Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.A fter an encounter with antigen and subsequent migration into the germinal centers of the secondary lymphoid organs, B cells undergo a regulated cascade of mutational events that occur at a very high frequency and are largely restricted to the variable (V) and switch (S) regions of the Ig heavy chain locus and the V region of the light chain locus. These mutagenic events are responsible for the somatic hypermutation (SHM) of the V regions and the class switch recombination of the constant (C) regions that are required for protective antibodies (1, 2). Both SHM and class switch recombination are initiated by activationinduced deaminase (AID) that preferentially deaminates the dC residues in WRC (W = A/T, R = A/G) hotspot motifs at frequencies 2-10-fold higher than SYC (S = G/C; Y = C/T) cold spots (3-7). During V region SHM, the resulting dU:G mismatch can then be replicated during S-phase to produce transition mutations, be processed by uracil-DNA glycosylase 2 and apurinic/ apyrimidinic endonucleases through the base excision repair pathway to produce both transitions and transversions (8-10), or be recognized by MutS homolog (MSH)2/MSH6 of the mismatch repair (MMR) complex that recruits the low-fidelity polymerase eta (Polη) to generate additional mutations at neighboring A:T residues (11).The specificity of AID targeting to the Ig gene has been under intense investigation. Studies have shown that AID deamination and mutagenesis targets single-stranded DNA substrates generated during ...