Aberrantly resolved RAG-mediated DNA breaks inAtm-deficient lymphocytes target chromosomalbreakpoints in cisGrace K. Mahowalda, Jason M. Barona, Michael A. Mahowalda, Shashikant Kulkarnia, Andrea L. Bredemeyera,Craig H. Bassingb,c, and Barry P. Sleckmana,1

aDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Pathology and LaboratoryMedicine, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA19104;and cAbramson Family Cancer Research Institute, Philadelphia, PA 19104

Edited by Frederick W. Alt, Harvard Medical School, Boston, MA, and approved August 31, 2009 (received for review March 7, 2009)

Canonical chromosomal translocations juxtaposing antigen recep-tor genes and oncogenes are a hallmark of many lymphoid malig-nancies. These translocations frequently form through the joiningof DNA ends from double-strand breaks (DSBs) generated by therecombinase activating gene (RAG)-1 and -2 proteins at lympho-cyte antigen receptor loci and breakpoint targets near oncogenes.Our understanding of chromosomal breakpoint target selectioncomes primarily from the analyses of these lesions, which areselected based on their transforming properties. RAG DSBs arerarely resolved aberrantly in wild-type developing lymphocytes.However, in ataxia telangiectasia mutated (ATM)-deficient lym-phocytes, RAG breaks are frequently joined aberrantly, formingchromosomal lesions such as translocations that predispose (ATM)-deficient mice and humans to the development of lymphoidmalignancies. Here, an approach that minimizes selection biases isused to isolate a large cohort of breakpoint targets of aberrantlyresolved RAG DSBs in Atm-deficient lymphocytes. Analyses of thiscohort revealed that frequently, the breakpoint targets for aber-rantly resolved RAG breaks are other DSBs. Moreover, thesenonselected lesions exhibit a bias for using breakpoints in cis,forming small chromosomal deletions, rather than breakpoints intrans, forming chromosomal translocations.

ataxia telangiectasia mutated � chromosomal translocation �DNA double-strand break repair � V(D)J recombination

Double-strand breaks (DSBs) in DNA are generated bygenotoxic agents and cellular endonucleases as intermedi-

ates in several important physiologic processes including V(D)Jrecombination, Ig class switch recombination (CSR), DNAreplication, gene transcription, and meiosis. DNA DSBs activatea highly conserved cellular response that prevents cell cycleprogression, initiates repair of the broken DNA ends, andpromotes apoptosis of cells with persistent un-repaired DSBs (1,2). Broken DNA ends from a single DSB are usually rejoined;however, in some processes, such as V(D)J recombination andCSR, DNA ends arising from two DSBs are joined in a regulatedfashion, generating a new gene product (1–4). Broken DNA endsfrom distinct DSBs can also be joined aberrantly, leading to theformation of potentially dangerous chromosomal lesions such astranslocations, deletions, and inversions.

All developing lymphocytes generate programmed DNADSBs during V(D)J recombination, a process that joins variable(V), joining (J), and in some cases, diversity (D) gene segmentsto generate the second exon of antigen receptor genes (4). TheV(D)J recombination reaction is initiated by the recombinaseactivating gene (RAG)-1 and -2 proteins, which together formthe RAG endonuclease (5). RAG introduces DNA DSBs at theborder of two recombining gene segments and their f lankingRAG recognition sequences, termed recombination signals(RSs) (5). DNA cleavage by RAG occurs after an appropriateRS pair (12/23 compatible) forms a synaptic complex, generating

a pair of coding ends and a pair of signal ends (5). These DNAends are then processed and joined by the nonhomologousend-joining (NHEJ) pathway of DNA DSB repair to form acoding joint and signal joint, respectively (4). The generation andrepair of RAG-mediated DSBs occurs in developing lympho-cytes only during the G1-phase of the cell cycle (6).

Several lymphoid malignancies are associated with canonicaltranslocations involving antigen receptor loci and potentialoncogenes such as c-Myc and Bcl-2 (7, 8). Breakpoint analysesdemonstrate that many of these translocations form through theaberrant joining of RAG-mediated DNA breaks at antigenreceptor loci to specific chromosomal regions, hereafter referredto as breakpoint targets, near potential oncogenes. The forma-tion of these lesions likely also requires the generation of a DSBat the breakpoint target, which could potentially occur throughoff-target RAG activity (9–12). Because these lesions are se-lected based on their transforming ability, it is not possible toknow whether there is a significant mechanistic bias for the useof these breakpoint targets. However, analyses of mice in whichthe c-Myc-coding region was replaced with that of N-Mycdemonstrated that the bias for the targeting of c-Myc over N-Mycby IgH translocations in pro-B cell tumors is not due to selectionfor the specific oncogenic properties of c-Myc (13). Rather, theformation of pro-B cell tumors in these mice suggests that thereare mechanistic constraints that favor breakpoint targeting of thec-Myc locus in these cells.

Although the aberrant resolution of RAG DSBs occurs ex-tremely rarely in wild-type lymphocytes, these lesions are gen-erated at higher frequencies in lymphocytes with DNA repairdefects (14–16). In this regard, deficiency in the ataxia telangi-ectasia mutated (ATM) serine threonine kinase in both mice andhumans leads to an increased incidence of lymphoid tumors withRAG-dependent translocations involving antigen receptor loci(17–21). In addition, mature, nontransformed Atm-deficientlymphocytes have an increased frequency of translocations andother chromosomal aberrations involving antigen receptor loci(18, 19, 22–24).

Murine pre-B cells transformed through the expression ofthe viral abl kinase, hereafter referred to as abl pre-B cells,have been used to study the generation and repair of chro-mosomal RAG-generated DSBs (25). Inhibition of the ablkinase with STI571 leads to G1 cell cycle arrest, induction ofRAG expression, and robust rearrangement of the endoge-

Author contributions: G.K.M., J.M.B., C.H.B., and B.P.S. designed research; G.K.M., J.M.B.,and S.K. performed research; A.L.B. contributed new reagents/analytic tools; G.K.M.,J.M.B., M.A.M., and B.P.S. analyzed data; and G.K.M. and B.P.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: sleckman@wustl.edu..

This article contains supporting information online at www.pnas.org/cgi/content/full/0902545106/DCSupplemental.

www.pnas.org�cgi�doi�10.1073�pnas.0902545106 PNAS � October 27, 2009 � vol. 106 � no. 43 � 18339–18344

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nous Ig (Ig) � light (L) chain genes and chromosomallyintegrated retroviral recombination substrates (25–27). Induc-tion of V(D)J recombination by STI571 treatment of Atm�/�

abl pre-B cells leads to an accumulation of un-repaired codingends and to the aberrant resolution of these coding ends,analogous to what is observed in Atm-deficient lymphocytes invivo (19, 22–25). Here, we describe an experimental approachthat permits high-throughput cloning of breakpoint targetsfrom aberrantly resolved RAG DSBs generated during V(D)Jrecombination in Atm�/� abl pre-B cells. Our approach min-imizes selection biases, allowing for the elucidation of param-eters governing breakpoint targeting of chromosomal RAG-mediated DSBs that were not previously revealed through theanalysis of lymphoid tumors.

ResultsIsolation of Breakpoint Targets in Atm-Deficient Lymphocytes. Wehave previously analyzed chromosomally integrated retroviralV(D)J recombination substrates that undergo rearrangement bydeletion (pMX-DELCJ) or inversion (pMX-INV) in wild-type andAtm-deficient abl pre-B cells (Fig. S1) (25). After induction ofV(D)J recombination with STI571, these retroviral substrates un-dergo robust rearrangement with efficient coding joint formation inboth wild-type and Atm�/� abl pre-B cells (Fig. S2) (25). However,in Atm�/� abl pre-B cells. unrepaired coding ends accumulate in10–20% of cells, and approximately 10% of coding ends areresolved aberrantly as translocations or chromosomal deletions orinversions (Fig. S2) (25). In contrast, �1% of substrates exhibitaberrant joining in wild-type abl pre-B cells (25).

One of the challenges in elucidating the parameters governingbreakpoint target selection is the difficulty in obtaining largenumbers of breakpoint sequences. Because RAG breaks are ab-errantly resolved at such a high frequency in Atm�/� abl pre-B cells,we sought to isolate a large cohort of aberrantly resolved codingends from these cells. To this end, we generated seven Atm�/� ablpre-B cell lines, each with a single copy of the pMX-DELCJ orpMX-INV recombination substrate integrated at a unique chro-mosomal location. Cleavage of the substrate by RAG results in achromosomal coding end at the IRES that is either centromeric(Atm�/�:DELCJ-4, -21, -50, -84 and Atm�/�:INV-26) or telomeric(Atm�/�:DELCJ-46 and -70) (Fig. 1A and Fig. S1). In all of the cellsanalyzed, the retroviral substrate recombines robustly, leading tothe formation of normal coding joints and the accumulation ofun-repaired coding ends (Fig. S2).

We previously identified breakpoints in abl pre-B cell sub-clones isolated after the induction of V(D)J recombination (25).However, the breakpoint targets isolated by this approach couldbe biased by the impact of specific aberrant joints on cell division.In addition, aberrant joints that result in formation of dicentricor acentric chromosomes are unlikely to propagate efficiently individing cells, hindering our ability to recover these lesions. Tocircumvent these limitations, we developed a high-throughputinverse PCR (I-PCR) approach, described in detail in Materialsand Methods, to isolate the chromosomal breakpoint targets ofaberrantly resolved coding ends from retroviral substrates inG1-phase abl pre-B cells (Fig. 1B). Briefly, genomic DNA isdigested with MseI, which cuts at TTAA, and ligated at diluteconcentrations favoring circularization, followed by digestionwith KpnI to linearize the circularized products and PCRamplification of the chromosomal breakpoint targets (Fig. 1B).Most locations (86%) in the mouse genome are within 0.5 kb ofan MseI site; thus, the I-PCR was optimized to amplify productsof �1 kb in size.

I-PCR amplification of genomic DNA from Atm�/�:DELCJ

and Atm�/�:INV abl pre-B cells that had been treated withSTI571 to induce V(D)J recombination yielded a diversespectrum of products, primarily between 0.5 and 1.0 kb in size(Fig. 1C). As expected, I-PCR of genomic DNA from abl pre-B

cells that were not treated with STI571, and STI571-treatedwild-type abl pre-B cells (Atm�/�:DELCJ-119), yielded signif-icantly fewer products (Fig. 1C). These findings suggest thatthe I-PCR products from Atm�/� abl pre-B cells represent adiverse cohort of aberrantly joined coding ends. Importantly,because the RAG DSBs were generated and aberrantly joinedin cells arrested at the G1-phase of the cell cycle, we reason thatthe diversity of the recovered products will be minimallyaffected by selection.

Breakpoint Targets Are Concentrated in Antigen Receptor Loci. I-PCR products were cloned and sequenced from all seven Atm�/�

abl pre-B cell lines and from one wild-type abl pre-B cell line.

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Fig. 1. Strategy for breakpoint target analysis. (A) Chromosomal integrationsites (arrow) of pMX-DELCJ and pMX-INV in Atm�/� abl pre-B cells. (B) InversePCR (I-PCR) strategy. The pMX-DELCJ retroviral substrate is described in Fig. S1.The MseI (M) and KpnI (K) sites are shown, as is the breakpoint target sequence(gray rectangle) and the position of primers 1–3 used for PCR. (C) I-PCR andRag1 PCR of Atm�/�:DELCJ-50 and Atm�/�:DELCJ-119 abl pre-B cells treatedwith STI571 for the indicated times in hours. (Upper) I-PCR was performed onseparate aliquots of DNA from individual ligations (lanes 1–5, 6–10) or unli-gated (�) DNA. (Lower) Rag1 was amplified from 3-fold dilutions of ligated(�) or unligated (�) DNA as a loading control.

18340 � www.pnas.org�cgi�doi�10.1073�pnas.0902545106 Mahowald et al.

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Whereas approximately 65% (1903) of the sequences obtainedfrom Atm�/� cells were unique, only 14% (13) of those obtainedfrom wild-type cells were unique, consistent with our previousanalyses demonstrating that coding ends are rarely resolvedaberrantly in wild-type abl pre-B cells (25).

Of the 1903 unique breakpoint targets isolated from Atm�/�

abl pre-B cell clones, 1,542 (81%) are joined to a breakpoint atan antigen receptor locus (Fig. 2A and Table S1). Most of thesebreakpoints (95%) are at the IgL� locus in close proximity(�90% within 100 bp) of a V� RS, a J� RS, or cryptic RSs in theJ�-C� region (Fig. 2 B and C). Although only a small cohort ofaberrant joints was recovered from the wild-type abl pre-B cells,approximately 40% of these joints also involved breakpoints atthe IgL� locus in close proximity to an RS (Table S2). Together,these findings suggest that RAG DSBs at the IgL� locus arefrequent breakpoint targets for aberrantly resolved retroviralsubstrate coding ends.

Genotoxic DSBs Diversify Breakpoint Target Selection. The accumu-lation of un-repaired coding ends at the retroviral substrate maybe due, in part, to a paucity of DSBs that can serve as efficientbreakpoint targets in a subset of Atm�/� abl pre-B cells. If thisis the case, we reasoned that the introduction of DSBs throughexposure to ionizing radiation (IR) might provide additionalbreakpoint targets for these coding ends. Indeed, treatment ofAtm�/�:DELCJ-50 abl pre-B cells with 4 Gy of IR (�100 DSBsper cell) led to a significant decrease in the accumulation ofun-repaired pMX-DELCJ coding ends (Fig. 3A). Moreover,analysis of 1,622 breakpoints isolated from all seven Atm�/� ablpre-B cell lines exposed to 4 Gy IR revealed that on average, 81%(range 64% to 93%) target nonantigen receptor loci, as com-pared with 19% (range 10% to 29%) observed in un-irradiatedcells (Fig. 3B). As expected, there is an intermediate frequencyof breakpoints targeting nonantigen receptor loci in cells thatreceived 0.4 Gy IR (�10 DSBs per cell) (Fig. 3C). Thus, theintroduction of genotoxic DSBs leads to a decrease in theaccumulation of un-repaired substrate coding ends and to sig-nificant breakpoint target diversification for aberrantly resolvedRAG breaks in Atm�/� abl pre-B cells.

Preferential Use of Breakpoint Targets in cis. In un-irradiated ablpre-B cells, 60% (range 33–76%) of breakpoint targets atnonantigen receptor loci map to regions on the same chromo-some as the retroviral recombination substrate, which we referto as breakpoint targets in cis (Fig. 4A). Approximately 87%

(range 70–98%) of these breakpoint targets are located withina short distance (�200 kb) of the recombination substrate (Fig.4 B and C). The orientation of these breakpoints suggests thatthe majority of aberrant joints in cis form by deletion of theintervening sequence rather than inversion (Fig. 4C). Of thesmall cohort of aberrant joints isolated from wild-type abl pre-Bcells, those joints that did not use IgL� breakpoint targets alsoexhibited a bias for using breakpoints in cis (7 of 8), with all sevenbreakpoints in close proximity (�30 kb) to the retroviral sub-strate (Table S2). It is conceivable that the bias for formingaberrant joints in cis could be due, in part, to synapsis betweenan RS in the retroviral substrate and a cryptic RS at thebreakpoint target. In this regard, 32% of the 217 breakpointtargets in cis have a potential cryptic RS within 100 bp. However,most of these cryptic RSs are predicted to be poor substrates forRAG cleavage (mean RIC score �35.70 for potential 12 RSs and�56.56 for potential 23 RSs) (28).

The bias for the aberrant resolution of RAG DSBs usingbreakpoint targets in cis is particularly striking in Atm�/� abl pre-Bcells treated with IR. In these cells, the fraction of breakpointtargets recovered in cis with the retroviral substrate is 2–6-foldhigher than in un-irradiated cells (Fig. 4D). Moreover, the fre-quency of breakpoint targets in cis among cells receiving IR is 6- to10-fold higher than would be predicted based on the relativecontribution of the chromosome harboring the substrate to theentire genome (Fig. 4E). Together, these findings demonstrate thatthe aberrant joining of coding ends is significantly biased toward theuse of breakpoint targets at independently generated DSBs lying inclose proximity on the same chromosome.

DiscussionWe isolated and characterized a large cohort of breakpointtargets for aberrantly resolved coding ends generated duringV(D)J recombination in Atm�/� pre-B cells. Importantly, thesechromosomal lesions were generated in and isolated from G1-phase pre-B cells undergoing V(D)J recombination, thus mini-mizing potential selection biases. Our analyses reveal that inAtm�/� pre-B cells, the most common breakpoint targets foraberrantly resolved RAG DSBs are other DSBs. Moreover,there is a significant bias for using breakpoint targets in cis withthe RAG DSB.

In un-irradiated cells, breakpoint targets are most frequentlyfound at the IgL� locus near RSs, consistent with the notion thatthey are generated by RAG cleavage at the IgL� locus. Althoughthis could reflect a bias for the aberrant joining of two RAG

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Fig. 2. Breakpoint targets at antigen receptor loci. (A) Breakpoint targets from all Atm�/� pre-B cell lines. (B) Distribution of targeted antigen receptor loci.(C) Breakpoint targets within the IgL� locus, with an expanded view of four V� gene segments and the J�-C� region. V� and J� RSs are indicated by arrows, asare known cryptic RSs such as the intron recombining sequences (IRS) and recombining sequence (RS) 3� of C�.

Mahowald et al. PNAS � October 27, 2009 � vol. 106 � no. 43 � 18341

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breaks, it is unlikely that this occurs at a precleavage step due,for example, to synapsis of RSs in trans in the retroviral substrateand the IgL� locus. In this regard, the large cohort of aberrantjoints between the 3� coding end of the retroviral substrate andJ� gene segments would necessitate RS synapsis that violates the12/23 rule. Thus, we expect that most, if not all, of these lesionsare formed through the joining of DNA breaks generated byindependent RAG cleavage events at the retroviral substrateand at the IgL� locus. It is possible that the aberrant joining oftwo independently generated RAG breaks could be biased at apostcleavage step if, for example, the repair of RAG DSBs

generally occurs in specific regions of the nucleus, or if RAGDSBs associate with repair proteins that somehow promote theirjoining to other RAG DSBs.

It could be that RAG DSBs are normally the most abundantsource of breakpoint targets in G1-phase developing lympho-cytes. In this regard, aberrantly joined RAG DSBs between theT cell receptor �, �, and � loci, which all undergo rearrange-ment in CD4�:CD8� thymocytes, are found at increased levelsin Atm-deficient T cells (29, 30). Thus, the bias for breakpointtargets at the IgL� locus in abl pre-B cells could ref lect therobust rearrangement of this locus (25). Nevertheless, 19% ofthe breakpoint targets in Atm�/� abl pre-B cells involve regionsof the genome that do not house antigen receptor loci.Moreover, these regions do not have cryptic RSs that wouldmediate efficient RAG cleavage, although it is conceivablethat some may have features permitting RAG cleavage atsequences other than canonical RSs (9, 12). These findingssuggest that RAG DSBs can readily be joined to breakpointsnot generated by RAG-mediated DNA cleavage. In agreementwith this notion, treatment of Atm�/� abl pre-B cells with dosesof IR that should generate approximately 100 DSBs per cellleads to a dramatic reduction in the accumulation of un-repaired coding ends and an increase in the fraction ofaberrant joints involving nonantigen receptor loci. In thisregard, IR may generate DSBs in cells that do not otherwisehave breakpoint targets, or may generate additional break-point targets that can be used more efficiently, such as thosethat lie in cis with the coding end (see below).

Chromosomal translocations involving antigen receptor lociare common transforming lesions in lymphoid tumors. How-ever, we have shown that in Atm�/� abl pre-B cells, theaberrant resolution of RAG breaks favors the use of DNAbreakpoint targets on the same chromosome, forming smalldeletions rather than inversions or translocations. The bias forthe aberrant joining of a RAG break to a proximal DNA breakon the same chromosome may be due to general mechanismsthat promote the joining of broken DNA ends from distinctDSBs in cis, as proposed for the DSBs generated during Ig CSR(31). Interestingly, the joining of two DNA breaks fromdifferent switch regions during Ig CSR also appears to favordeletion over inversion, as observed here for the aberrantjoining of RAG breaks to breakpoint targets in cis. This couldref lect the activity of a directional tracking mechanism, al-though it could also be due to Atm deficiency because Atmfunctions in maintaining the stability of DSB complexes (25).In this regard, instability at two DSB complexes in cis couldlead to loss of the intervening sequence, with deletional repairremaining the only option for maintaining the integrity of thechromosome.

The preferential joining of DNA breaks in cis could be due, inpart, to their close physical proximity. In addition, this bias couldrely on other factors, such as chromatin modifications like �-H2AX,which spread for significant distances surrounding a DSB (32, 33).Such chromatin modifications could functionally ‘‘bridge’’ twoDSBs that lie in cis within several hundred kilobases of each other.By whatever mechanism, the joining of DNA ends at two DSBs incis could be important for maintaining genomic stability whilelimiting perturbation of the genome. In this regard, joining of DSBsin trans generates potentially dangerous chromosomal transloca-tions, including unstable lesions such as acentric or dicentricchromosomes. In contrast, breaks joined in cis would lead tochromosomal deletions or inversions, preserving the structuralintegrity of the chromosome.

Materials and MethodsCell Culture. Abl pre-B cells were generated, maintained, transduced withretroviruses, and treated with STI571 as described (25). Cells were irradiated

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Fig. 3. Breakpoint targets in cells with genotoxic DSBs. (A) Southern blotanalysis of pMX-DELCJ in Atm�/�:DELCJ-50 abl pre-B cells treated with STI571and IR as indicated. (B) Percentage of breakpoints targeting nonantigenreceptor loci (dark gray) in cells receiving 0 or 4 Gy IR. For all comparisons, P �0.0001 by �2 test. (C) Percentage of breakpoints targeting antigen receptorloci in STI571-treated Atm�/�:DELCJ-50 and Atm�/�:INV-26 abl pre-B cellsreceiving 0, 0.4, or 4 Gy of IR. *, P � 0.0001 or **, P � 0.01 by �2 analysis.

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24 h after the addition of STI571. DNA was recovered 96 h after the additionof STI571.

Southern Blot Analysis. Analysis of retroviral substrate rearrangement wascarried out on EcoRV-digested or EcoRV/NcoI double-digested genomic DNAusing probe C4 as described (25).

Inverse PCR. MseI-digested genomic DNA from abl pre-B cells was purified usingthe QIAquick PCR purification kit (Qiagen) and ligated at 5 ng/�L, followed bydigestion with KpnI. Five nanograms of this DNA was amplified with 5 pmol eachof primer 1 (CAT AGC GAA TTC CAT TGT ATG GGA TCT GAT CTG G) and primer2 (CCC TTG TTG AAT ACG CTT G) in a 25-�L reaction with 1 mM MgCl2 and 100 �MdNTPs. PCR conditions were 17 cycles of 92 °C for 60 s, 55 °C for 60 s, and 72 °C for60 s. One microliter of this reaction was then amplified with primer 1 and primer3 (AGT TGC GGA TCC CTC TTT CCA CAA CTA TCC) using identical conditions asabove except for an increase in the cycle number to 30. The combined productsof 22 independent PCRs were digested with BamHI and EcoRI and divided into

two fractions (0.5–1 kb, 1–1.5 kb) by fractionation on a 1% agarose gel. Eachfraction was gel extracted using the QIAquick gel extraction kit (Qiagen) andcloned separately into pCR2.1-TOPO (Invitrogen).

Sequence Analysis. Ligations were submitted to the Genome Sequencing Centerat Washington University and sequence reads were obtained with the M13and/or M13 REV primers using standard techniques. Sequences were aligned atthe 5� and 3� ends with the known pCR2.1 vector and 3� retroviral coding end(IRES) sequences.Thosewithanalignment lengthof�45ntoneitherend,orwithalignments that failed to extend beyond the primer 1 and primer 3 sequences inthe IRES, were eliminated. The sequence between the 5� end of the IRES and thefirst upstream MseI site was aligned to the mouse genome by BLASTN searchagainst the reference assembly (July, 2007). BLASTN results were considered truealignments only if the alignment began at query position 1 and ended within 20nt of the query end.

Loading Control PCR. A 452-bp region of the Rag1 gene was amplified usingserial 3-fold dilutions of the KpnI-digested ligation from I-PCR as template.

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Atm :DEL -21

-/-

CJAtm :

DEL -46

-/-

CJAtm :

DEL -50

-/-

CJAtm :

DEL -70

-/-

CJAtm :

DEL -84

-/-

CJAtm :INV-26

-/- Atm :DEL -4

-/-

CJAtm :

DEL -21

-/-

CJAtm :

DEL -46

-/-

CJAtm :

DEL -50

-/-

CJAtm :

DEL -70

-/-

CJAtm :

DEL -84

-/-

CJAtm :INV-26

-/-

Fig. 4. Bias for breakpoint targets in cis. (A) Percentage of breakpoint targets in cis among breakpoints not involving antigen receptor loci (red). (B) Percentage ofbreakpoints in cis within 200 kb of the retroviral substrate (blue). (C) The distribution of breakpoint targets in cis within 200 kb of the retroviral substrate integrationsite. The IRES coding end generated by RAG cleavage at the substrate is indicated by the arrow tip. (D) Percentage of breakpoint targets in cis in cells receiving 0 or 4Gy IR. (E) Fold overrepresentation of breakpoint targets in cis. Calculated as (# of breakpoints in cis/# total breakpoints)/(size of chromosome/size of genome).

Mahowald et al. PNAS � October 27, 2009 � vol. 106 � no. 43 � 18343

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PCR was performed with the R15P primer (AGA AGG AGA AGG ATT CCT CAGAGG GG) and R1IP primer (TTG GGA AGT AGA CCT GAC TGT GGG). PCRconditions were 92 °C for 3:00, followed by 30 cycles of 92 °C for 45 s, 60 °C for60 s, and 72 °C for 90 s.

Positional Cloning of Retroviral Integration. Inverse PCR was performed onTsp509I-digested genomic DNA ligated at 10 ng/�L. The primary PCR wasperformed with the hCD4 –1 primer (GGG CAG AAC CTT GAT GTT GGA) andLTR-f primer (ATC AGA TGT TTC CAG GGT GC), followed by secondary PCR

with the hCD4 –2 primer (CCA GTT TCA AGC TCA GCA TCA) and LTR-f, underthe conditions described above for I-PCR. The secondary PCR product wasgel purified and sequenced directly.

ACKNOWLEDGMENTS. We thank Beth Helmink for critical review of themanuscript. B.P.S is supported by National Institutes of Health GrantAI074953 and AI047829. J.M.B. was supported by Howard Hughes MedicalInstitute. G.K.M. is supported by a predoctoral training grant (NIH/NIAIDT32 AI007163).

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