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Induction of A:T Mutations Is Dependent on Cellular Environment but Independent of Mutation Frequency and Target Gene Location

Akiko Ukai^*, Konomi Ishimaru^*, Rika Ouchida^*, Hiromi Mori^*, Chie Kano^*,, Toshiyuki Moritan and Ji-Yang Wang^1,*

^* Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan; and Faculty of Medical Engineering, Suzuka University of Medical Science, Suzuka, Mie, Japan

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 (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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Ji-Yang Wang, Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. E-mail address: oh{at}rcai.riken.jp

² Abbreviations used in this paper: GC, germinal center; AID, activation-induced cytidine deaminase; EXO, exonuclease; IRES, internal ribosome entry site; MMR, mismatch repair; MSH, MutS homolog; PCNA, proliferating cell nuclear Ag; POLH, DNA polymerase ; SHM, somatic hypermutation; Tet, tetracycline.

³ The online version of this article contains supplemental material.