AID in Antibody Diversification: There and Back Again
Authors: Yuqing Feng, Noé Seija, Javier M. Di Noia, Alberto Martin
Journal: Trends in Immunology (2020)

Subverted base excision repair (BER) and mismatch repair (MMR) pathways act concertedly to generate antibody sequence diversity during SHM.In CSR, DNA DSBs are repaired by the nonhomologous end-joining pathway involving the 53BP1–Rif1–Shieldin axis, and by an alternative end-joining pathway involving HMCES (5-Hydroxymethylcytosine binding, ES-cell-specific) that binds and protects resected DSB ends.Genomic targeting of AID appears to be multilayered, with inbuilt redundancy, but robust enough to ensure that most of the genome is spared from AID activity.Cis elements and genome topology act together with trans-acting factors involved in transcription and RNA processing to determine AID activity at specific Ig regions. Other loci sharing genomic and transcriptional features with the Ig are collaterally targeted during SHM and CSR. 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. 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. Antibodies are essential to clearing viral, bacterial, and parasitic infections, as well as neutralizing toxins. The ability of Activation-Induced cytidine Deaminase (AID) to enhance antibody affinity through somatic hypermutation (SHM) (see Glossary) and alter the effector function of antibodies through class switch recombination (CSR) is vital to these tasks (see later). In addition, the generation of a long-lasting antigen-specific antibody memory response is crucial to establishing immunity against pathogens, and AID plays a key role in this endeavor by engraving memories of past pathogen experience through genetic modifications of immunoglobulin (Ig) genes. Here, we review the most essential findings on AID since its discovery two decades ago by providing a timely update on the molecular mechanisms of AID in B cell antibody production. As this review was written during the period that the world was engulfed by the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), we hope it will provide background to inspire vaccine design and the generation of therapeutic antibodies against SARS-CoV-2, as well as inevitable future pathogens. Vertebrates have evolved elegant genetic mechanisms to produce a repertoire of antibodies capable of recognizing and neutralizing a potentially unlimited number of foreign pathogens. Prior to antigen exposure, site-specific recombination of germline Ig variable (V), diversity (D), and joining (J) segments during B cell development provides the primary antibody repertoire [1.Schatz D.G. et al.The V(D)J recombination activating gene, RAG-1.Cell. 1989; 59: 1035-1048Abstract Full Text PDF PubMed Scopus (911) Google Scholar]. The processes of SHM, gene conversion (GCV), and CSR further diversify this antibody repertoire upon B cell-cognate antigen interactions. In antigen-activated B cells, SHM introduces point mutations in the Ig V region of both Ig heavy (Igh) and light chains to enable the process of affinity maturation in the germinal center of secondary lymphoid organs. GCV is an alternative mechanism of altering antibody specificity and affinity in select vertebrate species, including rabbits and birds. GCV involves the production of single-stranded (ss) DNA breaks or DSBs within the rearranged V(D)J region, triggering a homologous recombination-like process that repairs the lesion using Ig V pseudogenes. This produces templated sequence alterations within the V region. CSR is a region-specific deletional recombination event whereby the exons encoding for the IgM constant region within the Igh are replaced with a downstream constant region without changing the antigen-binding specificity, thereby altering the effector functions of the antibody. While all three processes are distinct genetic transactions, they share some common features, such as transcription of their target sequence [2.Peters A. Storb U. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation.Immunity. 1996; 4: 57-65Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar], and preferential mutation of RGYW/WRCY motifs (where W=A/T, R=A/G, Y=C/T), wherein the underlined G/C is mutated [3.Rogozin I.B. Diaz M. Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process.J. Immunol. 2004; 172: 3382-3384Crossref PubMed Google Scholar]. While not demonstrated until the discovery of AID, these and other specific similarities suggested a common inception to each of these processes. AID was identified by Tasuku Honjo's group in 1999 [4.Muramatsu M. et al.Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.J. Biol. Chem. 1999; 274: 18470-18476Crossref PubMed Scopus (865) Google Scholar], and shown to be essential for SHM and CSR in both mice and humans in 2000 [5.Revy P. et al.Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2).Cell. 2000; 102: 565-575Abstract Full Text Full Text PDF PubMed Google Scholar,6.Muramatsu M. et al.Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.Cell. 2000; 102: 553-563Abstract Full Text Full Text PDF PubMed Google Scholar], and later for GCV in chicken DT40 B lymphoma cell line [7.Arakawa H. et al.Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion.Science. 2002; 295: 1301-1306Crossref PubMed Scopus (361) Google Scholar]. Variable lymphocyte receptor diversification in ancient jawless vertebrates also depends on an AID-like cytidine deaminases named CDA1 and CDA2 [8.Morimoto R. et al.Cytidine deaminase 2 is required for VLRB antibody gene assembly in lampreys.Sci. Immunol. 2020; 5Crossref PubMed Scopus (1) Google Scholar], indicating that diversification of antigen receptors by deamination is an ancestral mechanism in vertebrates. AID was originally proposed to be an RNA editing enzyme due to sequence homology to its paralog, the RNA-cytosine deaminase apolipoprotein B mRNA editing catalytic polypeptide 1 (APOBEC 1) [4.Muramatsu M. et al.Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.J. Biol. Chem. 1999; 274: 18470-18476Crossref PubMed Scopus (865) Google Scholar]. However, the DNA deamination model which proposes that AID promotes antibody diversity by deaminating deoxycytidine (dC) to deoxyuridine (dU) within Ig genes is currently supported by significant experimental evidence [9.Methot S.P. Di Noia J.M. Molecular mechanisms of somatic hypermutation and class switch recombination.Adv. Immunol. 2017; 133: 37-87Crossref PubMed Scopus (76) Google Scholar]. Purified AID preferentially mutates dC within the WRCY motif [10.Pham P. et al.Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation.Nature. 2003; 424: 103-107Crossref PubMed Scopus (495) Google Scholar,11.Larijani M. Martin A. The biochemistry of activation-induced deaminase and its physiological functions.Semin. Immunol. 2012; 24: 255-263Crossref PubMed Scopus (0) Google Scholar]. As this motif is preferentially mutated during SHM in vivo [12.Rogozin I.B. Kolchanov N.A. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis.Biochim. Biophys. Acta. 1992; 1171: 11-18Crossref PubMed Scopus (387) Google Scholar], this result provides strong evidence supporting the DNA deamination model for AID function. Biochemical data suggest that AID is catalytically inefficient [13.Larijani M. et al.AID associates with single-stranded DNA with high affinity and a long complex half-life in a sequence-independent manner.Mol. Cell. Biol. 2007; 27: 20-30Crossref PubMed Scopus (62) Google Scholar,14.Pham P. et al.Analysis of a single-stranded DNA-scanning process in which activation-induced deoxycytidine deaminase (AID) deaminates C to U haphazardly and inefficiently to ensure mutational diversity.J. Biol. Chem. 2011; 286: 24931-24942Crossref PubMed Scopus (0) Google Scholar]. The low catalytic rate has been proposed to act as an evolutionary safeguard to limit the mutagenic activity of AID [11.Larijani M. Martin A. The biochemistry of activation-induced deaminase and its physiological functions.Semin. Immunol. 2012; 24: 255-263Crossref PubMed Scopus (0) Google Scholar]. Moreover, the accessibility of AID's catalytic pocket appears to be a key determinant of AID's catalytic rate [15.King J.J. et al.Catalytic pocket inaccessibility of activation-induced cytidine deaminase is a safeguard against excessive mutagenic activity.Structure. 2015; 23: 615-627Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar]. AID has been suggested to act on ssDNA by inducing multiple deaminations per binding event [10.Pham P. et al.Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation.Nature. 2003; 424: 103-107Crossref PubMed Scopus (495) Google Scholar]. A single-molecule resolution Förster resonance energy transfer (FRET) study visualized the ssDNA scanning motion of AID, and suggested that the enzyme was able to move bidirectionally while remaining bound to the same ssDNA [16.Senavirathne G. et al.Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution.Nat. Commun. 2015; 6: 10209Crossref PubMed Scopus (15) Google Scholar]. This behavior of AID on ssDNA may play a role in enhancing mutagenic repair by overwhelming faithful DNA repair (see later). Modeling AID on pre-existing structures of APOBEC enzymes [15.King J.J. et al.Catalytic pocket inaccessibility of activation-induced cytidine deaminase is a safeguard against excessive mutagenic activity.Structure. 2015; 23: 615-627Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar] and crystallizing truncated AID variants [17.Pham P. et al.Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification.DNA Repair (Amst). 2016; 43: 48-56Crossref PubMed Scopus (0) Google Scholar,18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar] have provided valuable structural details. X-ray structure analysis with human monomeric AID reveals that AID prefers to bind and deaminate structured substrates, such as G-quadruplex (G4) structures that can form at the switch (S) region, over linear ssDNA substrates [18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19.Duquette M.L. et al.AID binds to transcription-induced structures in c-MYC that map to regions associated with translocation and hypermutation.Oncogene. 2005; 24: 5791-5798Crossref PubMed Scopus (100) Google Scholar, 20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar] (Box 1). However, as such structures are rare, especially over diverse Ig V exons, it is likely that other structures are also acted upon by AID, such as short patches of melted DNA [21.Larijani M. Martin A. Single-stranded DNA structure and positional context of the target cytidine determine the enzymatic efficiency of AID.Mol. Cell. Biol. 2007; 27: 8038-8048Crossref PubMed Scopus (0) Google Scholar]. These X-ray structure studies suggested that AID contains a bifurcated nucleic acid binding surface [15.King J.J. et al.Catalytic pocket inaccessibility of activation-induced cytidine deaminase is a safeguard against excessive mutagenic activity.Structure. 2015; 23: 615-627Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]: one required for catalysis, and another positively charged surface in AID helix 6, thought to play a role in recognizing structured substrates. A similar bifurcated nucleic acid binding surface has been identified in APOBEC3H, mediating dimerization [22.Bohn J.A. et al.APOBEC3H structure reveals an unusual mechanism of interaction with duplex RNA.Nat. Commun. 2017; 8: 1021Crossref PubMed Scopus (3) Google Scholar]. Besides facilitating substrate binding, specific arginine residues in AID helix 6 may underpin a licensing mechanism that links AID to transcription elongation and warrants productive targeting [23.Methot S.P. et al.A licensing step links AID to transcription elongation for mutagenesis in B cells.Nat. Commun. 2018; 9: 1248Crossref PubMed Scopus (5) Google Scholar]. Nevertheless, biochemical, biophysical and structural approaches have provided valuable knowledge on AID targeting mechanisms during SHM and CSR (see later). A detailed summary of the structure–function description of AID can be found in [9.Methot S.P. Di Noia J.M. Molecular mechanisms of somatic hypermutation and class switch recombination.Adv. Immunol. 2017; 133: 37-87Crossref PubMed Scopus (76) Google Scholar].Box 1Role of Nucleic Acid Structures in Targeting AIDThe transcriptional units encoding the different constant domains of Igh have revealed roles for RNA structures and RNA processing in CSR and AID targeting. These elements are transcribed from a cytokine-driven promoter, which produces a sterile transcript to allow AID access. The primary transcript contains a large intron encompassing the S region (1–12 kb), made up of repeats rich in WRCY motifs. The G-rich composition of the S regions enables the formation of secondary structures such as DNA–RNA hybrids producing R loops and G4 quadruplexes during transcription [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar]. One model envisages that cotranscriptional formation of DNA G4 on the nontemplate strand ssDNA stabilizes R loops on the template strand, while processing of these R loops by the RNA exosome exposes ssDNA and permits deamination of both strands [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar].Others have proposed a role for R loops in CSR resolution, rather than on attracting AID activity [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar]. AID shows high activity on G4 or branched DNA substrates in biochemical assays, allegedly due to a bifurcated substrate-binding surface that can recognize ssDNA overhangs on these structures [18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. G4 DNA additionally promotes AID oligomerization in vitro, which would facilitate DSB formation by increasing the deamination density at the S regions [18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]; however, this remains to be tested in vivo.Another model proposes that AID binds to G4-RNA formed in the debranched S-region intron, which would then guide AID back to the DNA [104.Zheng S. et al.Non-coding RNA generated following lariat debranching mediates targeting of AID to DNA.Cell. 2015; 161: 762-773Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], presumably by base complementarity. This model could account for the link of CSR to RNA splicing [110.Lorenz M. et al.Switch transcripts in immunoglobulin class switching.Science (New York, N.Y.). 1995; 267: 1825-1828Crossref PubMed Google Scholar] and the interaction of AID with splicing factors such as heterogeneous nuclear ribonucleoproteins or polypyrimidine tract-binding proteins [9.Methot S.P. Di Noia J.M. Molecular mechanisms of somatic hypermutation and class switch recombination.Adv. Immunol. 2017; 133: 37-87Crossref PubMed Scopus (76) Google Scholar]. However, the mechanism by which G4-RNA guides and hands off AID to the DNA is not clear. The unwinding of G4-RNA in S-region transcripts by the DEAD-box RNA helicase 1 (DDX1) to allow the formation of DNA:RNA R-loop hybrids at the Igh by base complementarity was recently proposed as a mechanism [105.Ribeiro de Almeida C. et al.RNA helicase DDX1 converts RNA G-quadruplex structures into R-loops to promote IgH class switch recombination.Mol. Cell. 2018; 70: 650-662.e8Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar]. However, ablation of DDX1 reduced CSR only by 25% when analyzed per cell division, indicating that this mechanism is not essential for CSR. Additional aspects of this model remain to be demonstrated, including evidence of AID transfer from RNA to DNA in vivo, and whether this transfer participates in targeting AID for SHM at the Ig V or at off-target loci. The transcriptional units encoding the different constant domains of Igh have revealed roles for RNA structures and RNA processing in CSR and AID targeting. These elements are transcribed from a cytokine-driven promoter, which produces a sterile transcript to allow AID access. The primary transcript contains a large intron encompassing the S region (1–12 kb), made up of repeats rich in WRCY motifs. The G-rich composition of the S regions enables the formation of secondary structures such as DNA–RNA hybrids producing R loops and G4 quadruplexes during transcription [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar]. One model envisages that cotranscriptional formation of DNA G4 on the nontemplate strand ssDNA stabilizes R loops on the template strand, while processing of these R loops by the RNA exosome exposes ssDNA and permits deamination of both strands [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar]. Others have proposed a role for R loops in CSR resolution, rather than on attracting AID activity [20.Pavri R. R loops in the regulation of antibody gene diversification.Genes (Basel). 2017; 8: 154Crossref PubMed Scopus (0) Google Scholar]. AID shows high activity on G4 or branched DNA substrates in biochemical assays, allegedly due to a bifurcated substrate-binding surface that can recognize ssDNA overhangs on these structures [18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]. G4 DNA additionally promotes AID oligomerization in vitro, which would facilitate DSB formation by increasing the deamination density at the S regions [18.Qiao Q. et al.AID recognizes structured DNA for class switch recombination.Mol. Cell. 2017; 67: 361-373.e4Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]; however, this remains to be tested in vivo. Another model proposes that AID binds to G4-RNA formed in the debranched S-region intron, which would then guide AID back to the DNA [104.Zheng S. et al.Non-coding RNA generated following lariat debranching mediates targeting of AID to DNA.Cell. 2015; 161: 762-773Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], presumably by base complementarity. This model could account for the link of CSR to RNA splicing [110.Lorenz M. et al.Switch transcripts in immunoglobulin class switching.Science (New York, N.Y.). 1995; 267: 1825-1828Crossref PubMed Google Scholar] and the interaction of AID with splicing factors such as heterogeneous nuclear ribonucleoproteins or polypyrimidine tract-binding proteins [9.Methot S.P. Di Noia J.M. Molecular mechanisms of somatic hypermutation and class switch recombination.Adv. Immunol. 2017; 133: 37-87Crossref PubMed Scopus (76) Google Scholar]. However, the mechanism by which G4-RNA guides and hands off AID to the DNA is not clear. The unwinding of G4-RNA in S-region transcripts by the DEAD-box RNA helicase 1 (DDX1) to allow the formation of DNA:RNA R-loop hybrids at the Igh by base complementarity was recently proposed as a mechanism [105.Ribeiro de Almeida C. et al.RNA helicase DDX1 converts RNA G-quadruplex structures into R-loops to promote IgH class switch recombination.Mol. Cell. 2018; 70: 650-662.e8Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar]. However, ablation of DDX1 reduced CSR only by 25% when analyzed per cell division, indicating that this mechanism is not essential for CSR. Additional aspects of this model remain to be demonstrated, including evidence of AID transfer from RNA to DNA in vivo, and whether this transfer participates in targeting AID for SHM at the Ig V or at off-target loci. Uracils introduced in DNA are normally repaired faithfully, restoring the original DNA sequence (Figure 1). This is not the case during SHM, GCV, or CSR, whereby the initiating AID-induced lesion promotes further mutagenesis that benefits affinity maturation by providing a wide range of amino acid changes, or causes DNA breaks that trigger recombination events. Why faithful repair of DNA uracils is subverted during SHM has been the subject of intense investigation. The majority of the uracils in the genome are faithfully repaired by the base excision repair (BER) pathway: excision of uracils by uracil DNA glycosylase (UNG); incision at the resulting abasic sites by apurinic/apyrimidinic endonuclease 1; processing of the abasic sites by DNA polymerase β, followed by sealing of the final nick by DNA ligase 3 [24.Krokan H.E. Bjoras M. Base excision repair.Cold Spring Harb. Perspect. Biol. 2013; 5: a012583Crossref PubMed Scopus (410) Google Scholar]. Uracils escaping detection by UNG or single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) can be copied over by the replication machinery, producing transition mutations at G:C base pairs (Figure 1) [25.Di Noia J. Neuberger M.S. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase.Nature. 2002; 419: 43-48Crossref PubMed Scopus (433) Google Scholar,26.Di Noia J.M. et al.SMUG1 is able to excise uracil from immunoglobulin genes: insight into mutation versus repair.EMBO J. 2006; 25: 585-595Crossref PubMed Scopus (77) Google Scholar]. However, during SHM, the later steps of BER are circumvented, for reasons that are not fully clear (see Outstanding Questions). Replication across the noninstructive abasic site by the translesional DNA polymerases REV1 and Pol η generates both transition and transversion mutations at G:C pairs [27.Krijger P.H. et al.Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway.Eur. J. Immunol. 2013; 43: 2765-2770Crossref PubMed Scopus (18) Google Scholar,28.Kano C. et al.Analysis of mice deficient in both REV1 catalytic activity and POLH reveals an unexpected role for POLH in the generation of C to G and G to C transversions during Ig gene hypermutation.Int. Immunol. 2012; 24: 169-174Crossref PubMed Scopus (14) Google Scholar] (Figure 1). Indeed, mice deficient in UNG (Ung-/-) exhibit reduced transversion mutations at G:C pairs in Ig V regions relative to wild-type (WT) mice, with a minor impact on mutagenesis at A:T base pairs [29.Rada C. et al.Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.Curr. Biol. 2002; 12: 1748-1755Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar]. Mismatch repair (MMR) also mediates repair of uracil lesions, detected as U:G mispairs. Replication-associated canonical MMR (cMMR) is traditionally a high-fidelity process that preserves genomic stability. cMMR begins with the recognition of a base mispair by the MutSα heterodimer complex (MSH2/MSH6), followed by the recruitment of MutLα complex (MLH1/PMS2) and the activation of the endonuclease activity of PMS2, and removal of a track of the DNA strand by exonuclease EXO1 [30.Jiricny J. The multifaceted mismatch-repair system.Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (788) Google Scholar,31.Chahwan R. et al.AIDing antibody diversity by error-prone mismatch repair.Semin. Immunol. 2012; 24: 293-300Crossref PubMed Scopus (45) Google Scholar]. The resynthesis of the ssDNA gap by high-fidelity replicative polymerases restores the original DNA sequences. Instead, during SHM, a subverted MMR pathway termed noncanonical MMR (ncMMR) using the translesion DNA polymerase η is largely responsible for mutations at A:T base pairs [31.Chahwan R. et al.AIDing antibody diversity by error-prone mismatch repair.Semin. Immunol. 2012; 24: 293-300Crossref PubMed Scopus (45) Google Scholar, 32.Cascalho M. et al.Mismatch repair co-opted by hypermutation.Science. 1998; 279: 1207-1210Crossref PubMed Scopus (136) Google Scholar, 33.Rada C. et al.Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.Immunity. 1998; 9: 135-141Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 34.Wiesendanger M. et al.Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern.J. Exp. Med. 2000; 191: 579-584Crossref PubMed Scopus (0) Google Scholar, 35.Bardwell P.D. et al.Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.Nat. Immunol. 2004; 5: 224-229Crossref PubMed Scopus (0) Google Scholar, 36.Kim N. et al.Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications.J. Exp. Med. 1999; 190: 21-30Crossref PubMed Scopus (0) Google Scholar, 37.Zeng X. et al.DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes.Nat. Immunol. 2001; 2: 537-541Crossref PubMed Scopus (0) Google Scholar] (Figure 1). Mutagenic ncMMR is largely linked to AID lesions, as non-AID targets are faithfully repaired by MMR [38.Green B. et al.The mismatch repair pathway functions normally at a non-AID target in germinal center B cells.Blood. 2011; 118: 3013-3018Crossref PubMed Scopus (8) Google Scholar]. Recent work by a number of groups has shed light on ncMMR, whereby the UNG/SMUG1 and MMR pathways converge and work collaboratively to facilitate A:T and G:C mutagenesis [39.Frieder D. et al.The concerted action of Msh2 and UNG stimulates somatic hypermutation at A. T base pairs.Mol. Cell Biol. 2009; 29: 5148-5157Crossref PubMed Scopus (0) Google Scholar, 40.Krijger P.H. et al.Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation.J. Exp. Med. 2009; 206: 2603-2611Crossref PubMed Scopus (41) Google Scholar, 41.Girelli Zubani G. et al.Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.J. Exp. Med. 2017; 214: 1169-1180Crossref PubMed Scopus (8) Google Scholar, 42.Thientosapol E.S. et al.Proximity to AGCT sequences dictates MMR-independent versus MMR-dependent mechanisms for AID-induced mutation via UNG2.Nucleic Acids Res. 2017; 45: 3146-3157PubMed Google Scholar, 43.Dingler F.A. et al.Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation.Eur. J. Immunol. 2014; 44: 1925-1935Crossref PubMed Scopus (25) Google Scholar]. This view is supported by findings that both UNG and ncMMR are able to function in the G1 phase of the cell cycle [41.Girelli Zubani G. et al.Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.J. Exp. Med. 2017; 214: 1169-1180C