AID in Antibody Diversification: There and Back Again

Abstract

Activation-Induced cytidine Deaminase (AID) initiates affinity maturation and isotype switching by deaminating deoxycytidines within immunoglobulin genes, leading to somatic hypermutation (SHM) and class switch recombination (CSR). AID thus potentiates the humoral response to clear pathogens. Marking the 20th anniversary of the discovery of AID, we review the current understanding of AID function. We discuss AID biochemistry and how error-free forms of DNA repair are co-opted to prioritize mutagenesis over accuracy during antibody diversification. We discuss the regulation of DNA double-strand break (DSB) repair pathways during CSR. We describe genomic targeting of AID as a multilayered process involving chromatin architecture, cis- and trans-acting factors, and determining mutagenesis - distinct from AID occupancy at loci that are spared from mutation.

Figure 1

Multifaceted Uracil Lesion Processing in Somatic Hypermutation (SHM). AID-Induced dU:dG mismatches can be resolved by various pathways, leading to a diverse mutation spectrum at G:C and A:T base pairs in immunoglobulin variable (Ig V) regions. AID-induced uracil lesions can be repaired faithfully by canonical BER and MMR, leading to no SHM. Replicating over uracils by replicative polymerases generates transition mutations at G:C base pairs. UNG, and to a lesser extent, SMUG1, from the BER pathway, detect and excise uracils from DNA, creating abasic sites. Replicating over abasic sites by translesion synthesis polymerase from short-patch ncBER pathway generates transition and transversion mutations at G:C base pairs. Noncanonical MMR pathway or long-patch BER can excise single-stranded DNA surrounding either the dU:dG mismatch, or the abasic site, respectively, to initiate patch excision that leads to the engagement of translesion synthesis polymerases, generating both transition and transversion mutations at A:T base pairs. Abbreviations: AID, Activation-induced Cytidine Deaminase; ncBER, noncanonical base excision repair; ncMMR, noncanonical mismatch repair; SMUG1, single-strand selective monofunctional uracil DNA glycosylase; UNG, uracil DNA glycosylase. This figure was created using BioRender (https://biorender.com/).

Figure 2

AID-Mediated DSB Formation within S Regions Drives CSR. (A) Sequence feature of S regions in mouse Ig heavy chain locus: S regions preceding different constant region exons are highly enriched with AID deamination hotspot 5′-AGCT-3′, a palindromic variant representing the canonical RGYW/WRCY sequences. Only Sμ and Sα are shown here for simplicity. (B) DSB ends are joined through NHEJ and Alt-EJ to complete CSR. NHEJ mediates DSB EJ with no or minimal microhomology, whereas Alt-EJ is facilitated by complementary base-paring interactions between microhomologous sequences present between the two synapsed S regions DSBs. Black bars represent homologous sequences near DSB ends. Abbreviations: 53BP1, p53-binding protein 1; AID, Activation-induced Cytidine Deaminase; Alt-EJ, alternative end joining; CSR, class switch recombination; CTIP, CtBP-interacting protein; DNA-PKcs, DNA-dependent protein kinase, catalytic subunit; DSB, double-strand break; ES cell specific; HMCES, 5-Hydroxymethylcytosine binding, ES-cell-specific; KU, Ku 70/80 complex; LIG3, ligase 3; LIG4, ligase 4; MRN, MRE11–RAD50–NBN complex; NHEJ, nonhomologous end joining; RIF1, replication timing regulatory factor 1; S, switch; SHIELDIN, protein complex composed of SHLD1, SHLD2, SHLD3, and REV 7; XLF, XRCC4-like factor; XRCC 1/4, X-ray repair cross-complementing protein 1/4. This figure was created using BioRender (https://biorender.com/).

Figure I

Stepwise Model for Productive Deamination of Selected AID Off-Targets. While AID may be able to associate to chromatin in different ways, it could accumulate at paused promoter regions of many genes by virtue of its interaction with SPT5, but access only a subset of those genes bodies, for instance by a licensing mechanism and/or the abundance of ssDNA substrate. Abbreviations: AID, Activation-induced Cytidine Deaminase; RNAPII, RNA polymerase II.

Figure 3

Multilayered Productive AID Targeting in B cells. (A) AID activity is restricted to type A chromatin compartments, which are accessible and transcriptionally active, versus transcriptionally inactive B compartments. (B) AID activity is also limited to some TADs of DNA, often affecting highly transcribed genes regulated by superenhancers, with clusters by long-range interactions between regulatory sequences. The Ig and some off-target genes additionally harbor cis-acting elements named DIVAC (diversification activators) that attract AID activity. (C, D) AID can be recruited to promoter or promoter-proximal paused RNAPII via SPT5. (C) AID accesses the gene body of susceptible loci through associations with the elongating RNAPII complex, via a putative licensing mechanism. At these loci, several mechanisms can provide ssDNA AID substrates (yellow boxes): DNA supercoiling, DNA secondary structures, and RNA processing of sense and antisense RNA. Transcriptional stalling, caused by nucleic acid secondary structures or by early termination due to antisense transcription, can also expose and favor AID jumping to ssDNA, possibly helped by the regulated association of AID to the ssDNA binding protein RPA. Stalling and SPT5 association may also allow time for AID to act on the small transcription bubble within the RNAPII. (D) At loci that are not susceptible to mutation, AID may fail its coupling with transcription elongation, and/or lack access to ssDNA substrates. Abbreviations: AID, Activation-induced Cytidine Deaminase; ncRNA, noncoding RNA; NELF, negative elongation factor (causes RNAPII pausing); PAF, RNAPII-associated factor complex; RNAPII, RNA polymerase II; ssDNA, single-stranded DNA; TAD, topologically associated domain; TSS, transcription start site.