Joining-Deficient RAG1 Mutants Block V(D)J Recombination In Vivo and Hairpin Opening In Vitro

Molecular Cell, Vol. 7, 65–75, January, 2001, Copyright 2001 by Cell Press

Joining-Deficient RAG1 Mutants Block V(D)J Recombination In Vivo and Hairpin Opening In Vitro Heather Yarnall Schultz,* Mark A. Landree,* Jian-xia Qiu,†‡ Sam B. Kale,† and David B. Roth*†‡§ * Interdisciplinary Program in Cell and Molecular Biology † Department of Immunology ‡ Howard Hughes Medical Institute Baylor College of Medicine Houston, Texas 77030

Summary The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination. Introduction Immunoglobulin and T cell receptor genes require assembly. The antigen binding domains of these molecules are created through a series of DNA rearrangements that bring together short gene segments, termed V (variable), D (diversity), and J (joining), during lymphocyte differentiation to create variable region genes that encode a diverse array of antigen binding sites. This process, termed V(D)J recombination, is initiated by the introduction of site-specific double-stranded DNA breaks adjacent to the V, D, and J gene segments. These so-called coding segments are recognized by the recombinase because of their location adjacent to specific DNA sequences, recombination signal sequences (RSS), which consist of conserved heptamer and nonamer elements separated by 12 or 23 nucleotides of spacer DNA. Double-strand breaks are created by the RAG1/RAG2 protein complex, a lymphoid-specific nuclease that binds to the RSS and catalyzes DNA cleavage by a two-step mechanism. First, the RAG proteins introduce a nick precisely between the RSS and the adjacent coding element. Second, the newly formed 3⬘ OH attacks a phosphodiester bond on the opposite strand, generating a blunt signal end and a hairpin coding end (McBlane et al., 1995). Cleavage normally occurs in a coupled fashion at a 12/23 RSS pair (Eastman et al., 1996; Steen et al., 1996; van Gent et al., 1996). Both in vivo data (Zhu § To whom correspondence should be addressed (e-mail: davidbr@

bcm.tmc.edu).

et al., 1996; Lewis et al., 1988; Agard and Lewis, 2000) and biochemical studies (Agrawal and Schatz, 1997; Hiom and Gellert, 1998) indicate that the coding and signal ends remain associated with the RAG proteins in a postcleavage complex. Although it has been suggested that the postcleavage complex may be important for end processing and joining (Zhu et al., 1996), there is as yet no evidence that the RAG proteins assist in forming either coding or signal joints in vivo. The joining steps of V(D)J recombination are complex and poorly understood. Signal ends are typically joined without loss of nucleotides, but extra nucleotides (nontemplated, or “N” nucleotides) may be added by the enzyme terminal deoxynucleotidyl transferase (TdT). Coding ends are subject to both nucleotide loss (by unknown mechanisms) and N nucleotide addition (reviewed in Lewis, 1994). Coding ends also undergo a unique, obligatory processing step: the covalently sealed hairpin termini must be opened by endonucleolytic cleavage to make them available for end processing and joining. This hairpin opening reaction often leaves a characteristic footprint: opening away from the “tip” of the hairpin generates single-stranded extensions that can be incorporated into the coding joint, producing short (generally less than 4 nt) palindromic insertions termed P nucleotides (Lafaille et al., 1989; Roth et al., 1992; Meier and Lewis, 1993). With the exception of TdT, the identities and the biochemical actions of the end processing and joining factors involved in V(D)J recombination remain unclear. Analysis of mice and cell lines bearing mutations in double-strand break repair factors has shown that several non-lymphoid-specific factors play critical roles in both signal and coding joint formation. These include the Ku heterodimer, DNA ligase IV, XRCC4, and the DNAdependent protein kinase catalytic subunit (DNA-PKcs, the product of the murine scid gene) (reviewed in Bogue and Roth, 1996; Grawunder et al., 1998). DNA-PKcs is unique in that its inactivation impairs coding joint formation much more than signal joint formation (Bogue et al., 1998; Gao et al., 1998; Lieber et al., 1988; Malynn et al., 1988). In addition to impairing the efficiency of joint formation, mutational inactivation of the factors listed above also usually affects the structure of the joints. Both signal and coding joints suffer aberrant deletions in DNA-PKcs-deficient cells (Schuler et al., 1986; Lieber et al., 1988; Malynn et al., 1988); signal joints also undergo deletions in the absence of Ku (Pergola et al., 1993; Taccioli et al., 1993; Bogue et al., 1997). Coding joints from Ku and DNA-PKcs mutants often show unusually long P nucleotide inserts, suggesting aberrant hairpin opening (Kienker et al., 1991; Schuler et al., 1991; Bogue et al., 1997). Furthermore, hairpin coding ends accumulate in thymocytes of both DNA-PKcs-deficient and Ku-deficient mice (Roth et al., 1992; Zhu et al., 1996). These data suggest that DNA-PK plays a critical role in processing hairpin coding ends, but the nature of that role remains enigmatic. Identifying the nuclease(s) responsible for hairpin opening in vivo is of central importance to understanding

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the mechanism of V(D)J recombination. “Single-strand specific” nucleases isolated from organisms that do not carry out V(D)J recombination are capable of efficiently opening hairpins in vitro, generating structures compatible with the production of P nucleotides (Kabotyanski et al., 1995). These nucleases are thought to recognize structural distortions that occur near the hairpin “tip” (Kabotyanski et al., 1995). Thus, many cellular nucleases that recognize single-stranded regions or distorted DNA structures might be capable of opening hairpins. This makes it difficult to predict which nucleases actually are important for V(D)J recombination in vivo. Recent biochemical studies have shown that both the Mre11 nuclease (part of a multiprotein complex involved in nonhomologous recombination) and the RAG proteins themselves are capable of opening artificial hairpins under certain conditions, yielding short single-stranded extensions compatible with P nucleotide formation (Besmer et al., 1998; Paull and Gellert, 1998, 1999; Shockett and Schatz, 1999). However, these nuclease activities are not specific for hairpins: cleavage also occurs at other sites, including in the vicinity of nonhairpin DNA ends (Besmer et al., 1998; Paull and Gellert, 1999; Shockett and Schatz, 1999). Furthermore, the RAG proteins cleave at single-strand/double-strand transitions (Santagata et al., 1999). These end processing and flap endonuclease activities have been proposed to play important roles in coding joint formation (Santagata et al., 1999). Nevertheless, compelling evidence that either the RAG proteins or Mre11 opens hairpins or contributes to other end processing events during normal V(D)J recombination in vivo is lacking. Here we report the identification and analysis of RAG1 mutants that are severely defective for coding and signal joint formation in vivo. Together with additional biochemical analysis of the mutant proteins, these data strongly support a role for the postcleavage complex in joining coding and signal ends and suggest that the complex may be important for recruiting end processing and joining factors. One mutant also shows substantially impaired hairpin opening in vitro, providing genetic and biochemical evidence linking the in vitro hairpin opening activity of the RAG proteins to in vivo coding joint formation. Results In Vivo Identification of RAG1 Joining Mutants To identify mutants defective in the joining step, we screened a library of 75 site-directed mutants of RAG1 that we had previously used to identify active site amino acids (Landree et al., 1999). In this collection, all evolutionarily conserved acidic amino acids (glutamates and aspartates) in the active core of RAG1 (amino acids 384–1008) were changed to their noncharged counterparts, glutamine and asparagine. Truncated active core RAG1 was employed because this protein is fully active for recombination of plasmid substrates (Sadofsky et al., 1993; Silver et al., 1993), and, unlike full-length RAG1, soluble truncated RAG1 can be purified and used in biochemical assays. The mutant proteins were screened using an in vivo transfection system for V(D)J recombination of plasmid substrates (Landree et al., 1999). Here we

describe two mutants, E547Q and E423Q, that exhibit specific joining defects. Two independent isolates of each mutant were examined in all assays, with identical results (data from only one isolate are shown in most figures). Both mutants yielded wild-type levels of RAG1 protein in transient transfection assays, as measured by Western blotting; levels of RAG2 were also wild-type (Figure 1A). To identify joining mutants, we first examined formation of coding joints using a standard plasmid V(D)J recombination assay (Steen et al., 1997). Substrates containing a 12/23 RSS pair (pJH290 or pJH299) were cotransfected into Chinese hamster ovary fibroblasts along with expression vectors encoding truncated core RAG1 (mutant or wild-type) and core RAG2 (wild-type) proteins. Semiquantitative PCR assays revealed that both E547Q and E423Q are severely defective for coding joint formation, with levels of coding joints consistently at least 100-fold below wild type (representative data are shown in Figure 1B, compare lanes 3–5). Similar results were obtained using quantitative bacterial transformation assays, which measure recombinational activation of a bacterial drug resistance marker (the chloramphenicol acetyl transferase gene) carried on the substrate plasmid (Hesse et al., 1987) (data not shown). Both mutants were also defective for signal joints: levels of these products were decreased up to 100-fold (Figure 1C). In more than ten independent transfections, both mutants consistently proved to be somewhat more impaired in formation of coding joints than signal joints. Effects of RAG1 Mutants on Signal and Coding Ends To determine whether the defects in coding and signal joint formation result from effects on cleavage or joining, we assayed for the presence of the excised linear fragments terminating in signal or coding ends directly by Southern blotting (Steen et al., 1997). This method provides quantitative information about levels of excised linear molecules resulting from dual RSS cleavage and about the ability of the mutants to perform coupled cleavage at an RSS pair. Analysis of undigested DNA recovered from multiple independent transfections of wild-type and mutant RAG proteins revealed that levels of excised molecules terminating in signal ends were slightly (3- to 5-fold) reduced (Figure 1D), in agreement with the results of ligation-mediated PCR assays for each end (data not shown). Both mutants also yielded similarly (3- to 5-fold) reduced levels of excised linear molecules terminating in coding ends (Figure 1E). Results of ligation-mediated PCR assays for coding ends were consistent with the presence of hairpins at the termini (data not shown). These results indicate that the mutants have only a modest effect on cleavage. Indeed, analysis of the purified mutant proteins revealed little, if any, cleavage defect (see below). The effects of the RAG1 mutants on coding ends contrast sharply with the increased abundance of coding ends observed in thymocytes of DNA-PKcs- and Kudeficient mice (Roth et al., 1992; Zhu et al., 1996). The behavior of the RAG1 mutants in our transient transfection assays is, however, in agreement with the results of transient transfections in DNA-PKcs- and Ku80-defi-

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Figure 1. Effects of RAG1 Mutations on Protein Expression, Cleavage, and Joining In Vivo (A) Western blot of RAG proteins in cell lysates from transient transfections. Separated proteins were probed with anti-c-myc antibodies and HRP-conjugated anti-mouse IgG1 antibodies and visualized with the ECL-PLUS kit. The asterisk denotes a degradation product that is occasionally seen with both mutants and wild-type RAG1 proteins. (B) In vivo analysis of coding joints. One three-hundredth of a transfection was used in a PCR reaction for coding joint formation using the DR99 and ML68 primers. Transfections of wild-type RAG proteins were assayed at 1⫻, 1:10, and 1:100 dilutions; mutants and mock transfections lacking RAG expression vectors (“no RAGs”) were assayed without dilution. Reactions were run on polyacrylamide gels, blotted, and probed with DR99. All lanes are from the same gel. Squares indicate coding segments; arrows indicate PCR primers. (C) In vivo analysis of signal joints. One three-hundredth of a transfection was used in a PCR reaction for signal joint formation using the DR55 and DR100 primers. The blot was probed with DR55. Open triangle indicates the 12-RSS; closed triangle denotes the 23-RSS. (D and E) Southern blot analysis of E547Q and E423Q. For each mutant, two independent transfections with wild-type RAG2 were performed. Five-sixths of the DNA harvested from a transfection was electrophoresed through an agarose gel without restriction digestion to allow visualization of the doubly cleaved signal end fragment from pJH290 (D) or the doubly cleaved coding end fragment from pJH289 (E). The unrearranged substrate and cleaved plasmid backbone fragments do not appear because these large molecules transfer poorly under the conditions used. The blot was probed with a radiolabeled PvuII fragment of the pJH290 that contains the unrearranged signal pair and spans the entire length of the excised fragment.

cient fibroblasts. Coding ends do not accumulate in scid, Ku80-deficient, or XRCC4-deficient cell lines transfected with RAG expression vectors, despite the observed decrease in coding joint formation (Han et al., 1998 and unpublished data). In fact, signal ends do not accumulate in thymocytes of Ku80-deficient mice, which are almost completely defective for formation of signal joints (Zhu et al., 1996). There is, therefore, no reliable correspondence between impaired joining and increased levels of broken-ended intermediates. The presence of excised linear fragments in vivo (and normal coupled cleavage at both RSS in vitro, see below) demonstrates that cleavage is occurring at both RSS. Thus, decreased numbers of joints cannot be explained by an inability of the mutant proteins to perform coupled cleavage. Together, these data demonstrate that the

E547Q and E423Q mutants exhibit severe defects in joining that cannot be attributed to effects on cleavage. Coding and Signal Joints Formed by RAG1 Mutants Lack Structural Abnormalities Since the rare junctions isolated from double-strand break repair mutants (DNA-PKcs, Ku, etc.) exhibit characteristic structural abnormalities such as excessive deletions at both coding and signal joints and long P nucleotide inserts at coding joints, we examined the joints produced in fibroblasts transfected with the RAG1 mutants. Analysis of signal joints was simplified by the fact that a perfect fusion of the two heptamer sequences creates an ApaL1 restriction site. ApaL1 digestion of PCR products from multiple transfections of each mutant and wild-type controls revealed that virtually 100%

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Figure 2. Nucleotide Sequence Analysis of Coding Joints Nucleotide sequences of junctions formed from multiple transfections of two different substrates, pJH290 (deletional recombination) and pJH299 (inversional recombination) are shown. (A), junctions formed by E547Q; (B), junctions formed by E423Q. Similar results were obtained with both substrates, so the data are shown together. The sequence of a perfect coding joint is shown at the top. The number of nucleotides deleted from each coding end is given for each sequence. Capital letters indicate P nucleotides. Lowercase letters indicate N nucleotides. The number of junctions isolated with the indicated sequence is given in parentheses. “H” indicates joints containing short sequence homologies; “⫹TdT” indicates the junctions were isolated from transfections in which terminal deoxynucleotidyl transferase was present.

of the signal joints were ApaL1 sensitive (data not shown). This demonstrates that these junctions are precise, without loss of a single nucleotide from either end. We sequenced rare coding joints recovered from chloramphenicol-resistant colonies derived from bacterial transformation of plasmid recombination substrates. Both mutants produced coding joint sequences that appear indistinguishable from those produced by wildtype RAG proteins (see Qiu et al., 2001 [this issue of Molecular Cell]), without excessive deletions, increased use of junctional homologies, or long P nucleotides (Figures 2A and 2B). Furthermore, TdT (terminal deoxynucleotidyl transferase) added N nucleotides normally to coding and signal joints (Figures 2A and 2B and data not shown), indicating that this end-processing enzyme is able to access at least some of the ends. These data indicate that the joining mechanisms involved in forming these rare joints that escape the RAG1 mutant defects do not alter the structure of the junctions. RAG1 Mutants Catalyze Efficient Nicking and Hairpin Formation In Vitro To assess the biochemical properties of the mutants, the truncated active core versions (amino acids 384– 1008) of both E547Q and E423Q were purified from mammalian cells (along with coexpressed core RAG2, amino acids 1–383) as GST fusion proteins. To examine formation of nicks and hairpins, we used a standard oligonucleotide cleavage assay in which a labeled 12-RSS sub-

strate is incubated with the RAG proteins in Mn2⫹ (McBlane et al., 1995). Neither of the mutant proteins showed a significant defect in formation of nicks and hairpins (Figure 3A). We next tested the ability of the mutants to perform coupled cleavage at an RSS pair in the presence of Mg2⫹ (Hiom and Gellert, 1998). Under these conditions, both mutants showed very slight decreases in nicking and hairpin formation (Figure 3B) consistent with the small decreases in levels of signal and coding ends observed in vivo. Similar results were obtained using proteins purified from baculovirus-infected insect cells (data not shown). These demonstrate that although E547Q and E423Q are profoundly defective for joining, there is no substantial defect in cleavage. Both RAG1 Mutants Can Form Hybrid Joints We next examined the ability of the RAG1 mutants to form hybrid joints, which are produced by joining a signal end to a coding end (Lewis et al., 1988) (Figure 4A). Hybrid joints can be formed by a transesterification mechanism in which the 3⬘ OH of a signal end attacks the hairpin coding end (Melek et al., 1998), thereby bypassing the end-joining machinery. Hybrid joint formation by this mechanism provides a measurement of the ability of the RAG proteins to remain associated with the coding and signal ends after cleavage. Using a previously described PCR-based assay for hybrid joint formation by purified RAG1 and RAG2 (Melek et al., 1998), we found no obvious decrease in the ability of E423Q

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Figure 3. Nicking and Hairpin Formation by Purified Proteins Cleavage activity was measured using GST-fusion proteins copurified from transfections with either wild-type or mutant GST-RAG1 and wild-type GST-RAG2. The 12-RSS oligonucleotide substrate (DAR39/40) was radiolabeled at the 5⬘ end of the top strand. Cleavage produces a 16 nucleotide nicked product and a 32 nucleotide hairpin product. (A) Cleavage at the 12 RSS was assayed in 5 mM MnCl2. (B) Coupled cleavage was assayed in 5 mM MgCl2 in the presence of an unlabeled oligonucleotide containing a 23-RSS (DG61/62). Equivalent amounts of RAG proteins (as determined by Coomassiestained SDS–PAGE) were used in each assay.

or E547Q to form hybrid joints (Figure 4B). We also examined hybrid joint formation in vivo, as previous work suggests that a significant fraction of these junctions is formed by RAG-mediated transesterification. In fact, hybrid joints are formed efficiently even in double-strand break repair-deficient cells that are severely defective for formation of both coding and signal joints (Bogue et al., 1997; Han et al., 1997, 1998). We found that neither mutant was substantially defective for hybrid joint formation in vivo (data not shown). The ability of these mutants to form hybrid joints efficiently in vivo and in vitro provides strong evidence that these proteins are capable of forming postcleavage complexes that contain both coding and signal ends. E423Q Is Defective for Hairpin Opening In Vitro To explore the basis of defective coding joint formation by E547Q and E423Q, we tested the ability of the purified mutant proteins to open hairpins. Previous hairpinopening assays (Besmer et al., 1998) employed a synthetic hairpin oligonucleotide, DR109, that was originally used to assess hairpin opening by single strand–specific nucleases (Kabotyanski et al., 1995). Incubation of wildtype RAG proteins with this substrate in the presence of Mn2⫹ yielded the expected cleavage products of 17 and 18 nucleotides (Besmer et al., 1998), which result from introduction of nicks 5⬘ to the hairpin (Figure 5A, lane 1). We also observed a much longer product resulting from cleavage near the 3⬘ end of the substrate (an activity that has been termed “end processing” [Besmer et al., 1998]), and a few less prominent cleavage products that migrate more rapidly on the gel than the products of hairpin opening. All these products have

Figure 4. Efficient Hybrid Joint Formation by Both E547Q and E423Q (A) Schematic of hybrid joint formation with pJH299. (B) Hybrid joints formed from the pJH299 substrate in vitro were measured by a PCR assay that detects hybrid joints formed on the excised circle. RAG GST-fusion proteins were incubated with the plasmid substrate. One-fifth of the reaction mix was assayed by PCR using the DR55 and ML68 primers. The blot was probed with the joint-specific DR98 oligonucleotide. Symbols are as in Figure 1. All lanes are from the same gel.

been observed previously (Besmer et al., 1998; Shockett and Schatz, 1999) and are generated by the RAG proteins—their formation requires both RAG1 and RAG2 (Besmer et al., 1998; Shockett and Schatz, 1999), and they are not produced by cleavage-deficient active site mutants of RAG1 (Fugmann et al., 2000). The E547Q mutant did not exhibit a detectable defect in hairpin opening in the standard assay (Figure 5A, lane 2). Time course experiments also failed to reveal a kinetic defect in hairpin opening for this mutant (Figure 5B, lanes 8–13). Several independent protein preparations of E547Q, however, exhibited a small but consistent decrease in end processing (Figure 5A, lane 2): both the large cleavage product resulting from nicking near the 3⬘ end and the smaller cleavage products were reduced (Figure 5A, lane 2). Analysis of several independent purified protein preparations revealed a substantial impairment (up to 10fold) in the ability of the E423Q mutant to perform both hairpin opening and end processing (representative data are shown in Figure 5A, lane 3). Kinetic analysis confirmed that E423Q is severely defective for hairpin opening (Figure 5B, lanes 14–19). Increased concentrations of Mn2⫹ (up to 50 mM) failed to rescue hairpin opening activity (data not shown). The possible relationship between this defect and impaired coding joint formation in vivo is discussed below (see Discussion). Postcleavage Complexes Containing RAG1 Mutants Are Capable of Transposition The defects in signal and coding joint formation exhibited by the two RAG1 mutants could be caused by ef-

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Figure 5. Hairpin Opening Is Impaired by E423Q (A) A 5⬘ end-labeled hairpin oligonucleotide (DR109) was used as a substrate. GST-fusion proteins were incubated with this substrate in 10 mM MnCl2 at 30⬚C for 90 min. Hairpin opening immediately 5⬘ of the hairpin tip liberates 18- and 17-nucleotide products. Products resulting from cleavage near the 3⬘ end of the substrate (3⬘ end processing) are also observed. (B) Kinetic analysis of hairpin-opening activity of E547Q and E423Q. Reactions were performed as in (A) except that they were terminated at 0, 15, 30, 60, 90, or 180 min. Equivalent amounts of RAG proteins (as determined by Coomassie-stained SDS–PAGE) were used in each assay. All lanes are from the same gel.

fects on the postcleavage complex. For example, an unstable postcleavage complex could fail to properly assemble the end-joining machinery or allow one or more ends to escape prematurely. To assess the ability of the RAG1 mutants to remain associated with signal ends, we examined transposition in vitro. In this reaction, a postcleavage complex containing a pair of signal ends bound to the RAG proteins attacks a target DNA molecule, covalently joining one 3⬘ end of each RSS to the target via a transesterification reaction (Agrawal et al., 1998; Hiom et al., 1998). We tested the ability of the purified mutants to catalyze transposition using an oligonucleotide-based assay in which a 12/23 pair of radiolabeled oligonucleotide RSS substrates is incubated with the RAG proteins and a circular plasmid target (Hiom et al., 1998). Coupled transposition of both 12 and 23 ends linearizes the target plasmid; single ended transposition (which occurs rarely) nicks the target (Figure 6A). This assay, therefore, provides a sensitive functional test for the ability of the postcleavage complex to retain both signal ends in the presence of competitor DNA (the target). Analysis of several purified protein preparations of E423Q revealed only a slight decrease (2- to 3-fold) in transposition activity (Figure 6B, lanes 5–7). Transposition by E547Q was not diminished; in fact, this mutant was consistently 2- to 3-fold more active than wild type (Figure 6B, lanes 8 and 9). Notably, most transposition

events catalyzed by both mutants linearized the target, indicating double-ended transposition; no increase in nicked products (indicative of single RSS transposition) relative to wild-type levels was observed. We obtained similar results using precleaved ends: again, transposition by E547Q was 2- to 3-fold more efficient than wild type (data not shown). These data indicate that the ability of complexes containing the RAG1 mutants to retain both signal ends after cleavage is not substantially impaired, indicating that the defect in signal joint formation is not attributable to gross instability of the postcleavage complex. Discussion We have identified and characterized two RAG1 mutants that impair formation of both coding and signal joints. These effects are not attributable to defects in cleavage, because both in vivo and in vitro studies revealed only small reductions—or no reductions—in levels of signal and coding ends. Furthermore, formation of hybrid joints (which requires coupled cleavage at both RSS) was not significantly decreased. These RAG1 mutations, therefore, specifically impair the formation of coding and signal joints. It is noteworthy that these RAG mutants have such a specific effect on the joining phase of the reaction without affecting the cleavage steps. This observation indicates that the cleavage and joining activities of RAG1

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Figure 6. Both E547Q and E423Q Are Capable of Efficient Transposition (A) Schematic of the in vitro transposition reaction. (B) RAG GST-fusion proteins were incubated at 37⬚C with labeled 12 and 23 RSS in the presence of CaCl2. Plasmid target and MgCl2 were added, and the reactions were again incubated at 37⬚C. Products were separated on a 4%–20% gradient gel. Nicked products indicate single-ended transposition events, and linear products indicate double-ended transposition events.

are not interdependent and could involve different domains of the protein. RAG1 Is Critical for Joining Signal and Coding Ends Our data clearly show that RAG1 plays an important physiological role in the joining step of V(D)J recombination and strongly implicate the postcleavage complex in signal and coding joint formation. These findings are supported by the discovery of RAG2 mutants that allow cleavage but abolish signal and coding joint formation (Qiu et al., 2001). Our biochemical studies indicate that the effects of E423Q on coding joint formation may be attributable, at least in part, to a defect in hairpin opening, as discussed below. As yet, it is not possible to define precise biochemical correlates for the effects of these mutations on signal joints because current assays for signal joint formation in vitro require the removal of RAG proteins by artificial means before substantial signal joint formation can be observed (Leu et al., 1997; Ramsden et al., 1997). Our analysis revealed no significant effects of the RAG1 mutants on the ability of the RAG1/RAG2/paired RSS complex to undergo transposition. Furthermore, neither of the mutants substantially affected the efficiency of hybrid joint formation either in vivo or in vitro.

These data suggest that the RAG1 mutations do not substantially alter the stability of postcleavage complexes containing coding and signal ends. This interpretation is supported by our observation that levels of V(D)J recombination intermediates in vivo are not significantly decreased. Based on these results, defective signal joint formation by both mutants is likely to result more from defects in recruitment of the joining machinery to the ends than from premature disassembly of the postcleavage complex. Similar effects may be responsible for impaired coding joint formation by E547Q (and possibly by E423Q, although this mutant exhibits a specific defect in hairpin opening in vitro). The ability of E547Q to catalyze transposition more efficiently than wild-type RAG1 raises the possibility that the postcleavage complex containing signal ends might actually be somewhat more stable than wild type. Impaired disassembly of the postcleavage complex could, therefore, contribute to this mutant’s joining defects. The apparent lack of structural abnormalities in the rare junctions produced by both E423Q and E547Q would seem to set them apart from double-strand break repair mutants. This ostensible difference should be viewed with caution, however. First, junctions formed at some loci in double-strand break repair (DSBR) mutants fail to exhibit excessive deletions (Hendrickson et al., 1990; Pennycook et al., 1993; Bogue et al., 1997). Alternatively, we could miss certain structural abnormalities; our PCR and bacterial transformation assays would not be able to detect very large deletions formed in our transfections. We should also bear in mind that unlike DSBR mutants, the two RAG1 mutants studied here do not behave as nulls; they each harbor a single conservative amino acid substitution and may retain residual activity. Lastly, because our assays are not carried out in DSBR mutant cells, the rare ends that can bypass the RAG joining defect can interact with a wildtype version of the repair machinery. Do the RAG Proteins Open Hairpins In Vivo? We found that both RAG1 mutants are severely defective for coding joint formation in vivo. E423Q is also quite defective for hairpin opening in vitro despite being perfectly capable of performing the basic chemical reaction involved in hairpin opening, hydrolysis (nicking). Furthermore, the ability of this mutant protein to form hybrid joints demonstrates that it can bind to hairpin ends and carry out transesterification using a 3⬘ OH as a nucleophile. These functional capacities argue that this mutant bears a highly specific defect: it is unable to attack hairpins using water as a nucleophile. If the magnitude of the defect in hairpin opening in vitro (approximately 10-fold) accurately reflects the in vivo situation, it could certainly account for a severe (100-fold) defect in coding joints, as this requires opening hairpins at each end. It is intriguing that the E547Q mutant does not affect hairpin opening in vitro, since it is as defective for coding joint formation as E423Q. One possible explanation is that the phenotype of this mutation is mitigated by Mn2⫹, which is known to suppress defects conferred by other mutations in RAG1 (Landree et al., 1999). Another possibility is that E547Q is defective not in its ability to catalyze hairpin opening but rather in its ability to respond

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to regulatory signals such as DNA-PK in vivo (see below). It is also conceivable that the mutation blocks another step in the joining reaction, perhaps because of defective interactions with joining factors. Determining the molecular basis of the behaviors of these mutants in vitro should provide valuable insight into the mechanism of coding joint formation. Our analysis of the E423Q mutant provides the strongest evidence to date that the RAG proteins may function as hairpin-opening nucleases in vivo. This hypothesis is supported by three additional lines of evidence: (1) the RAG proteins stimulate coding joint formation in vitro (Leu et al., 1997; Ramsden et al., 1997); (2) truncation mutants of RAG2 exhibit mild defects in coding joint formation in vivo (Steen et al., 1999); and (3) we have recently identified RAG2 mutants with severe defects in both coding joint formation and hairpin opening in vitro (Qiu et al., 2001). We do not yet know whether other nucleases also participate in the processing of coding ends in vivo. Based on the ability of the purified Mre11 protein to open hairpins, it has been suggested that this nuclease may open hairpin coding ends during V(D)J recombination (Paull and Gellert, 1998, 1999). It should be noted that purified Mre11 alone does not efficiently open fully self-complementary hairpins, which are the type made by the RAG proteins (Paull and Gellert, 1998); cleavage of this type of hairpin requires the addition of the Nbs1 protein (Paull and Gellert, 1999). While Nbs1 is important for hairpin opening in vitro, NBS patients generally produce normal levels of IgM and CD8⫹ T cells (The International Nijmegen Breakage Syndrome Study Group, 2000), and normal coding joint sequences have been recovered from an NBS lymphoblastoid cell line (Petrini et al., 1994). Furthermore, four patients bearing two different non-null Mre11 mutations did not exhibit immunodeficiency (Stewart et al., 1999). Finally, recent work has revealed no apparent defects in coding joint formation in cells or mice bearing hypomorphic mutations in NBS, Mre11, or Rad50 (J. Petrini, personal communication). What Is the Role of Double-Strand Break Repair Factors in Hairpin Opening? DNA-PKcs is required for efficient coding joint formation, and is also important for formation of signal joints (Bogue et al., 1998; Lieber et al., 1988). Furthermore, hairpin coding ends accumulate in thymocytes of mice lacking DNA-PK activity (Roth et al., 1992; Zhu et al., 1996). These data strongly suggest that DNA-PK is required for the proper processing of hairpin coding ends. Since DNA-PK has no known hairpin opening activity, we have postulated that it may help to remodel the postcleavage complex, recruit additional joining factors to the complex (Roth et al., 1995; Zhu et al., 1996; Bogue et al., 1997), or activate the hairpin opening nuclease (Roth et al., 1995; Zhu and Roth, 1995; Besmer et al., 1998). Our data raise the possibility that DNA-PK may modulate the hairpin opening activity of the RAG proteins. In summary, our data genetically link the in vitro hairpin opening activity of the RAG proteins to a defect in coding joint formation, providing the most compelling evidence to date that the RAG proteins play an essential

role in hairpin opening in vivo. Our observation that E547Q is defective for end processing also suggests that other endonucleolytic activities performed by the RAG proteins in vitro (Santagata et al., 1999) might also have important in vivo correlates. Experimental Procedures Plasmid Constructs and Mutagenesis All conserved glutamates and aspartates in truncated active core of RAG1 (amino acids 384–1008) were mutated to their noncharged counterparts (D to N and E to Q) as described previously (Landree et al., 1999). Two independent clones of each mutant were generated. Both clones were independently assayed, and both copies yielded identical results; results are shown for one clone. DNA sequence analysis of the entire open reading frames of the E547Q and E423Q mutants revealed no other mutations. RAG1 GST-fusion proteins containing the E547Q and E423Q mutations were constructed using the previously described RAG1 GST fusion vector pEBG-1⌬N (kindly provided by D. Schatz) that encodes amino acids 384–1008 (Spanopoulou et al., 1996; Sawchuk et al., 1997). Mutations were introduced using a double-stranded mutagenesis protocol (Deng and Nickoloff, 1992). DNA was isolated from single clones, and the open reading frame was sequenced to confirm the mutation.

Transfections Chinese hamster ovary fibroblasts (RMP41 cells) were transiently transfected using the Fugene-6 transfection reagent (Roche) as described previously (Landree et al., 1999). Cells were transfected with 2.1 ␮g of wild-type or mutant RAG1 expression vector (pMAL-2), 2.5 ␮g wild-type RAG2 expression vector (pMAL-1) (Landree et al., 1999), and 5 ␮g of either the pJH290 or the pJH299 recombination substrate (Lieber et al., 1988; Hesse et al., 1989). Transfections with 4.6 ␮g of the pcDNAI/Amp vector were used as “no RAG” controls. Where indicated, 1.5 ␮g of SV40TdTs, an expression vector encoding the short form of murine terminal deoxynucleotidyl transferase (kindly provided by Dr. S. Gilfillan), was cotransfected. Robust expression of TdT was confirmed by Western blotting. Forty-eight hours posttransfection, DNA was harvested according to the method of Hirt (1967) and resuspended in a final volume of 30 ␮l TE. Western Blotting Proteins from transfection cell lysates were separated on 8% SDS– PAGE gels and transferred to a nylon membrane. RAG expression was determined using an anti-c-myc antibody (PharMingen) followed by an HRP-conjugated anti-mouse IgG1 antibody (PharMingen) and visualized using the ECL-Plus kit (Amersham Pharmacia).

Signal Joint Assays Signal joints were detected by PCR (24 cycles) using 0.1 ␮l of DNA harvested from a transfection. Primers DR55 and ML68 were used to detect signal joints derived from pJH290 (Steen et al., 1997), and DR55 and DR100 were used to detect signal joints from pJH299 (Han et al., 1998). Ten microliters of each PCR reaction was run on a 6% polyacrylamide gel, transferred to Genescreen Plus, and probed with a radiolabeled oligonucleotide, DR55.

Coding Joint Assays Coding joints were detected by PCR (24 cycles) using 0.1 ␮l of the DNA from a transfection. Primers DR99 and DR100 were used to detect joints derived from pJH290; DR99 and ML68 were used to detect junctions produced from pJH299. Ten microliters of each PCR reaction was run on a 6% polyacrylamide gel, transferred to Genescreen Plus, and probed with 32P end-labeled DR99. Coding joints were obtained for sequence analysis by isolation of chloramphenicol-resistant colonies derived from the recombination substrates, pJH290, or pJH299, as described (Hesse et al., 1987).

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Southern Blotting Undigested DNA samples from transfections with pJH290 or pJH289 as the substrate were subjected to Southern blot analysis and probed with an internally 32P-labeled 693 nucleotide PvuII fragment derived from pJH290 that hybridizes to the recombination intermediates (Steen et al., 1997). Purified GST-Fusion Proteins Wild-type or mutant GST-RAG1 expression vector (pEBG-1⌬N) and wild-type GST-RAG2 expression vector (pEBG-2⌬C) were transfected into RMP41 cells. After 48 hr, cells were lysed, and GSTRAG1 and GST-RAG2 proteins were copurified using a glutathione resin, as previously described (Spanopoulou et al., 1996; Sawchuk et al., 1997). Fusion proteins were dialyzed against 150 mM potassium glutamate, 25 mM HEPES (pH 7.5), 20% glycerol, 2 mM DTT. Protein concentrations were determined by Coomassie blue staining of polyacrylamide gels. Oligonucleotide Cleavage Assays Oligonucleotide substrates with the 12-RSS (DAR39/40) and the 23RSS (DG61/62) have previously been described (McBlane et al., 1995). All 12-RSS substrates were 32P end labeled on the top strand using T4 polynucleotide kinase. Substrates were annealed in 100 mM potassium glutamate. Oligonucleotide cleavage assays were performed as previously described (Hiom and Gellert, 1998). Briefly, 2 ␮l of purified RAG1 and RAG2 proteins was added to a reaction containing 25 mM MOPS, 2 mM DTT, 100 ␮g/ml BSA, 5 mM CaCl2, 19 mM KOAc, 25 fmol 32P end-labeled 12-RSS, 200 ng HMG-1, and, where indicated, 250 fmol unlabeled 23-RSS. Reactions were incubated at 37⬚C for 10 min. MgCl2 or MnCl2 was added to a final concentration of 5 mM. Reactions were then incubated at 37⬚C for 45 min and terminated by addition of 2 volumes of loading dye (95% formamide, 10 mM EDTA, 0.05% bromophenol blue). Products were resolved on a 10% acrylamide gel containing 30% formamide, 0.67⫻ TBE, 7 M urea, and 12.5 mM HEPES-K (pH 7.5). Hairpin-Opening Assay The hairpin-opening assay is based on previous work (Besmer et al., 1998). The DR109 oligonucleotide (5⬘-ATCCACTGGATCCCC GGGGA____TCCCCGGGGATCCAGTGGAT-3⬘) is completely selfcomplementary and forms a 20 base pair duplex terminating in one hairpin end (Kabotyanski et al., 1995). DR109 was 5⬘ 32P end labeled using T4 polynucleotide kinase. Hairpins were annealed prior to each experiment by heating the labeled oligonucleotide to 95⬚C for 5 min and then cooling immediately on ice for 15–30 min. Each hairpin-opening reaction contained 25 fmol of 32P-labeled DNA substrate (DR109), 10 mM MnCl2, 50 mM Tris (pH 8.3), 2 mM DTT, and 2 ␮l purified GST-RAG proteins in a 10 ␮l reaction. Concentrations of wild-type and mutant proteins were normalized by Coomassie staining so that each reaction contained the same amount of protein. Reactions were performed at 30⬚C for 90 min except where otherwise indicated and terminated by the addition of 10 ␮l of formamide loading dye (98% formamide, 0.05% bromphenol blue/xylene cyanol). Reactions were denatured at 95⬚C for 5 min and then run on a 12% polyacrylamide denaturing gel containing 7 M urea. Hybrid Joint Assays In vitro hybrid joint formation assays were performed as described (Melek et al., 1998) with slight modifications. The plasmid pJH299 (100 ng) was incubated with 2 ␮l of purified GST-RAG proteins and 20 ng HMG1. Reactions (10 ␮l) were incubated at 37⬚C for 60 min in 25 mM MOPS (pH 7.0), 2 mM DTT, 30 mM potassium glutamate, 30 mM KCl, 1% glycerol, 100 ng/ml BSA, and 4 mM MgCl2. Onefifth of the reaction mix was used for PCR (36 cycles) with the primers DR55 and ML68. Transposition Transposition reactions were performed essentially as previously described (Hiom et al., 1998). Both the oligo containing the 12-RSS (DAR39/40) and the oligo containing the 23-RSS (DG61/62) were 5⬘ 32 P end labeled on the bottom strand using T4 polynucleotide kinase. Each 10 ␮l transposition reaction contained 2 ␮l purified RAG proteins (50–100 ng each RAG1 and RAG2), 25 fmol of each labeled

RSS, and 30 ng HMG-1 in 37.5 mM HEPES (pH 7.5), 3 mM DTT, 50 mM potassium glutamate, 5 mM CaCl2, 0.006% NP-40, 60 ng/␮l BSA, and 10% glycerol. The reactions were incubated at 37⬚C for 20 min. Following this incubation, 100 ng target DNA (pUC 19) was added, and MgCl2 was added to a final concentration of 3 mM. The reactions were then incubated for 30 min at 37⬚C. An equal volume of stop buffer (100 mM Tris [pH 8.0], 10 mM EDTA, 0.2% SDS, and 350 ␮g/ml proteinase K) was added, and reactions were incubated at 37⬚C for an additional 30 min. Products were separated on a 4%–20% gradient polyacrylamide gel (Novex) and were visualized using a phosphorimager (Molecular Dynamics). Acknowledgments We thank David Schatz for providing wild-type RAG-GST fusion vectors and Susan Gilfillan for providing the TdT vector. Monica Calicchio and Suzanne Robertson provided technical and secretarial assistance, respectively. We also thank Wei-han Kan for providing technical help. We are grateful to Vicky Brandt for editorial help and for helpful discussions. We thank John Petrini for discussions and for communicating unpublished data. We thank Leslie Huye for purifying HMG-1 protein. Leslie Huye, Matt Neiditch, and Mary Purugganan provided critical comments on the manuscript. This work was supported by a grant from the National Institutes of Health (AI-36420). H. Y. S. was supported by a predoctoral fellowship from the National Institutes of Health (T32-AI07495). M. A. L. is supported by a National Institutes of Health Predoctoral Fellowship (T32-AI07495). D. B. R. is an Assistant Investigator of the Howard Hughes Medical Institute. Received July 17, 2000; revised December 14, 2000. References Agard, E.A., and Lewis, S.M. (2000). Postcleavage sequence specificity in V(D)J recombination. Mol. Cell. Biol. 20, 5032–5040. Agrawal, A., and Schatz, D.G. (1997). RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 89, 43–53. Agrawal, A., Eastman, Q.M., and Schatz, D.G. (1998). Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394, 744–751. Besmer, E., Mansilia-Soto, J., Cassard, S., Sawchuk, D.J., Brown, G., Sadofsky, M., Lewis, S.M., Nussenzweig, M.C., and Cortes, P. (1998). Hairpin coding end opening is mediated by RAG1 and RAG2 proteins. Mol. Cell 2, 817–828. Bogue, M., and Roth, D.B. (1996). Mechanism of V(D)J recombination. Curr. Opin. Immunol. 8, 175–180. Bogue, M.A., Wang, C., Zhu, C., and Roth, D.B. (1997). V(D)J recombination in Ku86-deficient mice: Distinct effects on coding, signal, and hybrid joint formation. Immunity 7, 37–47. Bogue, M.A., Jhappan, C., and Roth, D.B. (1998). Analysis of variable (diversity) joining recombination in DNA-dependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation. Proc. Natl. Acad. Sci. USA 95, 15559–15564. Deng, W.P., and Nickoloff, J.A. (1992). Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem. 200, 81–88. Eastman, Q.M., Leu, T.M.J., and Schatz, D.G. (1996). Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85–88. Fugmann, S.D., Villey, I.J., Ptaszek, L.M., and Schatz, D.G. (2000). Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Mol. Cell 5, 97–107. Gao, Y., Chaudhuri, J., Zhu, C., Davidson, L., Weaver, D.T., and Alt, F.W. (1998). A targeted DNA-PKcs-null mutation reveals DNA-PKindependent functions for Ku in V(D)J recombination. Immunity 9, 367–376.

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