Figure 1.
Initial steps of V(D)J recombination and structure of mouse TCRβ locus.
(A) According to the capture model initially proposed by Jones and Gellert [8], RAG1/2 complex binds to one RSS (step 1) and then captures the second RSS to form the PC (step 3). Within the PC, pairwise double-strand breakages occur via coupled transesterification reactions, thus leading to the production of SE and CE (step 5). Within this reactions pathway Curry et al. [9] proposed the order of the two nicking reactions; the first one occurs at the initiating RSS (black triangle) (step 2), the second one occurs at the captured RSS (white triangle) (step 4). An alternative model in which the first nick would occur at the captured RSS was considered in the Supplementary Text S1. (B) Schematic depiction of the TCRβ locus. 12- and 23RSSs are represented by black and white triangles, respectively. Gene segments are figured by grey rectangles. TCRβ locus rearrangements are ordered (Dβ-to-Jβ occur before Vβ-to-DJβ). The B12/23 constraint prohibits direct Vβ-to-Jβ rearrangements.
Figure 2.
RSS nicks at the TCRβ locus in mouse developing T cells.
(A) Strategy to detect RSS nicks in vivo using oligo-capture as described by [9]. Vertical and horizontal arrows schematize, respectively, sites for restriction enzyme digestion and primers for PCR amplification. The bar schematizes the hybridization probe used for Southern blot analysis. (B) Schematic view of the TCRβ regions analyzed in this study (not drawn to scale); 12- and 23RSSs are figured by black and white triangles, respectively; the single strand nick positions are indicated by vertical arrows. The locations of the PCR primers and hybridization probes are shown; H (HindIII); G (BglII); Ss (SstI); S (SphI); RV (EcoRV); RI (EcoRI). (C, D) Autoradiographs of Southern blots of oligo-captured DNAs. Total genomic DNA from WT, RAG1−/− or Eβ−/− DN thymocytes was investigated for single strand nicks at TCRβ RSSs or Cβ2 sequences (used as a negative control). Nicks at Vβ4 and Vβ16 genes were analyzed together; Vβ8 and Vβ5 corresponded to RSSs from three (Vβ8.1, Vβ8.2 and Vβ8.3) and two (Vβ5.1 and Vβ5.2) genes, respectively; single-strand nicks at Dβ2 12- and 23RSSs were analyzed conjointly as these two RSSs possess identical heptamers. Jβ1 and Jβ2 corresponded to all functional Jβ1 and Jβ2 12RSSs, respectively. They were analyzed in one single round using a mixture of specific heptamers followed by PCR amplification of a genomic fragment located at the 3′ end of the Jβ1 (or Jβ2) cluster. PCR reactions were carried out using increasing amounts of template DNA from the bead release (0.5, 1 and 2% of captured DNA) or the flow through (10, 25 and 50 ng of non-captured DNA). Additional controls used 2% of captured (C) and 50 ng of non captured (NC) fractions from genomic DNA treated in parallel except that the biotinylated oligonucleotide was omitted (No 7mer). Estimation of the amount of captured DNA. Assuming that the amount of genomic DNA is 6 pg per cell, the cellular equivalent of 10 ng of genomic DNA is 1650 cells or 3300 alleles. For the less efficient Dβ RSS (5′Dβ1 12RSS) the intensity of the band (when 2% of captured DNA is analyzed) is tenfold lower than the band of non-captured DNA (10 ng of DNA analyzed). Hence, for 2% of captured 5′Dβ1, we estimated that 330 Dβ1 alleles were amplified and that the total amount of captured 5′Dβ1 DNA is around 16500 Dβ1 alleles. We observed that the signal is well detected when approximately 80 copies were analyzed (0.5% of 5′Dβ1 captured DNA). Conversely, nicked Vβ and Jβ RSSs were not detected (even with an input of 10% of captured DNA, not shown), suggesting that there is less than 80 copies of Vβ or Jβ DNA in the PCR tube. If we considered that the efficiency of the oligocapture assay is similar for all DNA targets and that only the amount of nicked DNA varies, we estimated that the amount of nicked Vβ or Jβ RSSs is at least twentyfold lower than the amount of nicked Dβ.
Figure 3.
In vitro RAG1/2-mediated nicking assays.
As described on the left panel, the recombination substrate was first digested with NcoI and AccI restriction enzymes and the resulting 12/23 RSS-containing fragment was radio-labeled at the 5′ends (indicated by a star), then incubated for 5 min without (−) or with (+) the RAG1/2 extract. DNA samples were further digested with Eco0109I (Eo) and XbaI (X) enzymes and separated by denaturing PAGE. (Right) Autoradiographs of nicking assay analysis of the indicated recombination substrates. 12/23 RSS substrates were named according to the gene segments flanking the 23- and 12RSS (see Figure S6 for the construction of recombination substrates). The sizes of the intact 23- and 12RSSs (198 and 109 nt) and of the corresponding nicking products (27 and 38 nt) are indicated. Percentages of 12- and 23RSS single strand nicks (i.e., scanning intensity of individual nicked products vs. that of the corresponding original fragments) are shown below the gel image (und: undetected). For some substrates, two additional products of ∼35 and ∼45 nt in length (indicated by grey arrows) were detected in the RAG positive lane, they may correspond to non-hairpin CE DNA breaks (i.e., processed products of RAG1/2-generated hairpins) [46]. All results shown are representative of at least three separate experiments.
Figure 4.
Dβ1 12RSS nicks are not detected at germline Dβ1 locus.
Oligocapture assays were performed as described in Figure 2, except that the PCR primers and hybridization probe were specific for sequences in Dβ1-Jβ1 intervening DNA.
Figure 5.
The Dβ1 23RSS impairs RAG1/2-mediated cleavages at the adjacent Dβ1 12RSS in vitro.
(A) RAG1/2-mediated nicking assays using substrates comprised of the Dβ1 gene segment flanked by various combinations of 5′ and/or 3′ RSSs. The various Dβ-containing fragments were radio-labeled at the 5′ ends and incubated for 5 min without (−) or with (+) the RAG1/2 extract. (B) RAG1/2-mediated coupled cleavage assays of the substrates illustrated on the left. Depending on the substrate, Southern blot analysis used probes A or B, as indicated. (A and B) 12- and 23RSSs are depicted as black and white triangles respectively. Dβ1 RSSs are highlighted by a dot within the triangle. All results shown are representative of at least three separate experiments.
Figure 6.
Analysis of TCRβDMF minilocus rearrangement.
(A) TCRβwt contains germline Vβ14, Dβ1, Jβ1.1 and Jβ1.2 gene segments linked to the IgH intronic enhancer (Eμ) and constant region gene (Cμ). The locations of the Vβ14 probe (P), BglII (G), BamHI (B) and HindIII (H) sites are indicated. 23RSSs (white triangle) and 12RSSs (black triangle) are shown; dotted triangles correspond to the Dβ1 12- and 23RSSs. The TCRβDMF is similar to the TCRβwt except that the Dβ1 23RSS and the Jβ1.2 12RSS were replaced with the Vβ14 23RSS and the Dβ1 12RSS, respectively. Miniloci were not drawn to scale. (B) Southern blot analysis of BglII-digested genomic DNA. DNA was isolated from wild-type thymocytes (wt) and TCRβwt or TCRβDMF transgenic thymocytes. The expected size bands from the non rearranged endogenous TCRβ locus (end) and the TCRβwt or TCRβDMF minilocus in the non rearranged (GL), DJ, VD and VDJ/VJ configurations are indicated. The 2, 4, 6 and 9 kb markers are shown. (C) Sequences of some VDJ/VJ coding joints from TCRβDMF rearrangements. Germline coding sequences of Vβ14, Dβ1 and Jβ1.2 are indicated on the top. Nucleotide insertions (P/N) are indicated by capital letters. Presumptive P nucleotides are underlined.