B cells from Exo1-XRCC4-, Msh2-XRCC4- and Mlh1-XRCC4- mice and various control mice were stimulated in vitro for 4 days with αCD40 plus IL4 to induce IgG1 CSR. Following 4 days of culture, B cells were analyzed by flow cytometry to determine percent of B cells that had undergone CSR to IgG1. (A) Representative FACs dot plots of Exo1-XRCC4- and control B cells from one experiment. Gated population represents % of IgG1+ cells. (B) Same as in (A) with Mlh1-XRCC4- B cells. (C) Same as in (A) for Msh2-XRCC4- B cells

(A) Metaphases were prepared from B cells cultured with αCD40 plus IL4 for four days. Metaphases were hybridized with fluorescently labeled BAC199 and BAC207 which are specific for regions 3′ and 5′of the Igh locus. Picture shows chromosomes from an individual metaphase with an intact Igh locus (normal). (B) As in (A) where pictures show chromosomes from individual metaphases with an Igh locus associated break. (C) As in (A) where pictures show chromosomes from individual metaphases with Igh locus associated translocations. Cartoons depicting the status of each Igh locus shown in the metaphase pictures are displayed below the pictures. Pictures of Igh locus breaks and translocations are from the XRCC4-deficient strains analyzed. (D) Bar graphs represent percent of metaphases with Igh locus specific chromosomal aberrations from Exo1-XRCC4- and littermate controls. Total number of metaphases analyzed are in parentheses below each bar graph. 7 Exo1-XRCC4- mice, 6 WT, 6 XRCC4-, and 3 Exo1-mice were used. (E) As in (D), results are from 5 Mlh1-XRCC4 mice, 4 XRCC4-, 4 Mlh1- and 3 WT. (F) As in (D), results are from 9 Msh2-XRCC4- mice, 5 XRCC4-, 5 WT, and 4 Msh2- mice. Statistical significance was determined using a two-tailed T test. (G) Bar graphs represent the percent of DNA breaks observed that were chromosomal (unshaded) versus chromatid (black shaded) breaks. (H) Bar graphs represent the percent of abnormal chromosomes that had free (unshaded) versus translocated (black shaded) DNA ends.

B cells from WT, Msh2-, Mlh1- and Exo1- mice were isolated and stimulated in vitro for 4 days with αCD40 plus IL4 to induce CSR. Following 4 days of culture, B cells were fused to the NS1 myeloma cell line to generate hybridomas. IgM⁺ hybridomas were assayed by southern blot for internal Sμ recombination. Representative southern blots hybridized with a Cμ probe to detect ISR. Δ symbol signifies hybridomas with ISR. 4 WT, 3 Mlh1^−/−, 3 Exo1^−/−, and 2 Msh2^−/− mice were used for this analysis.

Sμ-Sγ1 junctions from XRCC4-, Exo1-XRCC4-, Mlh1-XRCC4-, and Msh2-XRCC4- B cells were amplified by PCR and aligned to μ and γ germline sequences. Amount of microhomology at switch junctions was determined by counting the number of uninterrupted shared nucleotide (nt) identity with Sμ and Sγ sequences at the junction. Cultures from 3 Exo1-XRCC4-, 3 Msh2-XRCC4-, and 2 Mlh1-XRCC4- mice were used to generate the Sμ-Sγ1 junctions. The slight difference in the percent of junctions with ≥5nts of microhomology observed for Msh2-XRCC4- and other XRCC4-deficient strains is not statistically different.

(A) Metaphases were prepared from B cells cultured with αCD40 plus IL4 for four days. Metaphases were hybridized with a fluorescently labeled telomere specific probe. Pictures show chromosomes from individual metaphases that are normal or contain DNA breaks or translocations. Cartoons depicting the status of the chromosomes shown in the metaphase pictures are displayed below the pictures. (B) Bar graphs represent percent of metaphases with chromosomal aberrations from 3 Exo1, 4 Mlh1-, 4 Msh2-, or 9 WT control mice. (C) As in (B), results are from 5 Exo1-XRCC4-, 3 WT and 4 XRCC4- mice. (D) As in (B), results are from 7 Mlh1-XRCC4 mice, 4 XRCC4- and 3 WT. (E) As in (B), results are from 9 Msh2-XRCC4- mice, 5 XRCC4- and 4 WT mice. Statistical significance was determined using a two-tailed T test.