title: Overlapping hotspots in CDRs are critical sites for V region diversification.
abstract: 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.
authors: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors: Lirong
authors: L
authors: Wei
authors: Department of Biosciences, University of Exeter, Exeter EX2 4QD, United Kingdom;
authors: Richard
authors: R
authors: Chahwan
authors: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors: Shanzhi
authors: S
authors: Wang
authors: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors: Xiaohua
authors: X
authors: Wang
authors: Department of Biological Sciences and Department of Chemistry, University of Southern California, Los Angeles, CA 90089;
authors: Phuong T
authors: PT
authors: Pham
authors: Department of Biological Sciences and Department of Chemistry, University of Southern California, Los Angeles, CA 90089;
authors: Myron F
authors: MF
authors: Goodman
authors: Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461; and.
authors: Aviv
authors: A
authors: Bergman
authors: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461; matthew.scharff@einstein.yu.edu thomas.maccarthy@stonybrook.edu.
authors: Matthew D
authors: MD
authors: Scharff
authors: Department of Applied Mathematics and Statistics, State University of New York, Stony Brook, NY 11794 matthew.scharff@einstein.yu.edu thomas.maccarthy@stonybrook.edu.
authors: Thomas
authors: T
authors: MacCarthy
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D022801:Complementarity Determining Regions; D003564:Cytidine Deaminase / Q000378:metabolism; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D009154:Mutation; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D022801:Complementarity Determining Regions; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D004247:DNA; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase
keywords: AID; IGHV3-23; complementarity-determining regions; cytosine deamination; somatic hypermutation
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