Assembly of TCRβ variable region genes is ordered during thymocyte development with Dβ to Jβ rearrangement preceding Vβ to DJβ rearrangement. The 5′Dβ 12-RSS is required to precisely and efficiently target Vβ rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vβ to Jβ rearrangement, this restriction ensures Dβ gene segment use in the assembly of essentially all TCRβ variable region genes. In this study, we show that rearrangement of Vβ 23-RSSs is significantly biased to the Dβ 12-RSS over Jβ 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vβ to Dβ rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.

Variable region genes are assembled during lymphocyte development from component variable (V), joining (J), and in some cases diversity (D) gene segments (1). This process, termed V(D)J recombination, occurs through DNA cleavage and joining steps and is conducted by an enzymatic complex collectively referred to as the V(D)J recombinase. Variable gene segments are flanked by recombination signal sequences (RSSs), which are composed of conserved heptamers and nonamers with intervening nonconserved 12- or 23-bp spacers (hereafter referred to as 12-RSSs and 23-RSSs, respectively) (1). The V(D)J recombination reaction is initiated by the lymphoid-specific recombinase-activating gene (RAG)3-1 and -2 proteins that introduce DNA double-strand breaks (DSBs) at the RSS-coding segment border (2, 3, 4). Generally expressed proteins of the nonhomologous end-joining pathway of the DNA DSB repair process join the coding and signal end pairs generating coding and signal joins, respectively (2, 5). Recombination only occurs between variable gene segments flanked by RSSs of dissimilar spacer lengths (1). This restriction, known as the 12/23 rule, is enforced by a requirement for synapsis of a 12/23 RSS pair before DNA cleavage (6, 7, 8).

Assembly of variable region genes exhibits lineage and developmental stage specificity and, in some loci, is regulated in the context of allelic exclusion (5, 9). In loci containing D segments, gene assembly can be ordered with D to J preceding V to DJ rearrangement. Regulation in these various contexts can be achieved through modulation of V, D, and J gene segment accessibility to the V(D)J recombinase by alterations in transcription, methylation, and chromatin structure of recombining gene segments (5, 9). In addition, regulation can occur at the level of the recombination reaction, for example, through differences in the ability of specific RSSs to serve as substrates for the V(D)J recombinase affected by RSS and/or adjacent coding sequence variations (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).

TCRβ variable region genes are assembled during thymocyte development from Vβ, Dβ, and Jβ gene segments (22). This process is ordered with Dβ to Jβ rearrangement preceding Vβ to DJβ rearrangement and allelic exclusion enforced at the Vβ to DJβ rearrangement step (22). The Vβ and Jβ gene segments are flanked by 23- and 12-RSSs, respectively, whereas Dβ gene segments are flanked 5′ and 3′ by 12- and 23-RSSs, respectively. Direct Vβ to Jβ rearrangement, which would satisfy the 12/23 rule, occurs rarely, if at all, due to a requirement for the 5′Dβ 12-RSS to target Vβ rearrangement (23, 24). This restriction, which is enforced at the DNA cleavage step, promotes rearrangement of Vβ gene segments from up to a megabase away precisely to the 5′Dβ 12-RSS and not the Jβ 12-RSSs that are <1 kb downstream (25). Furthermore, appending the 5′Dβ 12-RSS to a Jβ gene segment efficiently targets Vβ rearrangements specifically to this Jβ gene segment, suggesting that it can function in a position-independent fashion (23).

The mechanism by which the 5′Dβ RSS targets Vβ rearrangement is unknown, but may depend on common features of the V(D)J recombination reaction. Alternatively, it may rely on the expression of specific factors that promote use of the 5′Dβ RSS or Vβ/5′Dβ RSS pair by the V(D)J recombinase during thymocyte development. In this study, we have assayed recombination between Vβ, Dβ, and Jβ RSSs on extrachromosomal substrates in nonlymphoid cells. These extrachromosomal substrates were designed to allow for direct comparison of the efficiency of recombination between Vβ 23-RSSs and the 5′Dβ and Jβ 12-RSSs. Our findings suggest that targeting of Vβ to Dβ rearrangement is due, at least in part, to constraints imposed by the V(D)J recombinase without a requirement for lymphoid-specific factors other than the RAG-1/2 proteins.

pJH290 was modified by introducing restriction sites that allowed for cloning of RSSs at three positions, P1 (NcoI/XhoI), P2 (HpaI/NheI), and P3 (XmaI/SacII) (Fig. 1 A) (26). A 300-bp spacer fragment of nonspecific DNA was introduced between the RSS in P1 and the OOP sequence to increase the distance between the recombining RSSs. RSSs with four nucleotide-coding flanks were cloned into the different positions to generate the recombination substrates described in the text. All substrates used the Vβ14 23-RSS-coding flank (heptamer-TCTG) at P1 and the 5′Dβ1 12-RSS-coding flank (heptamer-GGGA) at P2. The 5′Dβ1 12-RSS coding flank was also used at P3 in pC:JκVκVκ. In all other substrates the Jβ1.1/Jβ1.2 12-RSS-coding flank (heptamer-CAAA) was used at P3.

FIGURE 1.

Recombination of the pC extrachromosomal substrate in CHO cells. A, Schematic of the unrearranged pC substrate showing the three positions where RSSs are introduced (P1, P2, and P3), the OOP transcriptional terminator, the β lactamase promoter (○), the genes encoding resistance to ampicillin (Amp), and chloramphenicol (Cam) and the 1233 and CAT3 primers used for PCR analyses. Also shown are schematics for pC substrates that have undergone P1 to P2 and P1 to P3 rearrangements. The expected size products for PCR of recombined substrates with the 1223 and CAT3 primers are indicated. The schematics are not drawn to scale. B, Sequences of RSSs. The consensus heptamer and nonamer sequences are shown in bold. C, PCR analysis of seven individual ampicillin- and chloramphenicol-resistant bacterial colonies generated from recombination of the pC:JκVκVκ substrate in CHO cells (lanes 1–7). Also shown is the 100-bp m.w. marker (MW) and a PCR in which template DNA was not added (−).

FIGURE 1.

Recombination of the pC extrachromosomal substrate in CHO cells. A, Schematic of the unrearranged pC substrate showing the three positions where RSSs are introduced (P1, P2, and P3), the OOP transcriptional terminator, the β lactamase promoter (○), the genes encoding resistance to ampicillin (Amp), and chloramphenicol (Cam) and the 1233 and CAT3 primers used for PCR analyses. Also shown are schematics for pC substrates that have undergone P1 to P2 and P1 to P3 rearrangements. The expected size products for PCR of recombined substrates with the 1223 and CAT3 primers are indicated. The schematics are not drawn to scale. B, Sequences of RSSs. The consensus heptamer and nonamer sequences are shown in bold. C, PCR analysis of seven individual ampicillin- and chloramphenicol-resistant bacterial colonies generated from recombination of the pC:JκVκVκ substrate in CHO cells (lanes 1–7). Also shown is the 100-bp m.w. marker (MW) and a PCR in which template DNA was not added (−).

Close modal

Transient recombination assays and analysis were performed in the Chinese hamster ovary (CHO) cell line as previously described using 3 μg each of RAG-1 and RAG-2 expression vectors (pJH548 and pJH549, respectively) and 2 μg of the recombination substrate with 6 μl of SuperFect (Qiagen, Valencia, CA) per microgram of DNA (27). Recovered plasmids were analyzed for rearrangement by PCR using the oligonucleotide primers 1233, 5′-AGCGGATAACAATTTCACACAGGA-3′ and CAT3, 5′-GGTGGTATATCCAGTGATTTT-3′. PCRs were conducted on colony stabs in 100-μl reactions containing 20 pmols of each primer, 50 mM KCl, 10 mM Tris (pH 8.0), 2 mM MgCl2, dNTPs, and Taq. PCR conditions were 3 min, 93°C for 1 cycle and 30 cycles of 1 min, 93°C; 1 min, 55°C and 1 min, 72°C. PCR products from rearrangements to P2 and P3 were resolved on 1.2% agarose gels.

Isolated recombination substrates were ligated to the BW linker using previously described conditions (25). Heminested PCRs were performed using the BW-1H, 5′-CCGGGAGATCTGAATTCCAC-3′ and 3P1, 5′-GATGAGAGGATCGACGAC-3′ in the primary LMPCR and the BW-1H and 3P2, 5′-GACGACATGGCTCGATTG-3′ primers in the secondary PCR. Primary and secondary LMPCR conditions were as previously described. LMPCR products were size-fractionated on 1% agarose gels and Southern blotting was performed with the 5P1 oligonucleotide probe, 5′-CTGCAGTCGACTCTCAT-3′, as previously described (25).

Plasmid-based extrachromosomal recombination substrates containing appropriate RSS pairs recombine in nonlymphoid cell lines upon expression of the RAG-1 and RAG-2 proteins. The prototypical extrachromosomal recombination substrate, pJH290, contains the Jκ1 23-RSS and the Vκ8 12-RSS pair flanking a prokaryotic transcription terminator and imparts bacterial resistance to ampicillin (26). Recombination between the two RSSs results in deletion of the transcription terminator and expression of the chloramphenicol acetyl transferase gene allowing for isolation of recombined plasmids based on their ability to impart bacterial resistance to ampicillin and chloramphenicol. To generate the competitive recombination substrate, pC, we modified pJH290 as described in Materials and Methods such that a single RSS could be introduced 3′ of the transcription terminator (position 1, P1) and two RSSs 5′ of the transcription terminator (positions 2 and 3, P2 and P3) (Fig. 1 A). Thus, RSSs introduced at P1 can rearrange to RSSs introduced at P2 and P3.

To determine whether rearrangement to P2 and P3 occurred in a position-independent manner, the pC:JκVκVκ substrate was generated by introducing the Jκ1 23-RSS at P1 and the Vκ8 12-RSS at P2 and P3 of pC (Fig. 1, A and B). The coding nucleotide flanks at P2 and P3 of the pC:JκVκVκ were held constant as described in Materials and Methods. The pC:JκVκVκ substrate was introduced into the nonlymphoid CHO cell line with and without RAG-1 and RAG-2 expression vectors followed by recovery of the substrate after ∼60 h. Substrates recovered from cells that did not express RAG-1/2 had not recombined; however, ∼2% of the pC:JκVκVκ substrate recovered from the RAG-1/2-expressing cells had recombined as evidenced by the fraction of ampicillin-resistant colonies that were also resistant to chloramphenicol (Table I). Analysis of individual ampicillin/chloramphenicol-resistant colonies by PCR revealed that essentially equivalent numbers had undergone Jκ1 23-RSS rearrangements to the Vκ8 12-RSS at either P2 or P3 (Table I and Fig. 1 C). Sequence analyses revealed that most isolated rearrangements were unique (data not shown). Together these data demonstrate that the pC substrate rearranges efficiently in CHO cells and without inherent biases for rearrangement to P2 or P3.

Table I.

Recombination of pC:JκVκVκ in CHO cellsa

RAG 1/2% Recombination% P2% P3
2.63± 1.48 50 50 
− — — 
RAG 1/2% Recombination% P2% P3
2.63± 1.48 50 50 
− — — 
a

Percentage of recombination was calculated as the fraction of recovered ampicillin-resistant bacterial colonies that were also resistant to chloramphenicol. Shown is the mean percentage of recombination and the SD from six independent experiments. The percentage of ampicillin- and chloramphenicol-resistant colonies that were rearranged to the RSSs at P2 and P3 is also indicated. Each experiment was carried out a minimum of four times.

The pC:V14DD substrate was generated by introducing the Vβ14 23-RSS at P1 and the 5′Dβ1 12-RSS at P2 and P3 of pC. The flanking coding nucleotides at P2 and P3 of pC:V14DD, and all subsequent substrates, corresponded to those at 5′ Dβ RSS and Jβ1.1 RSS, respectively, as described in Materials and Methods. Approximately 0.05% of pC:V14DD recovered from CHO cells expressing RAG-1 and RAG-2 had recombined with near equivalent use of the 5′Dβ 12-RSSs at P2 and P3 (Table II). The 5′Dβ1 12-RSS at P3 of pC:V14DD was replaced with the Jβ1.1 12-RSS to generate pC:V14DJ1.1. The efficiency of recombination of pC:V14DJ1.1 in CHO cells was similar to that of pC:V14DD; however, all Vβ rearrangements were to the 5′Dβ 12-RSSs at P2 with no detectable rearrangement to the Jβ1.1 12-RSS at P3 (Table II). Thus, the bias for Vβ to Dβ rearrangement observed in the endogenous TCRβ locus in thymocytes appears to be recapitulated on extrachromosmal recombination substrates in the CHO cell line.

Table II.

Recombination of TCRβ RSS containing pC substrates in CHO cellsa

SubstrateRSS% Recombination% P2% P3
P1P2P3
pC:V14DD Vβ14 5′Dβ1 5′Dβ1 0.05 ± 0.03 55 45 
pC:V14DJ1.1 Vβ14 5′Dβ1 Jβ1.1 0.05 ± 0.02 100 
pC:V14DJ1.2 Vβ14 5′Dβ1 Jβ1.2 0.03 ± 0.01 100 
pC:V2DJ1.1 Vβ2 5′Dβ1 Jβ1.1 0.13 ± 0.09 97 
pC:V15DJ1.1 Vβ15 5′Dβ1 Jβ1.1 0.08 ± 0.10 95 
pC:V14J1.1Vβ14 Jβ1.1 5′Dβ1 0.01 ± 0.00 100 
pC:V2J1.1Vβ2 Jβ1.1 5′Dβ1 0.11 ± 0.05 97 
pC:V14DVκ Vβ14 5′Dβ1 Vκ8 0.06 ± 0.01 12 88 
pC:V14VκVβ14 Vκ8 5′Dβ1 0.12 ± 0.05 89 11 
pC:JκDJ1.1 Jκ1 5′Dβ1 Jβ1.1 0.36 ± 0.14 98 
pC:JκJ1.1Jκ1 Jβ1.1 5′Dβ1 0.30 ± 0.13 100 
SubstrateRSS% Recombination% P2% P3
P1P2P3
pC:V14DD Vβ14 5′Dβ1 5′Dβ1 0.05 ± 0.03 55 45 
pC:V14DJ1.1 Vβ14 5′Dβ1 Jβ1.1 0.05 ± 0.02 100 
pC:V14DJ1.2 Vβ14 5′Dβ1 Jβ1.2 0.03 ± 0.01 100 
pC:V2DJ1.1 Vβ2 5′Dβ1 Jβ1.1 0.13 ± 0.09 97 
pC:V15DJ1.1 Vβ15 5′Dβ1 Jβ1.1 0.08 ± 0.10 95 
pC:V14J1.1Vβ14 Jβ1.1 5′Dβ1 0.01 ± 0.00 100 
pC:V2J1.1Vβ2 Jβ1.1 5′Dβ1 0.11 ± 0.05 97 
pC:V14DVκ Vβ14 5′Dβ1 Vκ8 0.06 ± 0.01 12 88 
pC:V14VκVβ14 Vκ8 5′Dβ1 0.12 ± 0.05 89 11 
pC:JκDJ1.1 Jκ1 5′Dβ1 Jβ1.1 0.36 ± 0.14 98 
pC:JκJ1.1Jκ1 Jβ1.1 5′Dβ1 0.30 ± 0.13 100 
a

The 23-RSS introduced at P1 and the 12-RSSs at P2 and P3 of pC are indicated. Each substrate was assayed a minimum of four times.

The bias for rearrangement between Vβ 23-RSSs and the 5′Dβ 12-RSS was also observed when the Jβ1.1 12-RSS of pC:V14DJ1.1 was replaced with the Jβ1.2 12-RSS (pC:V14DJ1.2) or when the Vβ14 23-RSSs was replaced with either the Vβ 2 or 15 23-RSS (pC:V2DJ1.1 or pC:V15DJ1.1, respectively) (Table II). Thus, the bias for Vβ to Dβ rearrangement on extrachromosomal substrates is not dependent on specific Vβ or Jβ RSSs. Finally, exchanging the position of the 5′Dβ and Jβ1.1 12-RSSs (pC:V14J1.1D and pC:V2J1.1D) did not alter the bias for Vβ 23-RSS to 5′Dβ 12-RSS rearrangement (Table II).

Targeting of Vβ to Dβ rearrangement by the 5′Dβ RSS in the endogenous TCRβ locus occurs at the DNA cleavage step of the V(D)J recombination reaction (25). DNA cleavage intermediates from recombination on extrachromosomal substrates were assayed by LMPCR. These analyses revealed similar levels of signal end intermediates from cleavage at the Vκ RSSs at P2 and P3 of the pC:JκVκVκ substrate consistent with the equal level of recombination to these two positions in this substrate (Table I and Fig. 2). As expected, detection of these signal ends was dependent on the expression of RAG-1/2 and the addition of ligase (Fig. 2). Analysis of signal end cleavage intermediates from the RSSs at P2 and P3 of the pC:V2DJ1.1 and pC:V2J1.1D substrates revealed detectable signal ends only from cleavage at the 5′Dβ1 12-RSS and not the Jβ1.1 12-RSS. These findings demonstrate that, as in the endogenous TCRβ locus, targeting of Vβ rearrangement by the 5′Dβ RSS on extrachromosomal substrates occurs at the DNA cleavage step of the V(D)J recombination reaction.

FIGURE 2.

Analysis of Vβ to Dβ rearrangement DNA cleavage intermediates from the pC extrachromosomal substrate. A, Schematic of signal end cleavage intermediates from cleavage at P1 and P2 or P1 and P3. Shown is the oligonucleotide linker used for ligation to the signal ends, the BW-1H, 3P1, and 3P2 primers used for heminested PCR and the 5P1 oligonucleotide used to probe PCR products. B, Signal ends from cleavage at P2 and P3 from the pC:JκVκVκ, pC:V14DD, pC:V2DJ1.1, and pC:V2J1.1D substrates isolated from CHO cells that expressed (+) or did not express (−) RAG-1/2 and assayed by LMPCR in the presence (+) or absence (−) of ligase. LMPCR was conducted with the BW-1H, 3P1, and 3P2 oligonucleotides and products probed with the 5P1 oligonucleotide as described in Materials and Methods.

FIGURE 2.

Analysis of Vβ to Dβ rearrangement DNA cleavage intermediates from the pC extrachromosomal substrate. A, Schematic of signal end cleavage intermediates from cleavage at P1 and P2 or P1 and P3. Shown is the oligonucleotide linker used for ligation to the signal ends, the BW-1H, 3P1, and 3P2 primers used for heminested PCR and the 5P1 oligonucleotide used to probe PCR products. B, Signal ends from cleavage at P2 and P3 from the pC:JκVκVκ, pC:V14DD, pC:V2DJ1.1, and pC:V2J1.1D substrates isolated from CHO cells that expressed (+) or did not express (−) RAG-1/2 and assayed by LMPCR in the presence (+) or absence (−) of ligase. LMPCR was conducted with the BW-1H, 3P1, and 3P2 oligonucleotides and products probed with the 5P1 oligonucleotide as described in Materials and Methods.

Close modal

Previous analyses have demonstrated that RSSs with consensus heptamer/nonamer sequences generally function better than those that deviate from consensus (20). In this regard, the heptamer/nonamer sequences of the 5′Dβ1 12-RSS are closer to consensus than those of the Jβ 12-RSSs. The bias for Vβ to Dβ rearrangement could be due to features of the 5′Dβ1 12-RSS that make it a better substrate than the Jβ 12-RSSs regardless of its 23-RSS partner, or to specific constraints imposed by the Vβ/Dβ RSS pair. To distinguish between these possibilities, we initially generated the pC:V14DVκ substrate by replacing the Jβ1.1 12-RSS of pC:V14DJ1.1 with the Vk8 12-RSS (Table II). The heptamer/nonamer sequence of the Vκ8 12-RSS is closer to the consensus than that of the 5′Dβ 12-RSS (Fig. 1,B). In CHO cells, pC:V14DVκ and pC:V14DJ1.1 rearrange with equal efficiency (Table II). However, in striking contrast to pC:V14DJ1.1, rearrangement of the Vβ14 23-RSS is no longer specifically targeted to the 5′Dβ 12-RSS on pC:V14DVκ as 83% of recombined plasmids exhibit Vβ14 23-RSS rearrangement to the Vκ8 12-RSS (Table II). Similar results were obtained when positions of the Vκ8 and 5′Dβ 12-RSSs were exchanged (pC:V14VκD) (Table II). Furthermore, the pC:JκDJ1.1 and pC:JκJ1.1D substrates, in which the Vβ14 23-RSS was replaced with the Jκ1 23-RSS, exhibit a bias for rearrangement of the Jκ1 23-RSS to the 5′Dβ over Jβ 1.1 12-RSS (Table II). Together, these findings suggest that the preferential targeting of Vβ rearrangement to the 5′Dβ over Jβ RSSs is due to intrinsic properties of these RSSs rather than a specific requirement for Vβ/Dβ RSS synapsis.

In developing thymocytes, Vβ gene segment rearrangement is targeted by the 5′Dβ 12-RSS (23, 24). In this study, we show that on extrachromosomal substrates in the nonlymphoid CHO cell line, rearrangement of Vβ 23-RSSs is also preferentially targeted to the 5′Dβ 12-RSS over Jβ 12-RSSs. Similar to what was observed in the endogenous TCRβ locus, this constraint is mediated at the DNA cleavage step of the V(D)J recombination reaction on extrachromosomal substrates (25). These findings demonstrate that targeting of Vβ rearrangement by the 5′Dβ RSS may be enforced in part by the features of the V(D)J recombination reaction that do not require substrate chromosomal context or lymphoid-specific factors other than the RAG-1/2 proteins.

The generation of DNA DSBs during V(D)J recombination occurs through the initial binding of the RAG-1/2 proteins to RSSs followed by synaptic complex formation and DNA cleavage. The bias for use of the 5′Dβ 12-RSS over Jβ 12-RSSs could be mediated at any, or all, of these steps. Importantly, our findings demonstrate that this bias is due to independent features of the 5′Dβ 12-RSS and not to a specific requirement for synapsis with Vβ 23-RSSs. This is evidenced by the bias for rearrangement of the Jκ 23-RSS to the 5′Dβ 12-RSS over the Jβ 1.1 12-RSS and the bias for rearrangement of Vβ 23-RSSs to the near consensus Vκ 12-RSS over the 5′Dβ 12-RSS.

The ordered assembly of TCRβ variable region genes (Dβ to Jβ preceding Vβ to DJβ) may be determined, in part, by processes that promote accessibility of DJβ gene segments before Vβ gene segments during thymocyte development. Targeting of Vβ gene segment rearrangement to the Dβ gene segment, instead of otherwise accessible downstream Jβ gene segments, could then be determined by the more efficient use of the 5′Dβ 12-RSS, as compared with the Jβ 12-RSSs. Notably, recent in vitro analyses have demonstrated that synapsis and cleavage occurs most efficiently through complexing of a RAG-1/2-bound RSS with an unbound complementary RSS (28, 29). Furthermore, these studies demonstrated that faithful preservation of the 12/23 restriction is more readily observed when the RAG-RSS complex forms initially on a 12-RSS (28). Thus, targeting of Vβ rearrangement by the 5′Dβ RSS in vivo could occur through the preferential binding of the RAG-1/2 proteins to the 5′Dβ 12-RSS, over the Jβ 12-RSSs, followed by synaptic complex formation with a Vβ 23-RSS. However, our findings are not inconsistent with the possibility that additional factors promote Vβ to Dβ rearrangement. Specifically, the Vβ/5′Dβ RSS pair catalyzes recombination on extrachromosomal substrates much less efficiently than the Vκ/Jκ RSS pair. However, a large fraction of mature T cells have two complete VDJβ rearrangements, suggesting that Vβ to Dβ rearrangement proceeds efficiently during thymocyte development (B. Khor and B. P. Sleckman, unpublished observations) (23, 30). Thus, additional factors may directly or indirectly promote Vβ to Dβ rearrangement in developing thymocytes.

RSSs can affect the relative use of specific gene segments in the formation of variable region genes. Strikingly, the differential ability of the 5′Dβ vs Jβ 12-RSSs to be used by the V(D)J recombinase would affect the assembly of all TCRβ variable region genes. This constraint ensures Dβ gene segment use which may be important for the generation of a TCRβ chain repertoire with a normal CDR3 loop length distribution. In addition, it ensures that assembly occurs through two rearrangement steps, Dβ to Jβ followed by Vβ to DJβ, thus increasing the potential for variable region gene diversification due to the imprecise joining process. As such, this restriction is likely important for the generation of a diverse repertoire of functional αβ TCRs.

We thank Dr. Wojciech Swat for critical review of the manuscript and Dr. Frederick W. Alt for advice regarding the generation of the competitive recombination substrate.

1

This work is supported in part by the National Institutes of Health Grant AI47829 (to B.P.S.). B.P.S. is a recipient of an Investigator Award in General Immunology and Cancer Immunology from the Cancer Research Institute. R.E.T. is supported by a predoctoral training grant in tumor immunology from the Cancer Research Institute. A.L.W. is supported by a National Institutes of Health graduate training grant.

3

Abbreviations used in this paper: RAG, recombinase-activating gene; DSB, DNA double-strand break; CHO, Chinese hamster ovary; LMPCR, ligation-mediated PCR.

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