Somatic hypermutation patterns are shaped by both motif position and sequence grammar.

Somatic hypermutation (SHM) in variable regions of immunoglobulin genes by activation-induced deaminase (AID) is essential for the maturation of protective antibodies against pathogen and vaccine antigens. AID preferentially mutates cytosines within WRCH motifs (wherein W = A/T, R = A/G, and H = A/C/T) in single-stranded DNA, yet these motifs show large but reproducible variation in mutation frequency, suggesting a crucial role for sequences flanking the WRCH motifs (i.e., a sequence grammar) in determining mutational outcomes. However, the nature of this sequence grammar is poorly understood. Here, we demonstrate that identical sequence contexts can exert significantly varying effects on the mutagenesis of different WRCH motifs. Molecular dynamics simulations reveal that both the sequence context and the specific WRCH motif modulate AID activity by altering the mode and strength of AID's interactions with single-stranded DNA. Repositioning a motif and its context within the variable region significantly alters its mutability. Therefore, the mutability of AID target cytosines is determined by a motif-specific sequence grammar that determines, in part, how activation-induced deaminase binds single-stranded DNA, as well as the motif position.