B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Sμ-Sγ3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Sμ or Sγ3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2.

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Sm segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but ®nd very few mutations in GL Sg3 and Sg1 regions in activated B cells. These data suggest that switch recombination initiates in the Sm segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and speci®city of mutations in GL and recombined Sm segments differ, suggesting different repair mechanisms. Mutations in recombined Sm regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Sm do not. Additionally, induction conditions affect mutation speci®city within the GL Sm segment. Mutations are most frequent near the S±S junctions and decrease rapidly with distance from the junction. Finally, we ®nd that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S±S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities.

Deficiencies of the Msh2 protein or the Smu tandem repeat (SmuTR) sequences each reduce isotype switching in mice by about 2- to 3-fold. We find that switching in mice deficient for both Msh2 and SmuTR is nearly ablated. We propose that the SmuTR provides closely spaced cleavage sites that can undergo switch recombination independent of Msh2, whereas cleavages in sequences flanking the SmuTR require Msh2 processing to allow recombinational joining. We also find that changes in Smu sequences alter the focus of switch junctions within Sgamma sequences, indicating that sequences of switch regions act together in the choice of switch recombination junctions. These findings help to explain the conservation of tandemly repeated switch regions associated with heavy chain constant genes in species capable of switching.

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Sμ-Sγ3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3′ single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1 − / − splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Sμ segments and recombined Sμ-Sγ3 segments cloned from Ercc1 − / − splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1 − / − cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S–S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.