SμTR^−/− and wild-type mice have similar numbers of upstream CSR events. (a) DC-PCR measures CSR events upstream of the SμTR region. A BsrGI site distinguished CSR junctions located upstream and downstream of the site. Only CSR sites in sequences upstream of BsrGI generate PCR signals. Total CSR events are measured by EcoRI DC-PCR (E, EcoRI; BGI, BsrGI). PCR primers are indicated by arrowheads. (b) Twofold dilutions of genomic DNAs were assayed by Sμ/Sγ1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR reactions. (c) CSR events in wild-type or SμTR^−/− mice as percentages of total wild-type CSR events (set to 100%). Mean values and SEM are from six independent experiments with two mice per group.

CSR within sequences upstream of the SμTR are reduced in Msh2^−/− mice. (a) Twofold dilutions of genomic DNAs were assayed by Sμ/Sγ1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR. (b) CSR sites in wild-type or Msh2^−/− mice measured relative to total wild-type CSR (set to 100%). Mean values and SEM of at least four independent experiments are presented. (c) CSR events in Msh2^−/− mice measured by EcoRI, HindIII, and XbaI real-time DC-PCR and compared with wild-type mice. Mean values and SEM are from six independent experiments with two Msh2^−/− mice and seven independent experiments with four wild-type mice.

Model depicting the targeting domain for CSR in the JH-Cμ region for wild-type and SμTR^−/− mice. The JH-Cμ intron region is shown with small circles cartooning WRC motifs (potential AID cleavage sites) in the JH-Cμ region. Open ovals represent domains accessible to CSR cleavage; regions not accessible to CSR are indicated by shaded ovals.

SμTR^−/− mice have increased levels of CSR events targeted to downstream sequences. (a) DC-PCR measuring CSR junction sites downstream of the SμTR region. HindIII and XbaI sites downstream of the SμTR region were used in DC-PCR assays to measure CSR junctions in sequences downstream of each site. Total CSR sites were determined by EcoRI DC-PCR (E, EcoRI; H1 and H2, HindIII; X, XbaI). Primer pairs used for the different DC-PCR assays are indicated by small arrows of different types. (b) Wild-type and SμTR^−/− CSR events measured by EcoRI, HindIII, and XbaI real-time DC-PCR. Copy numbers for CSR events were derived from pSμ/Sγ3 standard curves. Rag-1 PCR normalized CSR events relative to Rag-1 copy number. Mean values and SEM are from seven independent experiments with four mice per group. (c) Percentage of SμTR^−/− switch events downstream of the H2 relative to events downstream of H1 (set to 100%). Mean values and SEM are from three independent experiments per group.

CSR junction sites and AID target sites within different segments of the JH-Cμ intron. Histograms of CSR site, WRC motif, and WGCW motif frequencies are shown for different segments of the JH-Cμ region. (a) Comparisons of wild-type with Msh2^−/− mice that both have wild-type Sμ regions. (b) Comparisons of SμTR^−/− mice with a shortened Sμ region. DNA segments are drawn in proportion on the x axis. Areas of boxes in the histogram are proportional to frequencies in each segment, resulting in box heights that indicate relative recombinational frequencies per nucleotide. Boxes for mutant mice are drawn to scale relative to wild-type mice. Switching reductions in SμTR^−/− varied slightly in different DC-PCR assays and were averaged. Junction site distributions in each segment were assumed to be uniform. Wild-type CSR frequencies are set to 100%; other CSR frequencies are normalized to this total. Total WRC and WGCW motif frequencies were set to 100% for each mouse strain. The SμTR is only partially sequenced (available from GenBank/EMBL/DDBJ under accession no. AC073553). Densities of motifs are assumed conserved throughout the SμTR. The total numbers of each motif in the Iμ–Cμ region are indicated in the top right of each graph.