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Proc Natl Acad Sci U S A. 2015 Feb 17;112(7):E728-37. doi: 10.1073/pnas.1500788112. Epub 2015 Feb 2.

Overlapping hotspots in CDRs are critical sites for V region diversification.

Author information

1
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
2
Department of Biosciences, University of Exeter, Exeter EX2 4QD, United Kingdom;
3
Department of Biological Sciences and Department of Chemistry, University of Southern California, Los Angeles, CA 90089;
4
Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461; and.
5
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461; matthew.scharff@einstein.yu.edu thomas.maccarthy@stonybrook.edu.
6
Department of Applied Mathematics and Statistics, State University of New York, Stony Brook, NY 11794 matthew.scharff@einstein.yu.edu thomas.maccarthy@stonybrook.edu.

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.

KEYWORDS:

AID; IGHV3-23; complementarity-determining regions; cytosine deamination; somatic hypermutation

PMID:
25646473
PMCID:
PMC4343087
DOI:
10.1073/pnas.1500788112
[Indexed for MEDLINE]
Free PMC Article

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