Fig 2: B-cell death depends on UNG-processed AID lesions.Cell numbers (a) and percentage of apoptotic population (b) with(+)/without(-) cytokine stimulation (CIT) at Day 3 are showed for indicated genotypes. UM-/-: Ung-/-Msh2-/-, UMR-/-: Ung-/-Msh2-/-Rev7-/-. Colored dots indicate individual clones. In a, n = 5 for parental CH12F3 cells; n = 6 for Rev7-/-, Ung-/-Rev7-/-(with CIT) and Msh2-/-Rev7-/-; n = 3 for Ung-/-Rev7-/-(without CIT); n = 4 for the other genotypes. In b, n = 6 for parental CH12F3 cells, Ung-/-Rev7-/- and Msh2-/-Rev7-/-; n = 5 for Rev7-/-; n = 4 for the other genotypes. Three independent clones for each genotype were assayed, and n represents independent experiments. Data are represented as mean ± SD. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed for all panels. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns: p > 0.05. P-values and sample sizes are provided in Supplementary Table 2. Source data are provided as a Source Data file.

Fig 3: AID deficiency rescues the cell death of REV7-deficient B cells in CSR.a Assessment of Rev7 deletion efficiency. Schematic illustration of genotyping strategy of Rev7-floxed allele (upper). Q-PCR primers are marks as arrows. A locus in Gapdh (chr6) was assayed as the loading control (blue arrows). Percentage of flowed allele are shown for Cd19creRev7fl/fl B cells at Day 3 and 4 after stimulation (lower). Colored dot indicates data from same mouse. n = 5 for each genotype, n represents independent mice. Cell numbers (b) and percentage of apoptotic population (c) upon LPS/IL4 (upper) or LPS (lower) are plotted. d Genotyping of indicated CSR-activated B cells. e Percentage of cells in G2/M phase is plotted for the indicated genotypes. A-/-: Aicda-/-, A5-/-: Aicda-/- 53bp1-/-, ACR-/-: Aicda-/- Cd19creRev7fl/fl. n = 3 for each genotype in b–e and n represents independent mice. Data are represented as mean ± SD. Two-tail paired t-test was performed for a and d, and one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for b, c, and e. ACR-/- mice are used as reference group in comparison for b, c, and e. **p < 0.01, *p < 0.05, ns: p > 0.05. P-values and sample sizes are provided in Supplementary Table 2. Source data are provided as a Source Data file.

Fig 4: REV7 HORMA domain is required for B cell survival upon AID expression.a Schematic illustration of critical residues on REV7 protein (left). Representative western blot of REV7 mutant proteins in B cells from three replicates are shown at right. EV: empty vector control. CSR levels to IgG1 (b), end resection level in S?1 region (c), and percentage of C > G transversion in 5'Sµ region (d) after LPS/IL4 stimulation at Day 4 of REV7-complementated primary B cells. CSR levels to IgA (e), cell numbers (f), and percentage of apoptotic population (g) of indicated CH12F3 cells with cytokine stimulation (CIT) at Day 3 are shown. n = 3 for each genotype in b–g and n represents independent experiments. Data are represented as mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for b–g. Data from cells complemented with WT REV7 protein are used as reference group in comparison. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns: p > 0.05. P-values and sample sizes are provided in Supplementary Table 2. Source data are provided as a Source Data file.

Fig 5: REV7 is required for B-cell viability during CSR.a CSR levels to IgG1 after LPS/IL4 stimulation at Day 3 and 4. n = 5 for WT and 53bp1-/-; n = 4 for Cd19cre and Cd19cre Rev7fl/fl. b Growth curve of indicated B cells upon LPS/IL4 (left) or LPS (right) stimulation. Cell numbers at Day 4 of four sets of mice are subjected to statistical calculation. n = 4 for all genotypes. c Percentages of apoptotic cell (AnnexinV+PI-) at Day 4 after stimulating with LPS/IL4 (upper) or LPS (lower) are plotted. n = 4 for WT and 53bp1-/-; n = 3 for Cd19cre and Cd19cre Rev7fl/fl. d Multiple roles of REV7. e End resection of AID-initiated breaks at S?1 is shown for indicated B cells. Resection ratio is defined as previously reported37. n = 4 for WT; n = 3 for 53bp1-/-, Cd19cre, and Cd19cre Rev7fl/fl. f Percentage of cells in G2/M phase is plotted for the indicated genotypes. n = 4 for WT, 53bp1-/-, and Cd19cre Rev7fl/fl; n = 3 for Cd19cre. g Mutation spectrum of C/G in the 5' Sµ region is shown for both strands of genomic DNA. n = 3 for all genotypes. n, independent mice. Data are represented as mean ± SD in a, b, c, e, f, and g. Two-tailed unpaired t-test was performed for b and one-way ANOVA followed by Dunnett’s multiple comparisons test was performed for a, c, e, f, and g. Data from Rev7 knockout are compared with those from other genotypes. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns: p > 0.05. P-values and sample sizes are provided in Supplementary Table 2. Source data are provided as a Source Data file.