title: Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.
abstract: Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.
authors.affiliation: Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Tsurumi, Yokohama, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ukai
authors.forename: Konomi
authors.initials: K
authors.lastname: Ishimaru
authors.forename: Rika
authors.initials: R
authors.lastname: Ouchida
authors.forename: Hiromi
authors.initials: H
authors.lastname: Mori
authors.forename: Chie
authors.initials: C
authors.lastname: Kano
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Moritan
authors.forename: Ji-Yang
authors.initials: JY
authors.lastname: Wang
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D053843:DNA Mismatch Repair / Q000235:genetics / Q000276:immunology; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000276:immunology*; D005347:Fibroblasts / Q000276:immunology*; D017930:Genes, Reporter / Q000235:genetics / Q000276:immunology; D018858:Germinal Center / Q000276:immunology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D041681:NIH 3T3 Cells; D017354:Point Mutation; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; D007136:Immunoglobulins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases