 2B). The Ku70/Ku80-depleted and control-depleted S300 fractions were then tested in the cleavage assay. Control-depleted S300 fractions were unchanged for activity, inhibiting RAG-mediated cleavage of substrates in the absence of HMGB1 and conferring 12/23 regulation in the presence of HMGB1, demonstrating that the depletion protocol itself does not considerably affect the inhibitory activity (Fig. 2C, lanes 10-13). In contrast, anti-Ku heterodimer depletion removed most of the inhibitory activity from the S300 fraction (Fig. 2C, lanes 2 and 3 versus lanes 6 and 7). When HMGB1 and Ku-depleted S300 were present in the reaction, regulated cleavage activity was lost, with high levels of cleavage of 12/12 and 23/23 substrates (Fig. 2C, lanes 8 and 9). We conclude that Ku70/Ku80 is required for the activity found in the S300 fraction.



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  		FIG. 2.
Ku70/Ku80 heterodimer is a factor contributing to the S300 cleavage-regulating activity. A, SDS-PAGE/Western blot analysis of the S300 and S600 fractions for components of the DNA-dependent protein kinase. Upper panel, anti-Ku Western blot; lower panel, anti-DNA-PKcs Western blot. B, immunodepletion of Ku70/Ku80 heterodimer from the S300 fraction. The S300 was sequentially depleted of Ku or keratin in total four times. The supernatants and beads were then analyzed by SDS-PAGE-Ku70/Ku80 Western blot. The upper panels show supernatants from the Ku- and keratin-depleted S300. Beads for the Ku- and keratin-depleted S300 fractions are shown in the lower panels. C, Ku70/80- or keratin-depleted samples were incubated with RAG1/2, in the presence or absence of HMGB, as indicated. Substrates and coding ends are schematically indicated at the left.

 
Reconstitution of the 12/23 Rule with Purified Components—We then addressed whether Ku70/Ku80 is exclusively responsible for the S300 activity. Immunodepletions may have codepleted other factors complexed with Ku70/Ku80, such as DNA-PKcs. We purified Ku70/Ku80 heterodimer and DNA-PKcs in order to reconstitute the reaction (Supplementary Fig. 2). Cleavage assays were performed with combinations of purified RAG1/2, HMGB1, Ku70/80, and DNA-PKcs, whereas protein combinations including the S300 fraction served as controls (Fig. 3). The addition of DNA-PKcs to RAG1/2 or to RAG1/2 with HMGB1 had little effect on the cleavage reaction (Fig. 3). The addition of Ku70/Ku80 to RAG1/2 or to RAG1/2 with HMGB1 inhibited cleavage of all substrates (Fig. 3). This result was identical to the effect of the S300 fraction (Fig. 3, lanes 5 and 6). Although purified Ku heterodimer could inhibit cleavage of the substrates, it could not be used in combination with HMGB1 to regain 12/23 specificity, demonstrating that purified Ku70/Ku80 was not sufficient to replace the activity found in the S300 fraction (Fig. 3, compare control lanes 7 and 8 with lanes 25-28). Thus, neither DNA-PKcs alone nor Ku70/Ku80 alone is sufficient to reconstitute 12/23-regulated cleavage in a reaction mixture containing RAG1/2 and HMGB1. In contrast, when both Ku70/Ku80 and DNA-PKcs were added to a reaction containing RAG1/2 and HMGB1, we observed high levels of cleavage of the 12/23 substrate and significant inhibition of cleavage of 12/12 and 23/23 substrates (Fig. 3, lanes 29-32). This combination of proteins achieved the same effect on RAG-mediated cleavage seen with the combination of S300 and HMGB1 (Fig. 3, lanes 7 and 8). Interestingly, increasing amounts of Ku70/Ku80 within this protein combination did not inhibit the high levels of 12/23-regulated cleavage that were observed (Fig. 3, lanes 29-32). In a reaction containing DNA-PKcs, Ku70/Ku80, and RAG1/2, but lacking HMGB1, regulated cleavage was not observed. Instead, inhibition of cleavage of all substrates was seen (Fig. 3, lane 33). In conclusion, Ku70/80 and DNA-PKcs, when used in combination with RAG1/2 and HMGB1, increase 12/23 specificity by preferentially inhibiting 12/12 and 23/23 cleavage.



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  		FIG. 3.
Purified components of the DNA dependent protein kinase can replace the cleavage-regulating activity found in the S300 fraction. Shown is a cis-cleavage assay examining the effects of purified Ku70/Ku80 and DNA-PKcs on RAG-mediated cleavage. Four-point titrations of DNA-PKcs, Ku70/Ku80, or DNA-PKcs plus Ku70/Ku80 were incubated with HMGB1 and RAG1/2 on the three cleavage substrates, as indicated. Substrates and coding ends are schematically indicated at the left.

 
In Vitro Cleavage Activity and 12/23 Rule of Thioredoxin-RAGs—Since GST dimerization may create nonfunctional RAG complexes that could interfere in our assays, we wanted to investigate whether the protein requirement for the 12/23 rule was independent of the tag used. For this, we generated Trx-core-RAG-expressing vectors. Thioredoxin was chosen, since it has been shown to increase the solubility of the fusion protein upon extraction from cells, similar to GST, yet does not dimerize. We also added the FLAG tag to the N terminus of Trx-core-RAG1 and the HA tag to the N terminus of Trx-core-RAG2. This protein combination was active in in vivo recombination assays (data not shown). Since the FLAG tag was present only in the RAG1 fusion, we took advantage of the FLAG purification system to purify the FLAG-Trx-core-RAG1·HA-Trx-core-RAG2 (Trx-RAGs) complex. As seen in Supplementary Fig. 3A, we successfully purified the Trx-RAG complex from 293T cells. This protein preparation has three major advantages over the GST-RAGs preparation. First, the RAG1-RAG2 complex is enriched, since RAG2 is purified based on its ability to interact with RAG1 and not with the matrix. Second, the possibility of having RAG1-RAG2 complexes through GST dimerization is eliminated; and third, the presence of degradation products was reduced (Supplementary Fig. 3A and data not shown).

We next tested the ability of Trx-RAGs to cleave DNA. To increase the sensitivity of the assay as well as to allow detection of the SE product (direct measurement of cleavage at both RSSs), we radiolabeled the linear DNA fragment by PCR as reported (16). This substrate was incubated with either GST-RAGs or Trx-RAGs. Trx-RAGs alone were very inefficient in cleaving DNA, yet when HMGB1 was added to the reaction, Trx-RAGs showed a higher DNA cleavage activity compared with GST-RAGs (Supplementary Fig. 3). Under these conditions, we asked whether Trx-RAGs maintain the 12/23 rule in vitro. For that, we incubated Trx-RAGs with 12/23, 12/12, and 23/23 substrates and quantitated the SE product. As observed in Fig. 4A, at 100 ng of HMGB1, Trx-RAGs cleave efficiently only the 12/23 substrate, although cleavage of 12/12 and 23/23 was also observed. Comparing the levels of SE product, we see a 74- and a 185-fold preference over 12/12 and 23/23 substrates, respectively (Fig. 4A). At higher amounts of HMGB1 (1500 ng), we clearly see an increase of DNA cleavage in all substrates. However, the increase of SE product was higher with 12/12 and 23/23 substrates, resulting in a very significant decrease in 12/23 specificity. The -fold preference over 12/12 and 23/23 substrates was 15 and 26, reflecting a decrease of 5 and 7 times in 12/23 regulation, respectively (Fig. 4A). GST-RAGs were less efficient in maintaining the 12/23 rule in the presence of 100 ng of HMGB1, with a -fold preference over 12/12 and 23/23 of 5.1 and 8.8, respectively. However, 12/23 specificity was not significantly affected by increasing the HMGB1 concentration (Fig. 4A). Overall, Trx-RAGs show higher activity in cleavage and 12/23 regulation when they are compared with GST-RAGs in the presence of HMGB1 (Fig. 4A).



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  		FIG. 4.
12/23 rule of thioredoxin-RAGs and HMGB1 effect in different substrates. A, cleavage assay using 12/23, 12/12, and 23/23 linear substrates. Linear DNA substrates were incubated with either GST-RAG or Trx-RAG proteins in the absence or presence of HMGB1 (100 or 1500 ng). Cleavage products as well as substrate fragments are indicated on the left. Bottom, percentages of SE product are shown. Bracket, slow migrating bands obtained upon 23/23 cleavage. The right panel represents the -fold preference obtained for GST- as well as Trx-RAG proteins in the presence of 100 and 1500 ng of HMGB1. The Trx-RAG/GST-RAG ratio is also shown. B, Same as in A, except that the amounts of HMGB1 used were 50, 150, and 500 ng. Bottom, graphs representing the 12/23 preference with respect to 12/12 and 23/23 substrates at different HMGB1 amounts.

 
The 12/12 and 23/23 substrates analyzed in the experiments described above have identical RSSs. To extent our observations to other 12/12 and 23/23 substrates with nonidentical RSSs, we generated two plasmid substrates containing two 12 or two 23 RSSs that differ in the spacer sequence. We chose 5' DFL16.1 12RSS to replace one of the RSS in pJH290 12/12 and VH81x 23RSS to change one of the RSS in pJH290 23/23. These RSSs have been shown to be functional in in vivo recombination assays (25). Thus, these two new plasmids were used to generate the corresponding linear DNA substrates (12/12B and 23/23B) (Fig. 4B). All substrates were incubated with Trx-RAGs and increasing concentrations of HMGB1. The level and type of cleavage were similar to the other 12/12 and 23/23 substrates at all concentrations of HMGB1, with uncoupled and coupled cleavage being increased when high amounts of HMGB1 were used (Fig. 4, A and B). This HMGB1 effect was also observed in our assays using GST-RAGs (Figs. 1, 2, 3). Interestingly, we observed the appearance of slower migrating bands only with both 23/23 substrates (Fig 4, A and B). These bands (indicated by a bracket), which migrate above the substrate, may represent transposition products and are currently being investigated. In terms of -fold preference, however, we see a detectable decrease in 12/23 regulation when there is an increase in the amount of HMGB1 in the reaction (except when 12/23 preference is compared with the substrate 12/12B). In summary, the Trx-RAG complex is able to maintain the 12/23 rule in vitro efficiently in the presence of a low concentration of HMGB1. However, when higher concentrations of HMGB1 are present in the reaction, 12/23 regulation is significantly decreased.

Effects of Ku and DNA-PKcs on RAG-mediated Cleavage of Linear and Circular DNA Substrates—Since we found that Ku70/80 in combination with DNA-PKcs preferentially inhibit 12/12 and 23/23 RAG-mediated cleavage when GST-RAGs and 1.5 µg of HMGB1 are used, we extended our analyses to reactions containing Trx-RAGs and different amounts of HMGB1. As found in our previous assays with GST-RAGs, Ku70/80 alone is a potent inhibitor of RAG-mediated cleavage, independent of HMGB1 concentration or the linear substrate utilized (Fig. 5, A and B). DNA-PKcs alone had a mild inhibitory activity only in reactions containing 12/12 or 23/23 substrates (Fig. 5, A and B). However when DNA-PKcs was used in combination with Ku70/80, there was a high inhibition of RAG-mediated cleavage in reactions containing 50 ng of HMGB1 and any type of DNA substrate (Fig. 5A). In reactions containing 12/23 substrate, SE product was reduced 94 and 92.5% when 100 and 400 ng, respectively, of each DNA-PKcs and Ku70/80 were added to the RAGs/HMGB combination. However, when the concentration of HMGB1 was increased 10 times (500 ng), there was a preferential inhibition of 12/12 and 23/23 RAG-mediated cleavage in the presence of Ku70/80 and DNA-PKcs (Fig. 5, A and B). For example, when 100 ng of each DNA-PKcs and Ku70/80 were added to reactions containing 500 ng of HMGB1, SE product was reduced 47% in 12/23 substrate-containing reactions, in contrast to the 94% observed in the presence of 50 ng HMGB1. In reactions containing 12/12 or 23/23 substrate and 500 ng of HMGB1, SE product was reduced 98.6% (12/12), 94.6% (12/12B), or 100% (both 23/23s). These results indicate that DNA-PKcs in conjunction with Ku70/80 inhibits RAG-mediated cleavage at low concentration of HMGB1. In contrast, in reactions containing 10-times more HMGB1, there is preferential inhibition of 12/12 and 23/23 RAG-mediated cleavage. Therefore, a clear increase in the 12/23 rule is observed when DNA-PKcs and Ku70/80 are present in the cleavage reaction. This increase in 12/23 regulation was also observed in our previous assay using GST-RAG1/2 and high concentration of HMGB1 (Fig. 3).



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  		FIG. 5.
Effect of Ku70/80 and DNA-PKcs on Trx-RAG-mediated cleavage of linear DNA. A, linear DNA substrates were incubated with Trx-RAGs, in the absence or presence of HMGB1 (50 (top gel) or 500 ng (bottom gel)), Ku70/80, and DNA-PKcs. Bottom, percentage of SE product is shown. Substrate and cleavage products are represented to the left. Bracket, as in Fig. 4. B, graphs representing the percentage of SE product compared with the 100% obtained with RAG-HMGB1 combination. Top, boxes in gray scale represent different combinations of proteins.

 
It is known that Ku70/80 has a high affinity for DNA ends; therefore, to exclude the possibility that the increase in 12/23 regulation was an artifact due to binding and translocation of Ku70/80 into the DNA, we performed experiments with supercoiled circular DNA. The use of this type of substrate will eliminate the possibility that Ku70/80 modulates RAG-mediated cleavage by binding to DNA ends and possibly activating DNA-PKcs kinase activity or preventing RAG binding and cleavage. Therefore, we analyzed RAG-mediated cleavage using pDS 12/23, pDS 12/12, and pDS 23/23 plasmid substrates, which contain the same substrate sequence as the ones present in the linear DNA. Fig. 6A shows that Trx-RAGs inefficiently cleave 12/23 as well as 12/12 and 23/23 substrates. Titration of HMGB1 results in a clear 12/23 preference, with 50 ng of HMGB1 being enough to obtain this preference. Higher amounts of HMGB1 resulted in a slight increase on 12/23 and 12/12 cleavage, but a considerable increase in 23/23 cleavage was observed (Fig. 6A). When Ku70/80 and DNA-PKcs (100 or 400 ng each) were added to the reaction, we observed an inhibition of RAG-mediated cleavage in all three substrates. This inhibition was overcome with increasing concentration of HMGB1 in 12/23 substrates but not in 12/12 or 23/23 substrates. Therefore, in conditions of high amounts of HMGB1, Ku70/80 and DNA-PKcs increase the preference for a 12/23 substrate by preferentially inhibiting 12/12 and 23/23 cleavage (Fig. 6B).



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  		FIG. 6.
Effect of Ku70/80 and DNA-PKcs on RAG-mediated cleavage of circular supercoiled DNA. A, pDS 12/23, 12/12, and 23/23 plasmid DNA substrates were incubated with Trx-RAGs in the absence or presence of HMGB1 and Ku70/80 plus DNA-PKcs. Cleavage products as well as substrate fragments are indicated on the left. The arrowhead corresponds to undigested supercoiled DNA. The arrow corresponds to an uncharacterized product of 23/23 cleavage. Bottom, -fold increase of SE product (over RAGs alone). B, percentage of SE product with respect to RAG-HMGB1 sample. For 23/23 substrate, the two bands migrating at the position of the SE product were quantitated.

 
To determine the individual contribution of Ku70/80 and DNA-PKcs on the regulation of RAG-mediated cleavage of circular DNA, we analyzed their effect using two concentrations of HMGB1. Cleavage of all DNA substrates by Trx-RAGs alone was very inefficient and does not show 12/23 preference, yet we observed no supercoiled DNA after the incubation (Fig. 7A, arrowhead). When 50 ng of HMGB1 were added to the reaction, a clear preference (~20-fold) for a 12/23 substrate is observed. However, this 12/23 preference is lower when 500 ng of HMGB1 are used (Fig. 7C). In contrast to the cleavage inhibition observed when Ku70/80 was added to reactions containing linear substrates (Figs. 3 and 5), we found no inhibition of RAG-mediated cleavage of circular DNA substrates (Fig. 7, A and B). On the other hand, we observed a mild inhibition of 12/23 and 23/23 DNA cleavage when DNA-PKcs is added to reactions containing RAGs and HMGB1. When both Ku70/80 and DNA-PKcs are combined with RAGs and 50 ng of HMGB1, a strong inhibition of 12/23, 12/12, and 23/23 DNA cleavage (76, 50, and 60%, respectively) was observed (Fig. 7, A and B). However, when 500 ng of HMGB1 are used in the reactions this inhibition is only 9% with 12/23 DNA, whereas a 65 and 91% inhibition is still observed with 12/12 and 23/23 DNA cleavage, respectively (Fig. 7, A and B). Therefore, under these conditions (500 ng of HMGB1 plus Ku70/80 and DNA-PKcs), we observed an increase in the preference for cleavage of a 12/23 DNA substrate by inhibiting preferentially 12/12 and 23/23 DNA cleavage (Fig. 7, B and C). Furthermore, under the same conditions, for the 12/23 substrate we observed only a delay in the cleavage of DNA when Ku70/80 and DNA-PKcs are present in the reaction, but no obvious difference in the pattern of cleavage is observed (Supplementary Fig. 4B, right graph). However, at 50 ng of HMGB1, a dramatic decrease in the cleavage activity was observed throughout the incubation (Supplementary Fig. 4B).



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  		FIG. 7.
Requirement for both Ku70/80 and DNA-PKcs to modulate RAG-mediated cleavage of supercoiled circular DNA. A, pDS 12/23, 12/12, and 23/23 plasmids were incubated with Trx-RAGs in the absence or presence of HMGB1, Ku70/80, and DNA-PKcs. Substrate and cleavage products are indicated on the left. The arrow and arrowhead are as in Fig. 6. Bottom, -fold increase of SE product (over RAGs alone). B, percentage of SE product with respect to RAG-HMGB1 sample. For 23/23 substrate, the two bands migrating at the position of SE product were quantitated. C, 12/23 -fold preference with RAGs/HMGB1 (50 or 500 ng) in the absence and presence of Ku/DNA-PKcs. The values were obtained by using the -fold increase values obtained in A.

 
In conclusion, we observe a modulatory effect on RAG-mediated DNA cleavage by Ku70/80 and DNA-PKcs in reactions using either linear or circular DNA. Under conditions that favor efficient cleavage, the addition of Ku70/80 and DNA-PKcs enhances 12/23 regulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The 12/23 rule is observed at the cleavage stage of the recombination reaction, with a strong preference for cleaving a 12/23 rather than a 12/12 or 23/23 RSS pair in vivo (14). In vitro, 12/23 regulation was achieved using lymphoid cell extracts containing overexpressed core RAG proteins with a 25-fold cleavage preference for a 12/23 RSS pairing (16). In contrast, purified core RAG proteins showed only a slight 12/23 RSS cleavage preference (17). Together, these observations suggested that other lymphoid-specific or ubiquitous factors might play a role in 12/23-regulated cleavage by RAGs. We have shown that 12/23 RSS cleavage of purified RAGs could be restored by the addition of whole cell extracts from nonlymphoid cells. This indicated that ubiquitous factors were necessary for 12/23 rule regulation (19). In the present study, we describe the characterization of the ubiquitous factors and showed them to correspond to the NHEJ factors DNA-PKcs and Ku70/Ku80 as well as the previously characterized HMGB2 protein.

We have used linear and supercoiled circular double-stranded DNA cis-RSS cleavage substrates derived from the well characterized pJH290 recombination plasmid for this study and observe that DNA-PKcs and Ku70/Ku80, in the presence of HMGB, enhance the 12/23 rule. We observe specific effects of Ku70/Ku80 in combination with DNA-PKcs and HMGB on RAG-mediated cleavage. In our assays using linear DNA, Ku70/Ku80 alone could have inhibited RAG-mediated cleavage by binding to the ends of the linear double-stranded DNA cis-cleavage substrates and translocating inward to the RSSs, precluding RAGs from accessing their target sites. This idea is supported by the lack of inhibition of RAG-mediated cleavage by Ku70/80 when supercoiled circular DNA is used as substrate. On the other hand, regulated cleavage specificity was recovered with the addition of DNA-PKcs to a reaction containing HMGB, Ku70/Ku80, and RAGs, allowing for high amounts of coupled cleavage of the 12/23 substrate, while inhibiting cleavage of the 12/12 and 23/23 substrates. Although it is possible that Ku70/Ku80 and DNA-PKcs are recruited by the DNA ends of the linear cleavage substrate used in our study, this cannot discount the 12/23 RSS-specific effect, since all three substrates possess identical 5'- and 3'-ends. Furthermore, these results are supported by our experiments using supercoiled circular DNA, in which we observe a clear preferential inhibition of 12/12 and 23/23 cleavage when HMGB1 is present in higher concentrations, conditions at which we can observe higher cleavage. This preferential inhibition is consistently observed in all our assays, regardless of the fusion proteins and DNA substrates used in the analysis. With the complete protein reconstitution, only the 12/23 substrate is highly competent for cleavage, suggesting that there is a specific cleavage complex containing Ku70/80, DNA-PKcs, and HMGB in addition to the RAGs structured on the double-stranded DNA substrate.

In vivo evidence for a role of DNA-PKcs in 12/23 regulation comes from a study using SCID/+ or SCID/SCID pre-B cells. Lew et al. (26) have reported an increase of V{lambda}1/J{kappa}1/2 interlocus joints in the SCID/SCID background, which are produced from RSS pairings in violation of the 12/23 rule. It was concluded that recombination intermediates from the different chromosomes were joined in a nonspecific manner. In light of our observations and the high levels of 23/23 joining reported by Lew et al. (26), it is possible that the 12/23 rule is specifically being violated in these cell lines due to the mutation in DNA-PKcs. Further studies are required to elucidate the role of DNA-PKcs as well as Ku70/Ku80 in 12/23 regulation in vivo. These studies will require the use of very sensitive substrates to specifically investigate cleavage, since DNA ends generated from 12/12 or 23/23 cleavage are likely to be unstable and be very inefficient at generating 12/12 and 23/23 joining products. Our preliminary experiments using DNA-PKcs- and Ku-deficient cells have not yet detected these joining products (data not shown).

What is the role of HMGB in 12/23 regulation? Previous studies have shown that RAGs and HMGB1/HMGB2 are the only proteins required for 12/23 regulation in vitro (13, 15, 20, 21). All of these experiments used either trans-cleavage substrates (13, 21), in which the target RSSs are on separate double-stranded DNA oligonucleotides, or modified forms of cis-cleavage substrates in which the 12RSS and 23RSS were in the same double strand DNA molecule but with a nick or single-stranded DNA gap between the RSSs (15, 20). In addition, these substrates have inter-RSS spacing completely absent or considerably shorter than the minimal inter-RSS spacing found in typical in vivo recombination substrates (15, 20).

The in vivo role of HMGB proteins in V(D)J recombination is still an unresolved issue. Transfection experiments using mammalian cells and exogenous recombination substrates have shown that overexpression of either HMGB1 or HMGB2 results in an increase of signal joint formation (27). However, HMGB1-/- or HMGB2 -/- knockout mice show no obvious abnormalities in lymphocyte development (28, 29). This may be due to redundancy in the HMGB family, to the participation of a different HMGB-like protein, or simply to the fact that there exist other unknown mechanisms responsible for the formation of the postulated 12/23 synaptic complex in vivo. Our results suggest that in addition to HMGB1 (2), DNA repair factors Ku70/80 and DNA-PKcs may be important components of the precleavage complex.

What would be gained by having NHEJ factors as an obligate part of the precleavage complex? In contrast to double strand breaks caused by IR, V(D)J recombination is a specific and intentional genomic insult. The cleavage reaction is only beneficial if it is correctly targeted and ultimately repaired properly. The 12/23 rule is a critical level of regulation for the reaction and is an indication of correct synaptic complex formation. By having DNA-PKcs and Ku70/Ku80 as part of this correct complex, RAG-mediated breaks would occur in the genome only where the DNA-PK is already situated, so that no further recruitment is necessary to effect its DNA-damage response. In the absence of DNA-PKcs or Ku70/80, RSS breaks could still occur, as is seen in mice or cell lines deficient for components of the DNA-PK. These RSS breaks could possibly be deregulated, at single RSS or improperly paired RSS targets.

In NHEJ-deficient mice, p53-dependent apoptosis occurs in lymphocytes attempting to rearrange their antigen receptors. In a p53-deficient background, the absence of XRCC4, of ligase IV, or of any component of the DNA-PK can result in the development of B cell lymphomas (see Ref. 30 and references therein). Pro-B cell lymphomas possessing t(12;15) translocations from mice with a SCID p53-/- background are due to RAG activity, since generation of these lymphomas is suppressed by a RAG2 null mutation (31). Interestingly, p53-deficient mice competent for NHEJ do not develop these types of B cell lymphomas or these types of translocation events (32). This alteration in lymphoma class may indicate that translocations may not be solely due to deficient NHEJ repair after RAG-mediated breaks (30). We suggest that in addition to the repair defect in these NHEJ-deficient mice, deregulated V(D)J cleavage leading to formation of anomalous and/or unstable postcleavage complexes should be considered as a precipitating factor for translocation events. Future in vivo studies should be directed to test this hypothesis.

Based on the presence of RAG-mediated double strand breaks found in Ku70-, Ku80-, and DNA-PKcs-deficient mice and cell lines, these NHEJ factors have been proposed to participate only in the second step of V(D)J recombination, the DNA repair and joining phase (33). Our work assigns new roles for DNA-PKcs and Ku70/Ku80 in V(D)J recombination. We propose that the RAGs and HMGB assemble with DNA-PKcs and Ku70/80 and the target RSSs to form a complex at the cleavage stage of the V(D)J recombination reaction. DNA-PK, as an obligate part of the synaptic cleavage structure, could assist in preventing aberrant RAG-mediated cleavage events and would be preemptively situated at the sites of proper RAG-mediated cleavage, allowing for a rapid and directed response to the DNA lesions.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI38890 and AI45996-05 (to M. C. N. and P. C., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains four additional figures. Back

b Supported by Natural Sciences and Engineering Research Council of Canada (NSERC) PGS scholarships. Present address: Chromocell Corp., North Brunswick, NJ 08902. Back

c These authors contributed equally to this work. Back

i An Investigator of the Howard Hughes Medical Institute. Back

j Supported by a Cancer Research Institute Investigator award and a Leukemia and Lymphoma Society Scholar award. To whom correspondence should be addressed. Tel.: 212-659-9443; Fax: 212-849-2525; E-mail: patricia.cortes{at}mssm.edu.

1 The abbreviations used are: RSS, recombination signal sequence; NHEJ, nonhomologous end-joining; HMGB1 and -2, high mobility group protein 1 and 2, respectively; WCE, whole cell extract; GST, glutathione S-transferase; Trx, thioredoxin; HA, hemagglutinin; SE, signal end; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit. Back


   ACKNOWLEDGMENTS
 
D. J. S. is grateful to Drs. Eric Meffre, Eva Besmer, and Xiao-Feng Qin for valuable discussions and suggestions. We thank Yaping Yu for purification of DNA-PKcs and Ku70/Ku80 and Claudia Tapia for assistance with experiments.



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 ABSTRACT
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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