Research Article: Activation-Induced Cytidine Deaminase Initiates Immunoglobulin Gene Conversion and Hypermutation by a Common Intermediate

Date Published: July 13, 2004

Publisher: Public Library of Science

Author(s): Hiroshi Arakawa, Huseyin Saribasak, Jean-Marie Buerstedde

Abstract: Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (ψV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, ψV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the ψV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence.

Partial Text: Immunoglobulin (Ig) genes are further diversified after V(D)J rearrangement by gene conversion, hypermutation, or a combination of the two. Surprisingly, even closely related species employ different strategies: mice and humans use exclusively hypermutation (Milstein and Rada 1995), whereas rabbits, cows, and pigs use mainly gene conversion (Butler 1998). The balance between the two phenomena can also shift during differentiation: for example, chicken B-cells first develop their Ig repertoire by gene conversion in the bursa (Reynaud et al. 1987; Arakawa and Buerstedde 2004) and later fine tune it by hypermutation in splenic germinal centers (Arakawa et al. 1996).

These results demonstrate that the deletion of the nearby pseudogene donors abolishes Ig gene conversion in DT40 and activates a mutation activity that closely resembles Ig hypermutation. The features shared between this new mutation activity and somatic hypermutation include (1) AID dependence, (2) a predominance of single nucleotide substitutions, (3) distribution of the mutations within the 5′ transcribed region, (4) a preference for hotspots, and (5) Ig gene specificity. The only differences between the mutation activity induced by loss of ψV and Ig hypermutation in vivo are the relative lack of mutations in A/T bases and a predominance of transversion mutations in the ψV knockout clones. However, these differences are also seen in hypermutating Epstein Barr virus–transformed B-cell lines (Bachl and Wabl 1996; Faili et al. 2002) and DT40 mutants of RAD51 paralogs (Sale et al. 2001), indicating that part of the Ig hypermutator activity is missing in transformed B-cell lines. Interestingly, the rate of Ig hypermutation in the AIDRψV– clone seems higher than the rate of Ig gene conversion in the DT40Cre1 progenitor. An explanation for this could be that some conversion tracts are limited to stretches of identical donor and target sequences and thus leave no trace. The ratio of transversion to transition was lower for the AIDRψVpartial clone (see Figure 4). Although we can only speculate about the cause of this difference, it might be due to the correction of point mutations by mismatch correction of one or more sites in gene conversion tracts.



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