V-region mutation in vitro, in vivo, and in silico reveal the importance of the enzymatic properties of AID and the sequence environment

Abstract

The somatic hypermutation of Ig variable regions requires the activity of activation-induced cytidine deaminase (AID) which has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) motif hot spots in in vivo and in vitro assays. We compared mutation profiles of in vitro assays for the 3' flanking intron of VhJ558-Jh4 region to previously reported in vivo profiles for the same region in the Msh2(-/-)Ung(-/-) mice that lack base excision and mismatch repair. We found that the in vitro and in vivo mutation profiles were highly correlated for the top (nontranscribed) strand, while for the bottom (transcribed) strand the correlation is far lower. We used an in silico model of AID activity to elucidate the relative importance of motif targeting in vivo. We found that the mutation process entails substantial complexity beyond motif targeting, a large part of which is captured in vitro. To elucidate the contribution of the sequence environment to the observed differences between the top and bottom strands, we analyzed intermutational distances. The bottom strand shows an approximately exponential distribution of distances in vivo and in vitro, as expected from a null model. However, the top strand deviates strongly from this distribution in that mutations approximately 50 nucleotides apart are greatly reduced, again both in vivo and in vitro, illustrating an important strand asymmetry. While we have confirmed that AID targeting of hot and cold spots is a key part of the mutation process, our results suggest that the sequence environment plays an equally important role.

Fig. 1.

Spatial AID-catalyzed mutation distribution in the Jh4 intron in vitro and in vivo. The distribution of mutations along the length of the sequence (horizontal axis) for the top (A) and bottom (B) strands is shown. Each graph compares in vivo (vertical axis, upwards) with in vitro (vertical axis, downwards) by showing the percentage of sequences mutated at each site. WRC hot spots and SYC cold spots (GYW and GRS on the bottom strand) are highlighted as shown. Note that both the leftmost 60 nt on the top strand and rightmost 60 nt on the bottom strand shown here were removed for the subsequent analysis.

Fig. 2.

Correlation distributions for simulated datasets. The dashed line in each graph represents correlations for the hot/cold spot model. Each curve is a normalized histogram with area under the curve equal to 1. The continuous lines (for the null model) and dotted lines (for the full model) are shown for comparative purposes. Simulation results are shown for top strand (A) and bottom strand (B) in vivo, and top strand (C) and bottom strand (D) in vitro.

Fig. 3.

Distribution of intermutation distances. Each graph shows the distribution of distances between mutations in sequences containing 2 or more mutations. Distributions for top strand in vivo (A) and in vitro (C) and for bottom strand in vivo (B) and in vitro (D) are shown. To represent the null model, a best-fit exponential curve (solid line) is also shown.