Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase η are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase η overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions.
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