Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Smicro 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 find very few mutations in GL Sgamma3 and Sgamma1 regions in activated B cells. These data suggest that switch recombination initiates in the Smicro segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Smicro segments differ, suggesting different repair mechanisms. Mutations in recombined Smicro regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Smicro do not. Additionally, induction conditions affect mutation specificity within the GL Smicro segment. Mutations are most frequent near the S-S junctions and decrease rapidly with distance from the junction. Finally, we find 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.