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Repair of Chromosomal RAG-Mediated DNA Breaks by Mutant RAG Proteins Lacking Phosphatidylinositol 3-Like Kinase Consensus Phosphorylation Sites This information is current as of October 2, 2021. Eric J. Gapud, Baeck-Seung Lee, Grace K. Mahowald, Craig H. Bassing and Barry P. Sleckman J Immunol 2011; 187:1826-1834; Prepublished online 8 July 2011;

doi: 10.4049/jimmunol.1101388 Downloaded from http://www.jimmunol.org/content/187/4/1826

Supplementary http://www.jimmunol.org/content/suppl/2011/07/08/jimmunol.110138 Material 8.DC1 http://www.jimmunol.org/ References This article cites 51 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/187/4/1826.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Repair of Chromosomal RAG-Mediated DNA Breaks by Mutant RAG Proteins Lacking Phosphatidylinositol 3-Like Kinase Consensus Phosphorylation Sites

Eric J. Gapud,*,1 Baeck-Seung Lee,*,1 Grace K. Mahowald,* Craig H. Bassing,†,‡ and Barry P. Sleckman*

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining Downloaded from gene segments and their ﬂanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modiﬁed to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or http://www.jimmunol.org/ RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination. The Journal of Immunology, 2011, 187: 1826–1834.

ymphocyte Ag receptor genes are assembled by the pro- phosphorylated signal ends to generate a coding joint and a signal cess of V(D)J recombination, whereby different V,D, and J joint, respectively (3, 4). L gene segments are appended to generate the second exon The ataxia telangiectasia mutated (ATM) and DNA dependent of all Ag receptor genes (1). The V(D)J recombination reaction protein kinase catalytic subunit (DNA-PKcs) proteins are mem- can be divided into DNA cleavage and joining steps. The DNA bers of the phosphatidylinositol 3-like family of serine/threonine by guest on October 2, 2021 cleavage step is carried out by the RAG1 and RAG2 proteins, kinases and are activated early in the DSB response (5–7). Once which together form the RAG endonuclease that introduces DNA activated, ATM and DNA-PKcs phosphorylate and regulate a host double-strand breaks (DSBs) at the borders of two recombining of downstream proteins that function in DNA damage responses gene segments and their associated RAG recognition sites, termed and DSB repair (5–10). ATM and DNA-PKcs specifically phos- recombination signals (RSs) (2). Proteins belonging to the non- phorylate serine and threonine residues that are directly followed homologous end-joining pathway of DNA DSB repair process and by glutamine (SQ/TQ motifs). Both kinases are activated by RAG join the resulting pair of hairpin-sealed coding ends and blunt DSBs and are integrally involved in the processing and joining of coding and signal ends (3, 4, 11–19). ATM functions to stabilize coding ends in postcleavage complexes until they can be joined *Department of Pathology and Immunology, Washington University School of Med- (16). DNA-PKcs promotes the hairpin-opening activity of the icine, St. Louis, MO 63110; †Department of Pathology and Laboratory Medicine, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Univer- Artemis nuclease (3, 4, 20, 21). ATM and DNA-PKcs also have sity of Pennsylvania School of Medicine, Philadelphia, PA 19104; and ‡Abramson overlapping activities that are critical for the efficient repair of Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104 signal ends (12, 13). 1 E.J.G. and B.-S.L. contributed equally to this work. A majority of the known functions attributed to ATM and DNA- Received for publication May 11, 2011. Accepted for publication June 13, 2011. PKcs during the process of V(D)J recombination depend on their This work was supported by National Institutes of Health Grants AI074953 (to B.P.S.), kinase activities, suggesting that they modulate downstream targets AI47829 (to B.P.S.), CA136470 (to B.P.S. and C.H.B.), and CA125195 (to C.H.B.). in DSB repair pathways (3, 4, 12, 13, 16). In this regard, many E.J.G. was supported by a Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Immunology training grant awarded to Washington University. C.H.B. is proteins involved in the repair of RAG DSBs can be phosphory- a Leukemia and Lymphoma Society scholar. lated by ATM or DNA-PKcs either in vitro or in vivo. In addition Address correspondence and reprint requests to Dr. Barry P. Sleckman, Depart- to ATM and DNA-PKcs themselves, these proteins include Ku70, ment of Pathology and Immunology, 660 South Euclid Avenue, Campus Box 8118, Ku80, XRCC4, DNA Ligase IV, Artemis, XLF, H2AX, and the Washington University School of Medicine, St. Louis, MO 63110. E-mail address: [email protected] components of the MRN complex (Mre11, Rad50, and Nbs1) (3, The online version of this article contains supplemental material. 4, 9, 19, 22–36). Abbreviations used in this article: abl, abelson; ATM, ataxia telangiectasia mutated; In addition to their central role in DNA cleavage, the RAG DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DSB, double-strand proteins also have been implicated in repairing the DSBs generated break; RS, recombination signal; SQ/TQ, serine and threonine residues directly fol- by their endonuclease function (37–39). After DNA cleavage lowed by glutamine; VkD, Vk oligonucleotide. in vitro, the RAG proteins remain closely associated with signal Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ends in postcleavage complexes (40, 41). Subsequent dissociation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101388 The Journal of Immunology 1827 of the RAG proteins from signal ends is required for the joining of The mutant RAG2 cDNA with the two SQ and lone TQ motifs converted 3AQ these DNA ends in vitro (42). In contrast, the role of the RAG to AQ (RAG2 ) was generated using a PCR-based mutagenesis ap- proteins during coding joint formation remains unclear. RAG2 proach. This was done through sequential overlapping PCR using complementary oligonucleotide pairs that contained the three mutations. These possesses 3 SQ/TQ motifs, and RAG1 has 10, any of which could sense and antisense pairs included S165A, T264A, and S365A oligonu- be phosphorylated by ATM and/or DNA-PKcs to modulate RAG cleotides (Supplemental Table I). A mutant BstXI/SacI 0.9-kb fragment function during the repair steps of V(D)J recombination. In this was generated using these oligonucleotides coupled with the 59 RAG2- HindIII and the PSP72DS oligonucleotide (Supplemental Table I). pSP72- regard, DNA-PKcs has been shown to phosphorylate RAG2 on 3AQ 3AQ 365 FLAG-RAG2 and pSP72-FLAG-RAG2 -GFP were generated by a conserved SQ motif (Ser ) in vitro (43). However, no signifi- replacing the 0.9-kb BstXI/SacI fragment in pSP72-FLAG-RAG2WT and cant effects on V(D)J recombination were observed in cells ex- pSP72-FLAG-RAG2WT-GFP with the mutant PCR fragment. pressing RAG2 containing a Ser365 to alanine mutation (44). In The mutant RAG1 cDNA with the six SQ and four TQ motifs changed to 10AQ addition, cells expressing a mutant form of RAG1 with two con- AQ (RAG1 ) was also generated using a sequential overlapping PCR served SQ motifs (S479 and S913) mutated to alanine also mutagenesis approach. The overlapping pairs of oligonucleotides included sense and antisense oligonucleotide pairs T76A, T136A, S191A, T250A, exhibited no defects in V(D)J recombination (44). Because only S479A, S635A, S738A, T859A, S913A, and S1034A oligonucleotides. a subset of RAG1 and RAG2 SQ/TQ motifs were analyzed, it These mutations were clustered in three fragments: BmtI/SphI 1.5-kb remains possible that phosphorylation of the RAG proteins by fragment (T76A, T136A, S191A, T250A, and S479A), SphI/MluI 1.5-kb ATM or DNA-PKcs at other SQ/TQ motifs is required for the fragment (S635A, S738A, T859A, and S913A), and an MluI/BglII 0.2-kb fragment (S1034A). The oligonucleotides used to flank the BmtI/SphI 1.5- normal repair of RAG-mediated DSBs. Moreover, in this study, kb fragment were RAG1-BmtI and S635A antisense. The oligonucleotides V(D)J recombination was analyzed on extrachromosomal plasmid used to flank the SphI/MluI 1.5-kb fragment were S479A sense and substrates in which the requirements for repair may be different PSP72DS. The oligonucleotides used to flank the MluI/SfiI 0.2-kb frag- from RAG DSBs generated within the context of the chromo- ment were S913A sense and PSP72DS. These fragments were shuttle Downloaded from cloned into pSP72-FLAG-RAG1WT and pSP72-FLAG-GFP-RAG1WT to some. Indeed, although neither ATM nor Mre11 deficiency 10AQ 10AQ generate pSP72-FLAG-RAG1 and pSP72-FLAG-GFP-RAG1 . leads to defects in the repair of RAG DSBs generated on extra- Conditions for all PCR reactions were 95˚C for 5 min followed by 15 chromosomal plasmid substrates, repair of chromosomal RAG cycles of 92˚C for 1 min, 57˚C for 1.5 min, and 72˚C for 1.5 min. The DSBs is defective in both mutant backgrounds (14–16, 18, 29, different RAG1 and RAG2 cDNAs were then subcloned into the pCST- 30, 45, 46). iThy1.1 retroviral vector and then introduced into abl pre-B cells by ret-

roviral transduction as previously described (16, 49). http://www.jimmunol.org/ We have previously developed an experimental approach that allows for the induction of chromosomal V(D)J recombination in Mice abelson-transformed pre-B cells, hereafter referred to as abl pre- Animals were housed in a specific pathogen-free animal facility at B cells (16). Treatment of abl pre-B cells with the abl kinase in- Washington University. Animal protocols were approved by the Washington hibitor STI571 leads to: 1) G1 cell-cycle arrest; 2) induction of University Institutional Animal Care and Use Committee. RAG gene expression; 3) rearrangement of the endogenous IgLk Generation and culture of abl pre-B cell lines locus; and 4) robust recombination at chromosomally integrated retroviral recombination substrates (16, 47). In this study, we V-abl–transformed pre-B cells were generated by culturing bone marrow 2/2 2/2 from 3–5-wk-old mice with the pMSCV v-abl retrovirus as described pre- modify this approach using RAG1 and RAG2 abl pre- 2/2 2/2 viously (16). To generate RAG2 :INV and RAG1 :INV cell lines, by guest on October 2, 2021 B cells to study RAG1 and RAG2 function. When treated with RAG22/2 and RAG12/2 abl pre-B cells were infected with pMX-INV, and 2/2 2/2 STI571, RAG1 and RAG2 abl pre-B cells undergo G1 cell- cells that had integrated the recombination substrate were sorted based on cycle arrest, but do not generate RAG DSBs. Constitutive ex- CD4 expression. These cells were then subcloned, and individual subclones pression of RAG1 or RAG2 by retroviral transduction of RAG12/2 were analyzed by Southern blotting to identify cells with single pMX-INV 2/2 integrants. Cells that underwent robust pMX-INV rearrangement when or RAG2 abl pre-B cells, respectively, restores STI571-in- reconstituted with RAG1 (for RAG12/2:INV abl pre-B cells) or RAG2 (for ducible chromosomal V(D)J recombination. By comparing cells RAG12/2:INV abl pre-B cells) were chosen for analysis. RAG22/2:DELSJ 2 2 that have been reconstituted with either wild-type or mutant ver- and RAG1 / :DELSJ cell lines were made identically, except cells were SJ sions of each RAG, we now analyze the effects of distinct mu- infected with pMX-DEL . Retroviral transduction and maintenance of abl pre-B cell cultures was carried out as previously described (16). Cells were tations in RAG SQ/TQ motifs on chromosomal V(D)J recom- treated with 3.0 mM STI571 (Novartis Pharmaceuticals) for the indicated bination. times at 106 cells/ml as previously described (16). Materials and Methods Southern blotting and PCR analysis Generation of retroviral vectors encoding wild-type and Southern blot analyses for pMX-INV and pMX-DELSJ rearrangements mutant RAG proteins were carried out using the C4 and C4b probes as previously described (13, 16). Quantitative PCR analyses were carried out for VkJk rearrangements N-terminal FLAG-tagged versions of RAG1 and RAG2 were generated in using the Vk oligonucleotide (VkD) and Jk2-39 oligonucleotides (Sup- the pSP72 shuttle vector (P2191; Promega). To this end, we first generated plemental Table I). PCR products were hybridized with the probe Jk2P a version of pSP72 (pSP72-FLAG) that contained a linker composed of the (Supplemental Table I). Quantitative loading control PCR for the IL-2 gene FLAG-sense and FLAG-antisense oligonucleotides that encode a single was performed as described previously (16). Conditions for both PCR FLAG tag (Supplemental Table I). The wild-type RAG2 cDNA was reactions were 95˚C for 5 min followed by 30 cycles of 92˚C for 1 min, modified by PCR to have a 59 HindIII site that allowed for cloning of the 60˚C for 1.5 min, and 72˚C for 1.5 min. Densitometric measurements of RAG2 cDNA in-frame and downstream of the FLAG sequence in pSP72- band intensities in Fig. 2D were determined using ImageJ software (Na- FLAG to generate pSP72-FLAG-RAG2WT. To generate pSP72-FLAG- tional Institutes of Health; http://rsbweb.nih.gov/ij/). RAG1WT, the wild-type RAG1 cDNA (RAG1WT) was similarly cloned into pSP72-FLAG except for use of BmtI restriction sites in RAG1WT and Northern blotting analysis pSP72-FLAG. To generate the FLAG-RAG2WT-GFP cDNA, a previously published Northern blot analysis was carried out as previously described using a 0.5- RAG2-GFP cDNA was digested with BstXI and BglII to release a fragment kb EcoRV/SphI RAG1 cDNA fragment or an 0.8-kb PstI RAG2 cDNA that contained the 39 portion of RAG2 in-frame with a complete GFP fragment (16). WT sequence (48). This fragment was then cloned into pSP72-FLAG-RAG2 Immunoprecipitation and Western blot analyses to generate pSP72-FLAG-RAG2WT-GFP. To generate the FLAG-GFP- RAG1WT cDNA, the GFP cDNA was modified to have 59 and 39 BmtI sites Immunoprecipitation was carried out from 1 3 109 abl pre-B cells using and was subcloned into pSP72-FLAG-RAG1WT to generate pSP72-FLAG- anti-FLAG mAb (F3040; Sigma-Aldrich) using a previous protocol, except GFP-RAG1WT. that anti-FLAG Ab (1:500) was prebound to protein A-Agarose resin beads 1828 FUNCTION OF RAG1 AND RAG2 SQ/TQ MUTANTS

2 2 (50). Immunoprecipitates were separated on a 10% SDS-PAGE gel and rearrangement is also inducible in RAG2 / :INV:R2WT and transferred onto Immobilon-P PVDF Membrane (IPVH00010; Millipore). RAG12/2:INV:R1WT abl pre-B cells treated with STI571, genomic Primary Abs for blotting were anti-RAG1, 1:500 (H-300 sc5599; Santa DNA from these cells was assayed by PCR using a degenerate Cruz Biotechnology) or anti-RAG2, 1:200 (C-19 sc7623; Santa Cruz 9 Biotechnology). Secondary Abs were used at the following concentra- VkD and an oligonucleotide downstream of Jk2 (Jk2-3 ). The tion dilutions: donkey anti-rabbit F(ab’)2 fragment, 1:5000 (45-000-683; combination of these two oligonucleotides detects Vk rear- Fisher) for anti-RAG1 primary; and goat anti-mouse, 1:5000 for anti- rangements to Jk1 and Jk2 (Fig. 3A). VkJk rearrangements were RAG2 primary (62-6520; Invitrogen). Development was performed using readily detected in STI571-treated RAG22/2:INV:R2WT and the ECL Plus HRP detection kit per the manufacturer’s instructions 2/2 WT (RPN2132; Amersham Biosciences). RAG1 :INV:R1 abl pre-B cells (Fig. 3B,3C). Prior to STI571 treatment, these cells exhibit only low levels of VkJk rearrange- Flow cytometry ments (Fig. 3B,3C). Finally, RAG22/2:INV and RAG12/2:INV abl Flow cytometric analyses were carried out using a BD FACSCalibur (BD pre-B cells had no detectable IgLk rearrangements regardless of Biosciences) and data analyzed using FlowJo 4.6.2 for Macintosh (Tree whether they were treated with STI571 (Fig. 3B,3C). Together, Star). Flow cytometric cell sorting was carried out using a BD Aria Cell these findings demonstrate that chromosomal V(D)J recombi- Sorter (BD Biosciences). Rat anti-Thy1.1–PE Ab, 1:2000 (#551401; BD nation at either pMX-INV or the endogenous IgLk locus is readily Biosciences), was used to detect cells expressing Thy1.1. inducible in RAG22/2:INV or RAG12/2:INV abl pre-B cells reconstituted with wild-type RAG2 or RAG1, respectively. Results Experimental approach to study RAG function in chromosomal Chromosomal V(D)J recombination catalyzed by RAG1 and DSB repair RAG2 GFP fusion proteins Our strategy to reconstitute inducible chromosomal V(D)J re- To assay the effects of different RAG1 and RAG2 mutations on Downloaded from combination in RAG22/2 abl pre-B cells is shown in Fig. 1. A V(D)J recombination, we required cells with equivalent levels of similar strategy was used for reconstituting inducible chromo- wild-type and mutant RAG proteins. To this end, we generated somal V(D)J recombination in RAG12/2 abl pre-B cells. Initially, a version of RAG2 with an N-terminal FLAG tag and a C-terminal several independently derived RAG12/2 and RAG22/2 abl pre- GFP fusion and introduced this cDNA into the pCST-iThy1.1 B cell lines were generated with single integrants of the pMX-INV retroviral vector (Supplemental Fig. 1D,1E). RAG2WT-GFP ex- retroviral recombination substrate (RAG12/2:INV and RAG22/2: pression was readily detected by flow cytometric analyses of http://www.jimmunol.org/ INV abl pre-B cells, respectively) (Fig. 2A) (16). pMX-INV has RAG22/2:INV pre-B cells infected with the pCST-RAG2WT-GFP- a single pair of RSs that flank an antisense GFP cDNA (Fig. 2A). iThy1.1 retroviral vector (RAG22/2:INV:R2WT-GFP) (Supple- Recombination of pMX-INV occurs by inversion, placing the GFP mental Fig. 1D). Moreover, treatment of RAG22/2:INV:R2WT-GFP cassette in the sense orientation, which permits GFP expression abl pre-B cells with STI571 led to inducible rearrangement of both as an indicator of successful rearrangement. Full-length wild-type pMX-INV and the IgLk locus (Figs. 2C,2D,3B). In contrast, the RAG1 and RAG2 cDNAs with single N-terminal FLAG tags were RAG1WT-GFP C-terminal fusion was not expressed at significant introduced into the pCST-iThy1.1 retroviral vector, generating levels and did not rescue rearrangement in RAG12/2:INV cells pCST-FLAG-RAG1WT-iThy1.1 and pCST-FLAG-RAG2WT-iThy1.1, (data not shown). Accordingly, we generated and expressed by guest on October 2, 2021 respectively (Supplemental Fig. 1A). pCST-iThy1.1 has an in- a FLAG-tagged N-terminal GFP-RAG1WT fusion protein in ternal ribosome entry site-Thy1.1 cassette, permitting flow cyto- RAG12/2:INV abl pre-B cells (RAG12/2:INV:GFP-R1WT) (Sup- metric purification of cells with retroviral integrants based on Thy1.1 plemental Fig. 1E). The N-terminal GFP-RAG1 fusion variant expression (Fig. 1, Supplemental Fig. 1A,1B). rescued STI571-inducible rearrangement, albeit at lower levels Thy1.1-expressing RAG22/2:INV abl pre-B cells infected with than those observed in wild-type or RAG12/2:INV:R1WT abl pre- pCST-FLAG-RAG2WT-iThy1.1 (referred to as RAG22/2:INV:R2WT B cells (Figs. 2C,2D,3C, Supplemental Fig. 1E). Together, these abl pre-B cells) constitutively express wild-type RAG2 tran- findings demonstrate that retroviral introduction of the relevant scripts from the retrovirus (Supplemental Fig. 1C). Treatment of RAG/GFP fusion into RAG-deficient abl pre-B cells rescues these cells with STI571 leads to G1 cell-cycle arrest and induction STI571-inducible rearrangement of pMX-INV and the IgLk locus. of RAG1 gene expression, which, when coupled with retroviral Importantly, the levels of wild-type GFP-RAG1WT,RAG2WT-GFP, RAG2 expression, leads to rearrangement of pMX-INV (Fig. 2B– and their corresponding mutants can be compared by flow cy- D, Supplemental Fig. 1). Rearrangement of pMX-INV is evi- tometry. denced by GFP expression in 18% and 49% of RAG22/2:INV:R2WT abl pre-B cells treated with STI571 for 48 and 96 h, respectively Chromosomal V(D)J recombination in cells expressing an (Fig. 2B). Moreover, Southern blot analyses revealed a 3-kb SQ/TQ RAG2 mutant EcoRV/NcoI C4 probe hybridizing fragment, indicative of pMX- The murine RAG2 protein is 527-aa long and contains two SQ INV coding joint formation in STI571-treated RAG22/2:INV:R2WT (S165 and S365) motifs and one TQ (T264) motif (Fig. 4A). These abl pre-B cells, but not in RAG22/2:INV abl pre-B cells (Fig. 2C, residues do not overlap with the plant homeo domain, which 2D). Rearrangement of pMX-INV in RAG22/2:INV:R2WT abl pre- functions to tether RAG2 to methylated histone H3 present in B cells approached levels observed in wild-type abl pre-B cells chromatin (Fig. 4A) (2, 51). A version of FLAG-tagged RAG2 (WT:INV) (Fig. 2B–D). Using an identical approach, RAG12/2: was generated in which the three SQ/TQ motifs were mutated to INV abl pre-B cells were reconstituted with wild-type RAG1 AQ (RAG23AQ). This mutant was introduced retrovirally into (RAG12/2:INV:R1WT abl pre-B cells) using the pCST-FLAG- RAG22/2:INV abl pre-B cells, yielding a cell line that expresses RAG1WT-iThy1.1 retroviral vector. Treatment of RAG12/2:INV:R1WT RAG23AQ (RAG22/2:INV:R23AQ) at levels equivalent to wild-type abl pre-B cells with STI571 also led to inducible rearrangement RAG2 in RAG22/2:INV:R2WT abl pre-B cells (Fig. 4B). After treat- of pMX-INV (Fig. 2B–D). ment with STI571, robust rearrangement of pMX-INV was ob- The abl pre-B cells undergo inducible rearrangement of the served in both RAG22/2:INV:R2WT and RAG22/2:INV:R23AQ abl endogenous IgLk locus upon inhibition of the abl kinase with pre-B cells, as evidenced by both flow cytometric analyses and STI571. The murine IgLk locus contains ∼250 Vk gene segments Southern blotting (Fig. 4C,4D). Analyses of another independently and 4 functional Jk gene segments. To determine if IgLk gene generated RAG22/2:INV abl pre-B cell line that expressed either The Journal of Immunology 1829 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Experimental approach for studying RAG2 mutations. RAG22/2 abl pre-B cells are ﬁrst infected with pMX-INV, and cell lines with single pMX-INV integrants (RAG22/2:INV) were isolated. RAG22/2:INV abl pre-B cells were then infected with retroviruses encoding wild-type RAG2 (or RAG2 mutants) with Thy1.1 as an indicator of retroviral transduction to generate RAG22/2:INV:R2 abl pre-B cells. These retroviruses permit constitutive expression of RAG2 (shown in gray). Treatment with STI571 leads to G1 cell-cycle arrest and induction of transcription at the RAG1 and RAG2 loci. As the RAG2 gene has been replaced by the neomycin resistance gene, treatment with STI571 leads only to RAG1 protein expression (shown in gray). This coupled with RAG2 expression from the retrovirus leads to rearrangement of the pMX-INV and the endogenous IgLk locus (gray arrow).

RAG2 or RAG23AQ yielded similar findings (Supplemental Fig. 2A, are linked to a loss of ATM-mediated RAG2 phosphorylation, 2B). Moreover, STI571-treated RAG22/2:INV:R2WT and RAG22/2: we performed Southern blot analysis of genomic DNA from INV:R23AQ abl pre-B cells have equivalent levels of IgLk Vk to RAG22/2:INV:R23AQ abl pre-B cells treated with STI571. We did Jk rearrangements (Fig. 3B). not detect unrepaired pMX-INV coding ends or pMX-INV hybrid During chromosomal V(D)J recombination, deficiency of ATM joints, which were both readily apparent in ATM-deficient abl pre- leads to a 10–20% loss of coding ends from postcleavage com- B cells (Fig. 4D,4E). Similar results were observed when ana- plexes (16). This dissociation results in diminished coding joint lyzing another independently derived RAG22/2:INV abl pre- formation, an accumulation of unrepaired coding ends, and the B cells reconstituted with RAG2WT or RAG23AQ (Supplemental formation of pMX-INV hybrid joints, the latter of which are Fig. 2B). Finally, V(D)J recombination was similar in RAG22/2:INV produced by aberrant ligation of chromosomal pMX-INV coding abl pre-B cells that express RAG23AQ-GFP (RAG22/2:INV-R23AQ- and signal ends (Figs. 2A,4D,4E). To test whether these defects GFP) or RAG2WT-GFP (RAG22/2:INV-R2WT-GFP) (Fig. 4D,4F). 1830 FUNCTION OF RAG1 AND RAG2 SQ/TQ MUTANTS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. IgLk rearrangement in abl pre-B cells expressing RAG1 and RAG2 mutants. A, Schematic of PCR strategy for amplifying VkJk1 and VkJk2 coding joints. The RSs (triangles) and relative positions of the VkD and Jk2-39 oligonucleotides used for PCR ampliﬁcation and the Jk2P oligonucleotide used as a probe are shown. VkJk1 and VkJk2 coding joint formation results in 800-bp and 400-bp PCR products, respectively. Southern blot analysis of VkJk1 and VkJk2 coding joint PCR products ampliﬁed from genomic DNA (5-fold dilutions) obtained before and after 96 h in culture with STI571 from RAG22/2:INV, RAG22/2:INV:R2WT, RAG22/2:INV:R23AQ, RAG22/2:INV:R2WT-GFP and RAG22/2:INV:R23AQ- GFP abl pre-B cells (B) and RAG12/2:INV, RAG12/2:INV:R1WT, RAG12/2: INV:R110AQ, RAG12/2:INV:GFP-R1WT,orRAG12/2:INV:GFP-R110AQ abl pre-B cells (C). IL-2 gene PCR is also shown as a loading control.

Together, these data demonstrate that phosphorylation of RAG2 at SQ/TQ motifs is dispensable for efﬁcient formation of chro- 2/2 FIGURE 2. Rescue of chromosomal V(D)J recombination in RAG1 mosomal coding joints. and RAG22/2 abl pre-B cells. A, Schematic of the pMX-INV retroviral recombination substrate. Unrearranged (UR) pMX-INV and pMX-INV with Coding joint formation in cells expressing SQ/TQ mutant a coding end intermediate (CE), completed coding joint (CJ), signal joint RAG1 (SJ), and hybrid joint (HJ) products are shown. The approximate positions of The mouse RAG1 protein is 1040-aa long and contains four TQ the EcoRV (E) and NcoI (N) sites and C4 probe are indicated. B, Flow (T76, T136, T250, and T859) and six SQ (S191, S479, S635, S738, cytometric analysis of GFP expression at various times (h) after treatment with STI571 for wild-type (WT:INV) abl pre-B cells, (RAG12/2:INV) pre-B cells transduced with pCST-FLAG-RAG1WT-iThy1.1 (RAG12/2:INV:R1WT), 2 2 and RAG22/2 abl pre-B cells transduced with pCST-FLAG-RAG2WT- R1WT) and RAG2 / pre-B cells transduced with pCST-FLAG-RAG2WT- 2 2 iThy1.1 (RAG22/2:INV:R2WT), each containing a single pMX-INV genomic GFP-iThy1.1 (RAG2 / :INV:R2WT-GFP) and treated with STI571 for the integrant. C, EcoRV + NcoI-digested genomic DNA probed with the C4 indicated times. The rearrangement products (HJ and CJ) and intermediates probe. DNA samples from abl pre-B cells from B and RAG12/2 pre-B cells (CE) for pMX-INV are shown. Molecular weight markers are shown. D, transduced with pCST-FLAG-GFP-RAG1WT-iThy1.1 (RAG12/2:INV:GFP- Relative quantiﬁcation of coding joints (percent CJ) formed in C. The Journal of Immunology 1831

S913, and S1034) motifs (Fig. 5A). Two serines (S635 and S738) reside within a region that mediates RS heptamer binding and RAG2 interactions with one of these serines (S738) present in a zinc finger region (2). To investigate the potential function of these sites in coding joint formation, we developed mutant versions of FLAG-tagged RAG1 (RAG110AQ) and GFP-RAG1 (GFP- RAG110AQ) in which all 10 of these SQ/TQ motifs were mutated to AQ. These proteins were expressed in RAG12/2:INV abl pre- B cells, and stable clones with equivalent levels of RAG110AQ (RAG12/2:INV:R110AQ) and RAG1WT (RAG12/2:INV:R1WT)or GFP-RAG110AQ (RAG12/2:INV:GFP-R110AQ) and GFP-RAG1WT (RAG12/2:INV:GFP-R1WT) were selected for analysis (Fig. 5B, 5F). After induction of V(D)J recombination, coding joint formation at pMX-INV and the IgLk locus proceeded with similar kinetics in RAG12/2:INV:R110AQ and RAG12/2:INV:R1WT abl pre-B cells (Figs. 3C,5C,5D). Similar results were obtained upon analysis of a second set of independently derived RAG12/2:INV:R110AQ and RAG12/2:INV:R1WT abl pre-B cell lines (Supplemental Fig. 2C, 2D). Moreover, neither unrepaired pMX-INV coding ends nor Downloaded from pMX-INV hybrid joints were detected in RAG12/2:INV:R110AQ abl pre-B cells (Fig. 5D,5E, Supplemental Fig. 2D). Similar results were obtained upon analysis of IgLk locus and pMX-INV rearrangement in RAG12/2:INV:GFP-R1WT and RAG12/2:INV: GFP-R110AQ abl pre-B cells (Figs. 3C,5D and data not shown). Thus, we conclude that phosphorylation of RAG1 at any of the 10 http://www.jimmunol.org/ SQ/TQ motifs is not essential for efficient coding joint formation. Function of RAG1 and RAG2 during signal joint formation ATM and DNA-PKcs have redundant functions during signal joint formation, suggesting that they phosphorylate common downstream targets important for the repair of signal ends (12, 13). To determine if RAG1 or RAG2 are important targets of ATM and DNA-PKcs during this process, we generated several RAG12/2 and RAG22/2 abl pre-B cell lines with single integrants of the by guest on October 2, 2021 pMX-DELSJ retroviral recombination substrate (RAG12/2:DELSJ and RAG22/2:DELSJ). pMX-DELSJ is identical to pMX-INV except that the RSs have been reoriented such that rearrangement results in the formation of a chromosomal signal joint (Supple- mental Fig. 3A). Southern blot analyses can be carried out to detect unrepaired pMX-DELSJ chromosomal signal ends. In this regard, inducing rearrangement in DNA Ligase IV-deficient abl pre-B cells containing pMX-DELSJ (LigIV2/2:DELSJ) leads to an accumulation of unrepaired signal ends due to the deficiency in DNA Ligase IV (Fig. 6, Supplemental Fig. 3). RAG12/2:DELSJ abl pre-B cells were transduced with retroviruses encoding RAG1WT and RAG110AQ, and RAG22/2:DELSJ abl pre-B cells were transduced with retroviruses encoding RAG2WT-GFP, and RAG23AQ-GFP. Robust pMX-DELSJ signal joint formation was observed after induction of V(D)J recombination in both RAG12/2:DELSJ:R1WT and RAG22/2:DELSJ:R2WT-GFP abl pre-B cells treated with STI571 (Fig. 6, Supplemental Fig. 3). Treatment of RAG12/2:DELSJ:R110AQ and RAG22/2:DELSJ:

FIGURE 4. SQ/TQ motifs in RAG2 are not required for efﬁcient coding joint formation. A, Schematic showing positions of the two SQ motifs and (D) or EcoRV (E) and probed with the C4 probe. Atm2/2:INV, RAG22/2: the single TQ motif in the RAG2 protein. The plant homeo domain is also INV, RAG22/2:INV:R2WT, RAG22/2:INV:R23AQ, RAG22/2:INV:R2WT-GFP, shown (black). B, Western blot analysis of RAG2 protein expression in and RAG22/2:INV:R23AQ-GFP abl pre-B cells were treated with STI571 2 2 2 2 2 2 RAG2 / :INV, RAG2 / :INV:R2WT,orRAG2 / :INV:R23AQ pre-B cells. for 0, 48, or 96 h prior to harvesting DNA. Bands representing pMX-INV Samples were immunoprecipitated with an anti-FLAG Ab followed by unrearranged (UR), coding joint (CJ), hybrid joint (HJ), and coding end immunoblotting with an anti-RAG2 Ab. C, Flow cytometric analysis of intermediate (CE) are indicated. Molecular weight markers are also 2 2 2 2 GFP expression after treatment of RAG2 / :INV, RAG2 / :INV:R2WT, and shown. F, Flow cytometric analysis of GFP expression in RAG22/2:INV 2 2 RAG2 / :INV:R23AQ abl pre-B cells with STI571 for 0, 48, or 96 h. (dotted line), RAG22/2:INV:R2WT-GFP (solid line), and RAG22/2:INV: Southern blot analysis of genomic DNA digested with EcoRV and NcoI R23AQ-GFP (dashed line) pre-B cells. 1832 FUNCTION OF RAG1 AND RAG2 SQ/TQ MUTANTS Downloaded from http://www.jimmunol.org/

FIGURE 6. Signal joint formation in abl pre-B cells expressing RAG1 or RAG2 SQ/TQ mutants. A and B, Southern blot analyses of EcoRV- digested genomic DNA probed with the C4b probe. RAG12/2:DELSJ abl pre-B cells and RAG12/2:DELSJ abl pre-B cells transduced with pCST- FLAG-RAG1WT-iThy1.1 (RAG12/2:DELSJ:R1WT) or a pCST-FLAG- RAG110AQ-iThy1.1 (RAG12/2:DELSJ:R110AQ)(A) and RAG22/2:DELSJ abl pre-B cells and RAG22/2:DELSJ abl pre-B cells transduced with pCST- FLAG-RAG2WT-GFP-iThy1.1 (RAG22/2:DELSJ:R2WT-GFP) or pCST- by guest on October 2, 2021 FLAG-RAG23AQ-GFP-iThy1.1 (RAG22/2:DELSJ:R23AQ-GFP)(B) pre-B cells were treated with STI571 for 0, 48, or 96 h. Bands representing unrearranged (UR) pMX-DELSJ and normal signal joint (SJ) and unrepaired signal end (SE) are indicated. Molecular weight markers are shown.

R23AQ-GFP abl pre-B cells also led to efﬁcient pMX-DELSJ signal joint formation with no detectable accumulation of unrepaired signal ends (Fig. 6, Supplemental Fig. 3). Thus, chromosomal signal joining appears unimpaired in the presence of RAG1 or RAG2 proteins that are crippled for phosphorylation at their SQ/ TQ motifs.

Discussion In this study, we have shown that constitutive expression of wild- type RAG1 in RAG12/2 abl pre-B cells and wild-type RAG2 in RAG22/2 abl pre-B cells rescues STI571-inducible V(D)J recombination. Indeed, the level of pMX-INV and IgLk rearrangement in these cells after treatment with STI571 is similar to what is observed in wild-type abl pre-B cells treated with STI571. Thus, this approach can be used to assess the activity of mutant forms of FIGURE 5. SQ/TQ motifs in RAG1 are not required for efﬁcient coding RAG1 and RAG2 during chromosomal V(D)J recombination. joint formation. A, Schematic showing positions of the six SQ motifs and the four TQ motifs in the RAG1 protein. The zinc ﬁnger A (gray) and B (black) regions are shown. The heptamer-binding/RAG2 interacting region 2 2 is also shown (bracket). B, Western blot analysis of RAG1 protein ex- R1WT, and RAG1 / :INV:GFP-R110AQ abl pre-B cells treated with STI571 pression in RAG12/2:INV, RAG12/2:INV:R1WT,orRAG12/2:INV:R110AQ for 0, 48, or 96 h. Analysis was carried out as described in Fig. 4D and 4E. pre-B cells. C, Flow cytometry of GFP expression at various times in Molecular weight markers are shown. F, Flow cytometric analysis of GFP 2 2 2 2 culture with STI571 in RAG12/2, RAG12/2:INV:R1WT, and RAG12/2:INV: expression in RAG1 / :INV, (dotted line), RAG1 / :INV:GFP-R1WT 2 2 R110AQ pre-B cells. D and E, Southern blot analysis of genomic DNA from (solid line), and RAG2 / :INV:GFP-R110AQ (dashed line) pre-B cells an- Atm2/2:INV, RAG12/2:INV, RAG12/2:INV:R1WT, RAG12/2:INV:GFP- alyzed in D. The Journal of Immunology 1833

We have used this approach to determine whether phosphory- Disclosures lation of any serine or threonine residues in the 3 SQ/TQ motifs in The authors have no financial conflicts of interest. RAG2 or the 10 SQ/TQ motifs in RAG1 may be responsible for any of the defects observed in signal and coding joint formation in cells deficient in ATM or DNA-PKcs. ATM-deficient abl pre- References 1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302: 575– B cells exhibit defects in coding joint formation with an accu- 581. mulation of unrepaired coding ends and significant levels of hybrid 2. Schatz, D. G., and Y. Ji. 2011. Recombination centres and the orchestration of joint formation during inversional rearrangements (16). In contrast, V(D)J recombination. Nat. Rev. Immunol. 11: 251–263. 3. Lieber, M. R., Y. Ma, U. Pannicke, and K. Schwarz. 2004. The mechanism of these defects were not observed during V(D)J recombination in vertebrate nonhomologous DNA end joining and its role in V(D)J re- 2/2 3AQ 2/2 10AQ RAG2 :INV:R2 or RAG1 :INV:R1 abl pre-B cells. combination. DNA Repair (Amst.) 3: 817–826. Additionally, although efficient repair of signal ends depends on 4. Rooney, S., J. Chaudhuri, and F. W. Alt. 2004. The role of the non-homologous end-joining pathway in lymphocyte development. Immunol. Rev. 200: 115–131. the overlapping activities of ATM and DNA-PKcs, analysis of 5. Zhou, B. B., and S. J. Elledge. 2000. The DNA damage response: putting 2 2 2 2 RAG2 / :DELSJ:R23AQ-GFP or RAG1 / : DELSJ:R110AQ abl checkpoints in perspective. Nature 408: 433–439. pre-B cells revealed no defects in signal joint formation (12, 13). 6. Durocher, D., and S. P. Jackson. 2001. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr. Opin. Cell Biol. 13: 225–231. Together, these findings demonstrate that the observed defects in 7. Shiloh, Y. 2003. ATM and related protein kinases: safeguarding genome in- the repair of RAG DSBs in cells deficient in ATM and/or DNA- tegrity. Nat. Rev. Cancer 3: 155–168. PKcs cannot solely reflect a requirement for these kinases to 8. Tomimatsu, N., B. Mukherjee, and S. Burma. 2009. Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells. EMBO phosphorylate RAG1 or RAG2 at consensus SQ/TQ motifs. ATM Rep. 10: 629–635. or DNA-PKcs could phosphorylate other nonconsensus serines or 9. Matsuoka, S., B. A. Ballif, A. Smogorzewska, E. R. McDonald, III, K. E. Hurov, 3AQ 10AQ J. Luo, C. E. Bakalarski, Z. Zhao, N. Solimini, Y. Lerenthal, et al. 2007. ATM Downloaded from threonines in RAG2 or RAG1 that exert either a direct and ATR substrate analysis reveals extensive protein networks responsive to or compensatory effect on RAG function during the joining step DNA damage. Science 316: 1160–1166. of the reaction. However, recent analyses of .700 ATM targets 10. Calleń, E., M. Jankovic, N. Wong, S. Zha, H. T. Chen, S. Difilippantonio, M. Di Virgilio, G. Heidkamp, F. W. Alt, A. Nussenzweig, and M. Nussenzweig. 2009. in response to ionizing radiation reveal that essentially all of Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis the target serines and threonines were part of SQ/TQ motifs (9). in ATM-deficient lymphocytes. Mol. Cell 34: 285–297. Our studies are in agreement with previous analyses of mutants of 11. Matei, I. R., C. J. Guidos, and J. S. Danska. 2006. ATM-dependent DNA damage

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