Fig. 3: TMEJ is responsible for the repair (long-)resected AID-induced recombination events.

Using primary B cells (CD19 + ) day 4 post stimulation with anti-IgD dextran/LPS/IL-4. a Schematic long-range PCR (LR-PCR) visualizing resection tracts during IgG1 switching. Primers (burgundy arrows) in intronic enhancer iEμ and Ighy1 exon 6, amplify deletional rearrangements. Productive Sµ-Sy1 joins yield 4.9–17.3 kb amplicons (S-S joints); resected, unproductive events yield fragments <4.9 kb. b LR- PCR profiles of Sµ-Sy1 junctions. IL-2 is a loading control. See Supplementary Fig. 3. * indicates nonspecific band. c Violin plots of read lengths obtained by PacBio sequencing of LR-PCR fragments, from independent samples WT (n = 5), Polq^−/− (n = 6), Xrcc4^−/− Polq^−/− (n = 3), Xrcc4^−/−, Shld1^−/− and Shld1^−/− Polq^−/− (n = 4). Mixed-model regression test used followed by post-hoc Tukey HSD test, WT vs. Xrcc4^−/− (p = 0.0027), WT vs. Shld1^−/− (p = 1.11e-08), Polq^−/− vs. Xrcc4^−/− (p = 7.46e-05), Polq^−/− vs. Shld1^−/− (p = 2.00e-11), Xrcc4^−/− vs. Xrcc4^−/−Polq^−/− (p = 0.00053) and Shld1^−/− vs. Shld1^−/− Polq^−/− (p = 5.65e-14). For all boxplots: minima is minimum value, maxima is maximum value, center is median and quartiles shown by box and whiskers. See Supplementary Data 2 for number of reads plotted. d Unique breakpoints obtained via LR-PCRseq, were pooled by genotype and distribution of AID-junctions situated at Sµ and Sy1 regions of WT, Polq^−/−, Xrcc4^−/−,Xrcc4^−/− Polq^−/−, Shld1^−/− and Shld1^−/− Polq^−/− cells was plotted. Coordinates of unique breakpoints were used to generate distributions and to determine the percentage of unique junctions across Sµ and Sy1 genomic regions, using 250 bp bins. Percentage of unique junctions located within and around Sµ and Sy1 genomic regions are indicated. The total number of plotted junctions (n) is specified between brackets in the bottom right corner of each graph. Primers used for LR-PCR are in dark red. e Percentage of resected joints, break junction located outside a S-region, obtained via LR-PCRseq using PacBio. Resected joints are defined as joints where either or both 5’ or/and 3’ coordinates are outside Sµ or/and Sy1 loci, amongst unique junctions. Bars represent mean ± SEM, Unpaired two-tailed T-test (*p < 0.05, **p < 0.01). See also Supplementary Data 3. Source data are provided as a Source Data file.