In Vitro Processing of the 3'-Overhanging DNA in the Postcleavage Complex Involved in V(D)J Joining

The postcleavage complex involved in V(D)J joining is knownto possess a transpositional strand transfer activity, whosephysiological role is yet to be clarified. Here we report thatRAG1 and RAG2 proteins in the signal end (SE) complex cleavethe 3'-overhanging structure of the synthetic coding-end (CE)DNA in two successive steps in vitro. The 3'-overhanging structureis attacked by the SE complex imprecisely, near the double-stranded/single-stranded(ds/ss) junction, and transferred to the SE. The transferredoverhang is then resolved and cleaved precisely at the ds/ssjunction, generating either the linear or the circular cleavageproducts. Thus, the blunt-end structure is restored for theSE and variably processed ends are generated for the syntheticCE. This 3'-processing activity is observed not only with thecore RAG2 but also with the full-length protein.

INTRODUCTION

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Introduction

V(D)J joining plays key roles in activating and diversifyingthe antigen receptor genes. In the initial phase of V(D)J joining,the protein products of recombination-activating genes (RAG1and RAG2) (39, 48) recognize the recombination signal sequences(RSS), each consisting of a conserved 7-mer (CACAGTG) and aconserved 9-mer (ACAAAAACC), separated by a spacer of constantlength of either 12 or 23 bp (6, 17, 35, 44, 45, 46, 52, 53).For the coordinate cleavage of RSSs, synaptic complex formationof the 12- and 23-RSSs is required (8, 18, 59). RSS DNA is cleavedby RAG proteins in two successive steps, nicking and hairpinformation (31, 58). A nick is first introduced at the codingand 7-mer border on the top strand. The resulting 3'-hydroxylgroup (3'-OH) then attacks the bottom strand to form a hairpinstructure at the coding end (CE) and a blunt end at the signalend (SE). After the cleavage of RSSs, the SEs stably stay withthe RAG proteins in vitro (2, 18, 21, 36). Physical associationof the CEs with the SE complex has been shown to occur in vitro(18, 56). This association in the presence of other repair proteinsappears to be necessary for the CE processing in vivo (18, 20,41, 56, 60).

In the joining phase of V(D)J recombination, CEs are processedand ligated to form a coding joint. Several factors are requiredin DNA end joining, including the Ku heterodimer (Ku70/80),the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs),Artemis, XRCC4, and DNA ligase IV (4, 11, 14, 15, 27, 28, 34,38, 42, 43, 54, 61). It has been shown that the Artemis/DNA-PKcscomplex opens the hairpin a few nucleotides from the tip, generatingthe 3'-overhanging structure (30, 50). After the hairpin opening,the CEs are modified by nucleotide deletions and additions forjunctional diversification (50). For nucleotide additions, terminaldeoxynucleotidyltransferase (TdT) has been reported to be responsiblefor the non-germ line encoded nucleotides (N) (13, 24). In contrast,the exact mechanism for the nucleotide deletions remains largelyunknown. The RAG proteins are known to mediate the strand transferof the SE to the hairpin CE or to the double-stranded (ds) DNA,resulting in the aberrant joining products in vitro (3, 19,26, 32). The 3' flap endonuclease activity was also reportedfor the RAG proteins and was suggested to be a candidate forthe CE processing activity (47). However, this activity wasSE independent and requires Mn²⁺.

In the present study, we examined the SE complex for the 3'-processingactivity using 3'-overhanging DNA. This is because the RAG proteinsremain bound to the SEs in vivo even after the RSS has beencleaved (2, 40). Here, we report that the RAG proteins in theSE complex can process and cleave the 3' overhang of the dsDNA in vitro in two successive steps.

MATERIALS AND METHODS

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Materials and Methods

Preparation of proteins. The glutathione S-transferase (GST)-tagged truncated RAG1 protein(amino acids 384 to 1040) was coexpressed either with the GST-taggedtruncated RAG2 protein or with the GST-tagged full-length RAG2protein (amino acids 1 to 383 or 1 to 527, respectively) inHEK-293T cells (56), purified with glutathione-agarose affinitychromatography (52), and dialyzed against 25 mM Tris-HCl (pH8.0)-2 mM dithiothreitol (DTT)-150 mM KCl-10% glycerol. Theproteins used in the experiments illustrated in Fig. 6 weredialyzed against a different buffer containing 25 mM Tris-HCl(pH 8.0), 2 mM DTT, 150 mM NaCl, and 20% glycerol. These twobuffers showed little difference in the 3'-processing activityof the RAG proteins. Porcine high-mobility-group 1 (HMG1) proteinwas prepared as described previously (1). The RSS cleavage activityof the full-length RAG2 was about 60% of that of the truncatedcore RAG2.

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FIG. 6. Full-length RAG2 has a 3'-processing activity comparable to that of the truncated RAG2. (A) Detection of the transposition activity. The reaction was performed with the labeled 23-SE and target plasmid as described in Materials and Methods. Transposed bands represent a two-ended insertion product schematically shown at the top. (B) Detection of the 3'-processing activity. The substrate DNA was labeled in the 3' overhang. The SE complex was reconstituted with the biotinylated 12-SE in the presence of 1 mM Mg²⁺ by using either the full-length RAG2 (FL) or the truncated RAG2 (core). The C-terminal region is missing in the core RAG2. The target or substrate DNA was allowed to react with the bead-isolated SE complex in the buffer containing 3 mM Mg²⁺ and 75 mM K⁺.

DNA substrates. Oligonucleotides were synthesized and purified with high-pressureliquid chromatography and/or by electrophoresis in a denaturingpolyacrylamide gel. The strands and their sequences were asfollows, with 7-mer and 9-mer signal sequences underlined: 12-SEtop strand (49-mer), 5'-CACAGTGCTCCAGGGCTGAACAAAAACCTCCTAGGGTTGCAGCTGACTC-3';23-SE top strand (60-mer), 5'-CACAGTGGTAGTACTCCACTGTCTGGGTGTACAAAAACCTCCTAGGGTTGCCATGGACTC-3';top strand of the 12-ST, 5'-CACAGTGCTCCAGGGCTGAACAAAAACCTCCTAGGGTTGCAGCTGACT-3';bottom strand of the 12-ST, 5'-AGTCAGCTGCAACCCTAGGAGGTTTTTGTTCAGCCCTGGAGCACTGTGGACCTAATAC-3';59-nucleotide (nt) top strand of the ds DNA, 5'-CTTAATACGACTCACTATAGGGCTATGTACTACCCGAACCACCAACCTAATACGACGAA-3'.Oligonucleotide substrates were 5' end labeled with [ ${gamma}$ -³²P]ATP(Amersham Biosciences) by using T4 polynucleotide kinase (NewEngland Biolabs) or 3' end labeled by annealing with appropriatecomplementary oligonucleotide and filling in 1 nt with [ ${alpha}$ -³²P]dGTP or [ ${alpha}$ -³²P]dCTP (Amersham Biosciences) by using a Klenowfragment (3' exo^–; New England Biolabs). To prepare the3'-dideoxy oligonucleotide, the oligonucleotide was extendedwith ddCTP or ddGTP (Roche Diagnostics) by using terminal deoxyribonucleotidetransferase (Roche Diagnostics). An internally labeled strandwas prepared by ligating the 5'-³²P-labeled 3' half (5'-GAGTCAGCTGCAACCCTAGGAGGTTTTTGTTCAGCCCTGGAGCACTGTG-3')to the unlabeled 5' half (5'-GACCTAATAC-3') in the presenceof the complementary strand by using T4 DNA ligase (New EnglandBiolabs). The labeled oligonucleotide was purified from a denaturingpolyacrylamide gel and reannealed to the indicated complementarystrand. Annealed DNA was further purified by electrophoresisin an 8% polyacrylamide gel, eluted from gel slices with anelution buffer (0.2 M NaCl, 1 mM EDTA, and 20 mM Tris-HCl [pH7.5]), and purified with reversed-phase column chromatography(Elutip-d; Schleicher & Schuell).

3'-Processing reactions. To examine the SE requirements, 12-SE DNA (8 nM) and 23-SE DNA(8 nM) were incubated with RAG1 (10 µg/ml), RAG2 (10 µg/ml),and HMG1 (8 µg/ml) proteins at 37°C for 120 min inbinding buffer (25 mM MOPS [morpholinepropanesulfonic acid]-KOH[pH 7.0], 5 mM Tris-HCl [pH 8.0], 2.4 mM DTT, 90 mM potassiumacetate, 30 mM KCl, 0.1 mg of bovine serum albumin [BSA]/ml,and 2% glycerol) containing 10 mM MgCl₂. The labeled ds DNA(200 cpm/µl) was added and incubated at 37°C for 60min. In all other experiments, the unlabeled or labeled 12-SEDNA (20 nM or 200 cpm/µl, respectively) and its partnerbiotinylated 23-SE DNA (20 or 8 nM, respectively) were incubatedwith RAG1 (10 µg/ml), RAG2 (10 µg/ml), and HMG1(8 µg/ml) proteins at 37°C for 100 min in 20 µlof the binding buffer containing 10 mM CaCl₂. Streptavidin-coatedmagnetic beads (Dynabeads M-280; 10 µg/µl) wereadded, and the reaction mixture was incubated at 37°C for20 min. After incubation, magnetic beads and supernatant wereseparated by use of a magnet stand. The beads were washed fourtimes at room temperature with 30 µl of binding buffercontaining 10 mM CaCl₂ and resuspended in the binding buffercontaining Mg²⁺ (10 mM if not otherwise specified). The labeledor unlabeled substrate ds DNA (200 cpm/µl or 5 nM, respectively)was added to the SE complex and incubated at 37°C. To examinethe effect of the Mg²⁺ concentration, the SE complex was reconstitutedand isolated with 1 mM CaCl₂ instead of 10 mM CaCl₂. DNA wasextracted from the reaction mixture by using phenol-chloroform-isoamylalcohol (25:24:1), precipitated with ethanol, washed with 70%ethanol, dissolved in formamide dye mix, and electrophoresedin a denaturing polyacrylamide gel.

Transposition. The SE complex was reconstituted by using the biotinylated 12-SEDNA (16 nM) and the labeled 23-SE DNA (12 nM) in a buffer containing1 mM MgCl₂, 25 mM MOPS-KOH (pH 7.0), 5 mM Tris-HCl (pH 8.0),2.8 mM DTT, 60 mM potassium acetate, 60 mM NaCl, 1 mg of BSA/ml,and 8% glycerol. The bead-bound SE complex was washed threetimes with wash buffer (25 mM MOPS-KOH [pH 7.0], 2.4 mM DTT,90 mM potassium acetate, 210 mM KCl, 1 mg of BSA/ml, and 2%glycerol) and once with a Tn buffer (25 mM MOPS-KOH [pH 7.0],2.4 mM DTT, 45 mM potassium acetate, 30 mM KCl, 0.1 mg of BSA/ml,and 2% glycerol). The amount of the isolated SE complex wasestimated by a count of the ³²P incorporated into the complex.The target plasmid (pBluescript II SK [Stratagene]) (25 ng/µl)was incubated with the SE complex (0.1 nM) at 37°C in theTn buffer containing 3 mM MgCl₂. After the transposition reaction,sodium dodecyl sulfate (0.5%) and proteinase K (1 mg/ml) (RocheDiagnostics) were added and incubated at 55°C for 60 min.DNA was extracted from the reaction mixture by using phenol-chloroform-isoamylalcohol (25:24:1), precipitated with ethanol, resuspended ina Tris-EDTA buffer, electrophoresed in a 1% Tris-acetate-EDTAagarose gel, transferred to a nylon membrane filter, and autoradiographed.The SE complex was reconstituted with the unlabeled 23-SE DNAin parallel and assayed for the 3'-processing activity in theTn buffer containing 3 mM MgCl₂.

Isolation of the complexes containing ST intermediates. After the 3'-processing reaction, the complex was reisolatedwith magnetic beads from the reaction mixture and washed oncewith the wash buffer containing 1 mM CaCl₂ and four times withthe Ca²⁺-free wash buffer. The isolated complex was resuspendedin the binding buffer containing 10 mM MgCl₂.

Enzymatic treatments of reaction products. For the calf intestine alkaline phosphatase (CIP) treatment,DNA extracted from the reaction mixture was incubated at 37°Cfor 30 min with 0.5 U of CIP (Roche Diagnostics) in the manufacturer'sbuffer. For the T4 polynucleotide kinase treatment, DNA wasincubated with 2 U of polynucleotide kinase in the manufacturer'sbuffer at 37°C for 30 min. For restriction enzyme treatment,DNA was incubated at 37°C for 120 min with 1 U of PvuIIor BsrGI (TaKaRa Biochemicals) in the manufacturer's buffer.For the exonuclease V (exoV) treatment, DNA was incubated at37°C for 120 min with 2 U of exoV (Amersham Biosciences)in 67 mM glycine-NaOH (pH 9.4)-10 mM ß-mercaptoethanol-6.7mM MgCl₂.

Purification and endonuclease treatment of each cleavage product. Each cleavage product was separated by electrophoresis in a15% denaturing polyacrylamide gel and eluted from the gel slicewith the elution buffer. DNA was precipitated with ethanol,washed with 70% ethanol, dissolved in a Tris-EDTA buffer, andtreated at 25°C for 10 min with mung bean nuclease (3.56,10.7, or 32 U/ml) (TaKaRa Biochemicals) in 50 mM Tris-HCl (pH5.2)-30 mM NaCl-10 mM ZnSO₄. DNA was extracted by phenol-chloroform-isoamylalcohol (25:24:1), concentrated by 1-butanol extraction, dissolvedin formamide dye mix, and electrophoresed in a 16.5% denaturingpolyacrylamide gel.

RESULTS

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Results

The SE complex processes the 3'-overhanging structure. To examine the CE processing activity of the SE complex, thecomplex was reconstituted with 12-SE, 23-SE, and RAG1/RAG2 proteinsin the presence of HMG1, and incubated with various ds DNA sampleslabeled at the 5' end of the top strand. As shown in Fig. 1A,ds DNA with the 3' overhang (4 nt or longer) was cleaved bythe SE complex in the presence of Mg²⁺. No cleavage was foundto occur near the blunt end or the recessed 3' end of ds DNA.In the ds DNA with a 15-nt 3' overhang, three major cleavagesites were detected in the ss DNA region, at or near the ds/ssjunction (Fig. 1B). The reaction was much reduced when eitherthe 12- or the 23-SE was omitted, and it was totally abolishedwithout the SEs (Fig. 1B). In the ds DNA region, cleavages wereweakly detectable, even in the absence of the SEs, at or nearthe ds/ss junction. These cleavages were probably due to the3' flap endonuclease activity, previously reported for the RAGproteins (47), because the reaction was enhanced by Mn²⁺ anddid not require the presence of SEs (Fig. 1B). We also examinedthe 3' CE processing by using uncleaved RSSs. However, sinceour reaction did not include repair factors such as Artemis,the 3'-overhanging CEs were not generated and unprocessed hairpinsaccumulated (data not shown).

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FIG. 1. 3'-Processing activity of the SE complex. (A) The SE complex cleaves the 3'-overhanging ds DNA near the ds/ss junction. Five ds DNAs with different end structures were labeled with ³²P (indicated by the asterisk) at the 5' end of the 59-nt top strand. The SE complex was reconstituted with HMG1, copurified RAG1/RAG2 proteins, and the 12-SE and biotinylated 23-SE DNAs; immobilized on the streptavidin-coated magnetic beads; and isolated by using a magnet stand (see Materials and Methods for details). The labeled substrates were incubated with the isolated SE complex in the Mg²⁺-containing buffer (see Materials and Methods) at 37°C for 60 min. DNA was extracted, electrophoresed in a 10% denaturing polyacrylamide gel, and autoradiographed. The 3'-overhanging ds DNA was cleaved near the ds/ss junction, while no cleavage was seen for the 5'-overhanging or the blunt-ended ds DNA. (B) The 3'-processing activity requires the 12- and 23-SEs. The SE complex was assembled with HMG1, RAG1/RAG2 proteins, and the 12-SE and 23-SE DNAs (complete) in the Mg²⁺-containing buffer (see Materials and Methods for details). The ds DNA with a 15-nt 3' overhang (labeled with ³²P at the 5' end of the top strand) was allowed to react with the SE complex in the presence of 10 mM Mg²⁺ at 37°C for 60 min. Omission (w/o) of either the 12- or 23-SE greatly reduced the cleavage of the substrate ( ${blacktriangledown}$ ). When Mg²⁺ (10 mM) was replaced with Mn²⁺ (2.5 mM) in the absence of the SEs, the 3' flap endonucleolytic cleavage ( ${triangledown}$ ) previously reported for the RAG proteins was enhanced.

The cleaved 3' overhang is transferred to the SE. To study the fate of the cleaved 3' overhang, the 3'-terminalnucleotide was labeled in the overhang (Fig. 2A) and the labeledds DNA was incubated in the presence of Mg²⁺ (Fig. 2B). Threetypes of reaction products were detected: one (Z) was a slowmigrating species, and two (X and Y) were fast migrating speciescompared with the uncleaved substrate (Fig. 2A). Since noneof these species were detected when the 5' end of the top strandwas labeled, they should represent the reaction products cleavedfrom the 3' overhang (data not shown). Formation of speciesX, Y, and Z was totally abolished when either RAG1 or RAG2 wasomitted (Fig. 2A). It should be mentioned that the SE complexcontaining the mutant RAG1 (D708A) (12, 23) failed to generateany of these reaction products (Fig. 2A, right). The substratewas efficiently cleaved at higher Mg²⁺ concentrations, e.g.,10 mM (Fig. 2B). With Ca²⁺, the subspecies of Z that are a fewnucleotides longer than those generated with Mg²⁺ were foundafter prolonged incubation (>2 h) (data not shown). At allMg²⁺ concentrations tested, species Z appeared first, peakedat an early time, and then slowly diminished, while speciesX and Y appeared later and continued to accumulate with time.The three bands of species Y, migrating at 13, 14, and 15 nt(designated Y1, Y2, and Y3, respectively), were the cleaved3'-overhanging structure. The faster-migrating species X wasassumed to be the circular form of the species Y, because itwas resistant to the exonucleolytic cleavage, as will be discussedlater. The third species, Z1 and Z2, each consisting of a clusterof three bands, migrated slower than the 12- and 23-SEs. Sincethe species Z appeared transiently during the cleavage, we assumedthese bands to be the reaction intermediates. To study the structuralnature of the species Z and its possible relationship to theSEs, we labeled the 12-SE at the 3'-terminal nucleotide of thebottom strand and reconstituted the SE complex. The complexwas incubated with the unlabeled 3'-overhanging ds DNA in theMg²⁺-containing buffer. After the reaction, DNA was extracted,electrophoresed, and autoradiographed. On the autoradiogram,slower migrating bands newly appeared behind the 12-SE, whosemobilities were identical to that of the Z1 band (Fig. 3A).These new bands, as well as the Z1 band, were cleaved into threesmaller fragments of 55, 56, and 57 nt by PvuII digestion. Sincethe 12-SE DNA contained a PvuII cleavage site 42 nt downstreamfrom the signal end, we assumed that the Z1 bands were generatedby the transfer of the 3' overhang from the ds DNA to the 12-SE(Fig. 3A). Similar results were obtained for Z2 by using the3'-labeled 23-SE (data not shown). We assume that transfer ofthe 3' overhang to the SE is due to the attack by the SE onthe 3' overhang. This is because the experiments with the 3'-dideoxySE revealed that the 3'-OH group of the 12-SE is required forthe generation of 12-STs (Z1), but not 23-STs (Z2), and viceversa (Fig. 2A). We also found that formation of the cleavageproducts Xs and Ys requires the 3'-OH of the 12- or 23-SE (Fig.2A). Henceforth, the strand transfer intermediate (Z) will bereferred to as ST DNA.

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FIG. 2. Cleavage of the 3'-overhanging DNA. (A) The substrate ds DNA was labeled with ³²P at the 3'-terminal nucleotide in the overhang and incubated with the SE complex in the presence of Mg²⁺ at 37°C as described for Fig. 1B. After the reaction, DNA was extracted, electrophoresed, and autoradiographed. Two sets of faster migrating products (X and Y) were detected. Behind the substrate band, two sets of faint bands (Z1 and Z2) appeared. (Left panel) When the 3'-terminal nucleotide of either the 12- or 23-SE, deoxyguanidine, was replaced with dideoxyguanidine (ddG), formation of species Z1 or Z2 was abolished. When both the 12- and 23-SEs had dideoxyguanidine at the end (ddG SEs), X, Y, and Z were all abolished. (Middle panel) Formation of X, Y, and Z are dependent on the presence of both RAG1 and RAG2. Reactions were performed with the separately purified RAG1 and RAG2. (Right panel) Reactions with the mutant RAG1. In contrast to the wild-type control (WT), the SE complex containing the mutant RAG1 (D708A) failed to generate any reaction products. The purified SE complex was used in these experiments. (B) Kinetics of the 3' overhang cleavage. The relative amounts of the three different reaction products, species X, Y, and Z, as well as of the unreacted substrate, were quantified by autoradiography. Reactions were performed for various lengths of time (1 to 100 min) by using the purified SE complex in three different Mg²⁺ concentrations (1, 3, and 10 mM).

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FIG. 3. Fate of the cleaved 3' overhang. (A) The 3' overhang is transferred to the SE. The substrate ds DNA with the 15-nt 3' overhang was incubated with the isolated SE complex in the presence of 10 mM Mg²⁺ for 10 min. Either the 12-SE or the 3'-overhanging ds DNA was labeled with ³²P at the 3'-terminal nucleotide. After the reaction, an aliquot of extracted DNA was digested with PvuII. Samples were electrophoresed ina 10% denaturing polyacrylamide gel and autoradiographed. A possible mechanism of the transfer of the 3' overhang is schematically shown on the right. A triangle indicates the RSS. (B) The transferred 3' overhang is cleaved from the SE. The SE complex was isolated and immobilized on avidin beads. The 3'-overhanging substrate DNA was allowed to react with the SE complex in the presence of 1 mM Mg²⁺ for 30 min. The reaction mix was separated into the bead-bound complex and unbound supernatant (sup). The isolated SE complex was extensively washed with the Mg²⁺-free buffer and incubated again with 10 mM Mg²⁺. Either the 3'-overhanging substrate DNA or the 12-SE was labeled with ³²P as indicated above for panel A. When the 3' overhang was labeled in the substrate, the ³²P label transferred to the STs and then appeared in the species X and Y. These pathways are schematically shown on the right.

The 3' overhang sequence transferred to the SE is precisely cleaved off. To examine whether the ST DNA is contained in the SE complex,we isolated the SE complex after the incubation with the 3'-overhangingds DNA (Fig. 3B). The SE complex was immobilized on streptavidin-coatedmagnetic beads with biotinylated 23-SE. The ds DNA with the³²P-labeled 3' overhang was allowed to react with the SE complexin the presence of 1 mM Mg²⁺ for 30 min. The bead-bound SE complexwas washed with the Mg²⁺-free buffer, and DNA was extractedfrom the complex, electrophoresed, and autoradiographed. Almostall of the unreacted substrate and species X and Y were washedout from the complex. However, the 12- and 23-STs remained withthe SE complex even after the washing (Fig. 3B). When the washedcomplex was reincubated in the Mg²⁺-containing buffer, the 12-and 23-ST bands disappeared, yielding the species X and Y bands(Fig. 3B). We performed a similar experiment with the unlabeled3'-overhanging ds DNA and the 12-SE labeled at the 3' end ofthe bottom strand. Again, most of the 12-ST DNA remained inthe complex after washing. When the complex was subsequentlyincubated in the Mg²⁺-containing buffer, the 12-STs graduallydisappeared. No cleavage products (species X and Y) were seenin this autoradiogram, because the 3' overhang of ds DNA wasnot labeled in this experiment. These results support the ideathat the species X and Y are generated from the 12- and 23-STsby the resolution reaction of the strand transfer intermediates.

To demonstrate the resolution process more directly, we reconstitutedthe SE complex with the 12-ST instead of the 12-SE. The 12-STDNA was labeled with ³²P at the 5' end of the bottom strand,paired with the 23-SE, and incubated with the RAG1, RAG2, andHMG1 proteins in the Mg²⁺-containing buffer. The ST band of58 nt (bottom strand) was converted to the 12-SE of 48 nt (Fig.4A). Even STs with overhangs as short as 1 nt were cleaved (datanot shown). We then labeled the 12-ST at the 3'-terminal nucleotideof the bottom strand and performed the same cleavage reaction.This time, two faster migrating cleavage products (X and Y)appeared (Fig. 4A). The band Y showed the electrophoretic mobilityexpected for the cleavage product of the 12-ST nicked at theds/ss junction. The other product, band X, was assumed to bethe circular form of the linear cleavage product, band Y (seebelow). Similar cleavage products were also generated from thelabeled 23-ST when it was paired with the 12-SE (data not shown).The cleavage of the 12-ST was significantly reduced when the12/23 rule was violated by pairing with the 12-SE (Fig. 4A).These results indicate that the 3' overhang in the ST, transferredfrom ds DNA to the SE, is indeed cleaved in the SE complex.We examined whether the resolution reaction is HMG dependent.It was found that the HMG protein is dispensable in the ST cleavagereaction (data not shown). Although HMG slightly stimulatesthe ST cleavage, this could be due to the stimulating effectof the HMG on the SE complex reconstitution rather than on thecatalysis.

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FIG. 4. The 3' overhang sequence is precisely cleaved from the ST. (A) Cleavage of the 12-ST. The 12-ST was labeled either at the 5' end or at the 3'-terminal nucleotide of the bottom strand. The labeled ST was paired with unlabeled partner SE and incubated with HMG1, RAG1, and RAG2 proteins in the presence of 10 mM Mg²⁺ at 37°C for 120 min. To block the circular DNA formation, the 3'-terminal nucleotide of the overhang, deoxycytidine (dC), was replaced with dideoxycytidine (ddC). The 12-ST was labeled at the ds/ss junction, paired with the 23-SE, and incubated with HMG1, RAG1, and RAG2 proteins. (B) Species X DNA is in a circular form. The 12-ST was labeled at the 3'-terminal nucleotide of the overhang and allowed to react as indicated above for panel A. Bands X and Y were isolated separately from a 15% denaturing polyacrylamide gel. Aliquots of X and Y samples were partially digested with mung bean nuclease (MBN) and electrophoresed in a 16.5% denaturing polyacrylamide gel. Digestion of X and Y are schematically shown.

Linear and circular DNA fragments as cleavage products. We then studied the structures of species X DNA by using variousenzymes that attack the DNA ends, e.g., alkaline phosphatase(which removes the terminal phosphates), T4 polynucleotide kinase(which phosphorylates the 5'-OH end), and exonuclease V (whichremoves linear DNA from both ends). None of these enzymes alteredthe mobilities or the intensities of the bands for X DNA (datanot shown). These results indicate that the species X DNA isin the circular form. To confirm this notion further, both Xand Y bands were purified from a denaturing polyacrylamide geland digested partially with mung bean endonuclease (Fig. 4B).Band Y was degraded quickly, generating a ladder of small nucleotides.In contrast, band X was converted to a slower-migrating band,whose mobility was the same as that of the band Y of 10 nt (Fig.4B). At higher concentrations of the mung bean nuclease, a ladderof degradation products appeared (Fig. 4B). When the phosphateat the ds/ss junction was labeled in the 3' overhang of the12-ST, bands X and Y were also generated after the incubationof the SE complex in the Mg²⁺-containing buffer (Fig. 4A). Theseresults indicate that the 3' overhang of 10 nt was preciselycleaved at the ds/ss junction in the 12-ST, resulting in thecircular (X) or linear (Y) product. We examined the length effectof the 3' overhang on the formation of circular DNA product(X). We found that only linear products (Y) were generated withshort 3' overhangs (2, 4, and 6 nt) (data not shown). With longer3' overhangs (8 and 10 nt), both linear (Y) and circular (X)products were generated. The terminal 3'-OH in the overhangappeared to be essential in generating the circular product(X). When the 3' end nucleotide, deoxycytosine, was replacedwith a dideoxycytosine, band Y was produced without the generationof band X from the 12-ST (Fig. 4A). Similar results were alsoobtained with the 3'-overhanging ds DNA when the end nucleotidein the overhang was replaced with a dideoxynucleotide (datanot shown). These results indicate that the ST is resolved intwo alternative pathways: one generating the circular product(X), and the other generating the linear form (Y). Neither reactionrequires an external energy source, such as ATP. The Y bandis probably generated by hydrolysis, while the X band appearsto be produced by intrastrand transesterification: a water moleculeor the terminal 3'-OH of the overhang attacks the phosphateat the ds/ss junction of the ST. To examine whether the resolutionof the ST molecule can occur with other nucleophiles, we tested1,2-ethanediol and glycerol in the 12-ST cleavage reaction.In a 20% denaturing polyacrylamide gel, the alcoholyzed Y migratedmore slowly than the hydrolyzed Y (Fig. 5). The shifted mobilitiescorrelated with the sizes of the alcohol molecules used in thereaction.

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FIG. 5. The ST can be resolved by alcoholysis. The SE complex was reconstituted with the labeled 12-ST and biotinylated 23-SE, isolated, resuspended in the Mg²⁺-containing buffer without glycerol, and divided into aliquots. The indicated amount (12.5 or 25%) of glycerol or 1,2-ethanediol was added, and the mixture was incubated at 37°C for 60 min. An aliquot of DNA extracted from the alcohol-free reaction mixture was treated with CIP. DNA samples were electrophoresed on a 20% denaturing polyacrylamide gel. An autoradiogram near the Y band is shown. Alcoholyzed products as well as the hydrolyzed Y band are indicated. A possible mechanism of alcoholysis is schematically shown on the bottom. HO-R indicates the alcohol.

Full-length RAG2 has a 3'-processing activity comparable to that of the truncated RAG2. It has been reported that the C-terminal region of the full-lengthRAG2 inhibits the RAG-mediated transposition activity (9, 57).Since the 3'-processing and the transposition reactions aremechanistically similar, we examined whether the C-terminalregion of the RAG2 also affects the 3'-processing activity.We reconstituted the SE complex either with the full-lengthRAG2 or with the truncated RAG2 lacking the C-terminal region.As shown in Fig. 6A, the transposition activity was significantlyreduced with the full-length RAG2 as reported previously (57).The double-ended insertion products predominate over the single-endedones under the Mg²⁺ condition without polyethylene glycol (Fig.6A and unpublished observation by T. Nishihara). The predominantgeneration of double-ended insertion products was also reportedby Neiditch et al. (37). In our experiments, the amount of transpositionproducts peaked before 10 min in 3 mM Mg²⁺ and then diminished.Transposition products did not accumulate even after 120 minwith either core RAG2 or full-length RAG2 (data not shown).Gellert and colleagues reported that the transposition productsincreased and then diminished in 25 mM Mg²⁺ but accumulatedin 5 mM Mg²⁺ (33). This discrepancy is probably due to the differencein the reaction procedures. According to their study, RSS cleavageand SE complex formation can proceed during the transpositionreaction (33). This would result in the accumulation of transpositionproducts during the reaction and mask the effect of disintegration.In our study, the SE complex was first purified and then usedfor the transposition assay. Thus, the transposition productsdid not accumulate. Unlike the transposition activity, the 3'-processingactivity under identical buffer conditions was not affectedby the presence of the C-terminal region of the full-lengthRAG2 (Fig. 6B). It should be mentioned that the full-lengthRAG2 also supported the cleavage of the 4-nt 3' overhang (datanot shown).

DISCUSSION

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Discussion

Mechanism of the 3' processing of the overhanging DNA with the SE complex. In the present study, we have found that the SE complex cleavesthe 3'-overhanging structure of the ds DNA under the physiologicalmetal condition. The 3'-overhanging structure is attacked bythe SE complex imprecisely near the ds/ss junction and transferredto the SE. The transferred overhang is then resolved and cleavedprecisely at the ds/ss junction. Thus, the blunt-end structureis restored for the SE and variably processed ends are generatedfor the CE DNA. The cleavage and transfer of the 3' overhangis likely to occur by transesterification: the 3'-OH group ofthe SE attacks the substrate DNA near the ds/ss junction, resultingin a covalent linkage of the SE to the 3' overhang (26). However,it is still possible that the 3' overhang is cleaved by a mechanismother than the strand transfer. Further study, e.g., on thestereochemical course of reaction, is needed to clarify thisissue. Unlike the RAG-mediated transposition (33), reversalof the strand transfer is not favored, and the 3'-processedds DNA is thus released from the SE complex (data not shown).The transferred 3' overhang is then cleaved from the SE by hydrolysis,alcoholysis, or circular DNA formation. This resolution is strikinglysimilar to the reaction mediated by the human immunodeficiencyvirus type 1 integrase, which removes two nucleotides from the3' end of the viral DNA via hydrolysis, alcoholysis, or circular-dinucleotideformation in vitro (10). A catalytic DDE motif, found in thetransposases and retroviral integrases, is also found in theRAG1 protein (12, 23, 25). We have found that the SE complexreconstituted with the mutant RAG1 (D708A), which has a mutationin the DDE motif (12, 23), failed to mediate both steps in the3'-processing reaction (data not shown).

The 3'-processing reaction is similar to but distinct from the transposition and disintegration reaction. The first step in the 3'-processing reaction can be regardedas the initial transposition reaction targeting the distortedDNA structure (26, 55). However, the 3'-processing and transpositionreactions are different in the subsequent steps. The transpositionalstrand transfer to the ds DNA is followed by the reversal reactionthat removes the SE from the transposition product and resealsthe target DNA (33). This reaction was proposed to explain atleast in part why the RAG-mediated transposition does not usuallyoccur in cells (33). In contrast, strand transfer to the 3'overhang is followed by the release of the 3'-processed ds DNAand by the hydrolysis-mediated resolution of the ST intermediate,accumulating the cleavage products.

It should be noted that the C-terminal region of the full-lengthRAG2 has different effects on the 3'-processing and the transpositionand disintegration reactions: the 3'-processing activity wasnot affected by the presence of the C-terminal region of RAG2,whereas the transposition activity was significantly reduced(Fig. 6) (57). We suppose that the presence of the C-terminalregion of RAG2 may interfere with the accessibility of the targetds DNA (9, 57), but not with the 3'-overhanging end. It is possiblethat the C-terminal region of the RAG2 may play a role in preventingthe harmful transposition in cells (9, 51, 57) while maintainingthe 3'-processing activity.

Implication for the junctional diversification in V(D)J joining. It is important to determine that the 3'-processing activitydetected in vitro in the present study plays a role in processingthe hairpin-opened CEs in vivo. It has been reported that thehairpin opening yields mainly the 3'-overhanging structure inthe CE (7, 29, 30, 49). Since the 3' overhang can be cleavedat variable sites near the ds/ss junction in vitro (Fig. 1),this cleavage, if it also occurs in vivo, may contribute tothe diversity at the junctional sequences of antigen receptorgenes. In contrast, the cleavage on the ST occurs preciselyat the ds/ss junction (Fig. 4). Restoration of the blunt-endstructure at the SE is consistent with the observation thatmost signal joints do not contain any nucleotide addition ordeletion (50).

In the double-strand break repair, nucleotide removal is usuallynecessary to tailor the DNA ends suitable for ligation. It hasbeen reported that the Artemis/DNA-PKcs complex possesses the3' and 5' overhang endonuclease activities that may be involvedin the repair and processing of the CEs (30). Variable CE processingmay require other nucleases with different cleavage site preferences.Since the SE complex tends to cleave at sites in the 3' overhangthat are more 5' than those of Artemis/DNA-PKcs (30), it couldcontribute more junctional diversity than the Artemis/DNA-PKcsalone, if the SE complex is actually involved in the CE processingin vivo.

To examine the CE processing by the SE complex in vivo, mutantRAGs defective in the 3'-processing activity will be helpful.Reconstitution of the postcleavage processes in vitro, whichdepend on repair factors such as Ku, will also be necessary.Although these attempts are now under way, some recent findingsare encouraging. First, studies with the mutant RAGs have suggestedthat the RAG proteins may play an architectural role in thepostcleavage phase of V(D)J recombination (20, 56). If thisis the case, the RAG proteins are likely to associate with orstay near the CEs in the SE complex (2, 18, 40). Second, unlikethe 3' flap endonuclease, the 3'-processing activity of theSE complex is high when Mg²⁺ is present in vitro (Fig. 1B).Third, among the known strand transfer activities of the SEcomplex, only the 3' processing could have a physiologic role,preventing a potentially harmful transposition in cells. Finally,the 3'-processing activity is not affected by the presence ofthe C-terminal region of the RAG2 (Fig. 6), while the transpositionactivity is significantly reduced (57). The C-terminal regionof the RAG2 may be important in preventing the harmful transposition(9, 51, 57) while maintaining the CE processing activity, althoughit has yet to be verified that this occurs in cells.

The RAG-mediated transposition can be found in Saccharomycescerevisiae cells (5) but rarely occurs in the mammalian cells.However, the SE complex appears to possess the strand transferactivity in the mammalian cells, because the RAG-mediated hybridjoining has been reported for the extrachromosomal recombinationwith the truncated RAG proteins (16, 51). A similar observationwas also made for the antigen receptor loci in the Ku gene knockoutmice where the hairpin CEs accumulate (4, 61). The hybrid joiningmay result from the unregulated strand transfer of the SE onthe CEs before hairpin opening.

V(D)J recombination may be a reversal of an accidental insertionof a transposable element into a primordial V gene, which wasexploited later by the vertebrate immune system during evolution(45). In the initial phase of V(D)J joining, the RAG proteinscleave the RSS by a mechanism similar to that of transpositionor that of retroviral integration (22, 58). The SE complex hasbeen reported to possess the transposition activity in vitro,while such harmful transpositional strand transfer is rarelyseen in vivo. It is tempting to assume that the strand transferactivity of RAG proteins is utilized to diversify the antigenreceptor genes in V(D)J joining by removing the 3' overhangfrom the hairpin-opened CEs. However, this, of course, mustbe verified in the in vivo system in the future.

ACKNOWLEDGMENTS

We are grateful to D. G. Schatz for providing us with the expressionvectors for RAG proteins. We also thank Hitomi Sakano for criticalreading of the manuscript and Takeshi Imai for discussion.

This work was supported by grants from Japan Science and TechnologyCorporation, the Ministry of Education, Culture and Science,and the Mitsubishi Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5689-7239. Fax: 81-3-5689-7240. E-mail: sakano{at}mail.ecc.u-tokyo.ac.jp.

Present address: Radiobiology Division, National Cancer CenterResearch Institute, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

REFERENCES

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