Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

Abstract

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination.

FIGURE 1

Antibody structure. The BCR is comprised of two immunoglobulin (Ig) heavy (IgH) chains encoded by the IgH heavy chain locus and two Ig light (IgL) chains. The rectangles represent Ig domains that constitute the structural units of the immunoglobulin heavy and light chains. The variable regions are assembled through V(D)J recombination of V_H, D_H, and J_H gene segments on the heavy chain and V_L and J_L gene segments on the light chain. Complementarity-determining regions (CDRs) are indicated as regions in dashed red boxes: CDR 1 and 2 are encoded in the V_H or V_L gene segments, and CDR 3 is encoded by the V_H D_H J_H junctional region or V_L and J_L junctional region. The heavy and light chain variable regions form the antigen-binding site. The constant region determines the class and effector function of the antibody molecule. doi:10.1128/microbiolspec.MDNA3-0037-2014.f1

FIGURE 2

Genomic alterations of the IgH locus. a. Organization of the IgH constant (C) region. Each C region is preceded by a switch (S) region and a noncoding “I” exon. Blue oval between V(D)J exon and Iμ represents IgH intronic enhancer (iEμ). Blue oval downstream of Cα represents IgH 3′ regulatory region (IgH 3′RR). μ and δ mRNAs are shown below the corresponding genes. Dashed line represents spliced transcript. b. AID generates point mutations and/or DNA double strand breaks (DSBs) at the V(D)J exon during somatic hypermutation (SHM). c. AID-initiated DSBs in Sμ and Sγ1 result in CSR to IgG1. μ and γ1 germline transcripts are initiated from promoters upstream of the corresponding I exons. doi:10.1128/microbiolspec.MDNA3-0037-2014.f2

FIGURE 3

Mechanisms of AID cytidine deamination in SHM and CSR. AID deaminates cytidine (C) to uridines (U). The U/G lesion may be repaired with high fidelity (i.e. to C/G) by conventional base excision repair (BER) or mismatch repair (MMR). Mutagenic outcomes during SHM and CSR are generated by the following processes. a. Replication over the U/G lesion produces transition mutations at C/G base pairs. b. Uracil-DNA-Glycosylase (UNG) of the BER pathway excises the U creating an abasic site. Replication over the abasic site generates transition and transversion mutations at C/G base pairs. N indicates any nucleotide A,G,C, or T. AP endonuclease 1 (APE1) may create a nick at the abasic site. Nicks on both DNA strands may lead to DSBs. c. MSH2-MSH6 of the mismatch repair pathway recognize the U/G mismatch. Exo1 excises the patch of DNA containing the mismatch. Error-prone polymerase resynthesizes the patch leading to spreading of mutations to A/T base pairs. Overlapping gaps may lead to DSBs. doi:10.1128/microbiolspec.MDNA3-0037-2014.f3

FIGURE 4

Transcriptional targeting of AID. a. R-loop structure. An R loop forms from G-rich RNA transcribed from the C-rich template strand forming a stable RNA-DNA hybrid with the C-rich template strand and looping out the G-rich nontemplate strand as ssDNA. b. A working model suggests that once AID is brought to a target via stalled Pol II and Spt5, the RNA exosome displaces or degrades the nascent RNA, thus making the template strand available for deamination, which may in vivo be further augmented by RPA association. Figure adapted from reference . doi:10.1128/microbiolspec.MDNA3-0037-2014.f4

FIGURE 5

Outcomes of DSBs in S regions. DSBs within a S region may be directly joined back together or be joined back together following end resection, leading to intra-switch region deletions. Alternatively, a DSB generated in one S region may join to a DSB in another S region over a long-range (60 to 160 kb), which may lead to CSR. In addition, DSBs generated in an S region may participate in chromosomal translocations by joining to other non-S-region DSBs on the cis chromosome or to DSBs on other chromosomes. doi:10.1128/microbiolspec.MDNA3-0037-2014.f5

FIGURE 6

Synapsis and end-joining. The roles of synapsis and tethering in promoting long-range joining are shown. We propose that S regions are synapsed by diffusion, and that synapsis is possibly enhanced by proximity of S regions resulting from chromatin organization into megabase/submegabase domains. Post-cleavage, synapsis may be maintained by general DSB response (DSBR) factors, promoting the joining of S-region DSB ends by classical nonhomologous end-joining (C-NHEJ) and possibly alternative end-joining (A-EJ). doi:10.1128/microbiolspec.MDNA3-0037-2014.f6