RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

DYW cytidine deaminase domains have a long-range impact on RNA recognition by the PPR array of chimeric plant C-to-U RNA editing factors and strongly affect target selection.

The protein factors for the specific C-to-U RNA editing events in plant mitochondria and chloroplasts possess unique arrays of RNA-binding pentatricopeptide repeats (PPRs) linked to carboxy-terminal cytidine deaminase DYW domains via the extension motifs E1 and E2. The E1 and E2 motifs have distant similarities to tetratricopeptide repeats known to mediate protein-protein interactions but their precise function is unclear. Here, we investigate the tolerance of PPR56 and PPR65, two functionally characterized RNA editing factors of the moss Physcomitrium patens, for the creation of chimeras by variably replacing their C-terminal protein regions. Making use of a heterologous RNA editing assay system in Escherichia coli we find that heterologous DYW domains can strongly restrict or widen the spectrum of off-targets in the bacterial transcriptome for PPR56. Surprisingly, our data suggest that these changes are not only caused by the preference of a given heterologous DYW domain for the immediate sequence environment of the cytidine to be edited but also by a long-range impact on the nucleotide selectivity of the upstream PPRs.

Unraveling the Enzyme-Substrate Properties for APOBEC3A-Mediated RNA Editing.

The APOBEC3 family of human cytidine deaminases is involved in various cellular processes, including the innate and acquired immune system, mostly through inducing C-to-U in single-stranded DNA and/or RNA mutations. Although recent studies have examined RNA editing by APOBEC3A (A3A), its intracellular target specificity are not fully characterized. To address this gap, we performed in-depth analysis of cellular RNA editing using our recently developed sensitive cell-based fluorescence assay. Our findings demonstrate that A3A and an A3A-loop1-containing APOBEC3B (A3B) chimera are capable of RNA editing. We observed that A3A prefers to edit specific RNA substrates which are not efficiently deaminated by other APOBEC members. The editing efficiency of A3A is influenced by the RNA sequence contexts and distinct stem-loop secondary structures. Based on the identified RNA specificity features, we predicted potential A3A-editing targets in the encoding region of cellular mRNAs and discovered novel RNA transcripts that are extensively edited by A3A. Furthermore, we found a trend of increased synonymous mutations at the sites for more efficient A3A-editing, indicating evolutionary adaptation to the higher editing rate by A3A. Our results shed light on the intracellular RNA editing properties of A3A and provide insights into new RNA targets and potential impact of A3A-mediated RNA editing.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the ß-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

AID function in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation of immunoglobulin (Ig) gene variable regions and class switch recombination (CSR) of Ig heavy chain constant regions. Two decades of intensive research has greatly expanded our knowledge of how AID functions in peripheral B cells to optimize antibody responses against infections, while maintaining tight regulation of AID to restrain its activity to protect B cell genomic integrity. The many exciting recent advances in the field include: 1) the first description of AID's molecular structure, 2) remarkable advances in high throughput approaches that precisely track AID targeting genome-wide, and 3) the discovery that the cohesion-mediate loop extrusion mechanism [initially discovered in V(D)J recombination studies] also governs AID-medicated CSR. These advances have significantly advanced our understanding of AID's biochemical properties in vitro and AID's function and regulation in vivo. This mini review will discuss these recent discoveries and outline the challenges and questions that remain to be addressed.

Aromatic disulfides as potential inhibitors against interaction between deaminase APOBEC3G and HIV infectivity factor.

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC 50 values close to or smaller than 1 µM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.

Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination.

The AID/APOBEC polynucleotide cytidine deaminases have historically been classified as either DNA mutators or RNA editors based on their first identified nucleic acid substrate preference. DNA mutators can generate functional diversity at antibody genes but also cause genomic instability in cancer. RNA editors can generate informational diversity in the transcriptome of innate immune cells, and of cancer cells. Members of both classes can act as antiviral restriction factors. Recent structural work has illuminated differences and similarities between AID/APOBEC enzymes that can catalyse DNA mutation, RNA editing or both, suggesting that the strict functional classification of members of this family should be reconsidered. As many of these enzymes have been employed for targeted genome (or transcriptome) editing, a more holistic understanding will help improve the design of therapeutically relevant programmable base editors.

The mRNA tether model for activation-induced deaminase and its relevance for Ig somatic hypermutation and class switch recombination.

Activation-induced deaminase (AID) only deaminates cytosine within single-stranded DNA. Transcription is known to increase AID deamination on duplex DNA substrates during transcription. Using a purified T7 RNA polymerase transcription system, we recently found that AID deamination of a duplex DNA substrate is reduced if RNase A is added during transcription. This finding prompted us to consider that the mRNA tail may contribute to AID action at the nearby transcribed strand (TS) or non-transcribed strand (NTS) of DNA, which are transiently single-stranded in the wake of RNA polymerase movement. Here, we used a purified system to test whether a single-stranded oligonucleotide (oligo) consisting of RNA in the 5' portion and DNA in the 3' portion (i.e., 5'RNA-DNA3', also termed an RNA-DNA fusion substrate) could be deaminated equally efficiently as the same sequence when it is entirely DNA. We found that AID acts on the RNA-DNA fusion substrate and the DNA-only substrate with similar efficiency. Based on this finding and our recent observation on the importance of the mRNA tail, we propose a model in which the proximity and length of the mRNA tail provide a critical site for AID loading to permit a high local collision frequency with the NTS and TS in the transient wake of the RNA polymerase. When the mRNA tail is not present, we know that AID action drops to levels equivalent to when there is no transcription at all. This mRNA tether model explains several local and global features of Ig somatic hypermutation and Ig class switch recombination, while integrating structural and functional features of AID.

Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases.

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddAtox, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Because ZFDs, unlike CRISPR-derived base editors, do not cleave DNA to yield single- or double-strand breaks, no unwanted indels caused by error-prone non-homologous end joining are produced at target sites. Furthermore, recombinant ZFD proteins, expressed in and purified from E. coli, penetrate cultured human cells spontaneously to induce targeted base conversions, demonstrating the proof-of-principle of gene-free gene therapy.

The optimal pH of AID is skewed from that of its catalytic pocket by DNA-binding residues and surface charge.

Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases. AID mutates immunoglobulin loci to initiate secondary antibody diversification. The APOBEC3 (A3) sub-branch mutates viral pathogens in the cytosol and acidic endosomal compartments. Accordingly, AID functions optimally near-neutral pH, while most A3s are acid-adapted (optimal pH 5.5-6.5). To gain a structural understanding for this pH disparity, we constructed high-resolution maps of AID catalytic activity vs pH. We found AID's optimal pH was 7.3 but it retained most (>70%) of the activity at pH 8. Probing of ssDNA-binding residues near the catalytic pocket, key for bending ssDNA into the pocket (e.g. R25) yielded mutants with altered pH preference, corroborating previous findings that the equivalent residue in APOBEC3G (H216) underlies its acidic pH preference. AID from bony fish exhibited more basic optimal pH (pH 7.5-8.1) and several R25-equivalent mutants altered pH preference. Comparison of pH optima across the AID/APOBEC3 family revealed an inverse correlation between positive surface charge and overall catalysis. The paralogue with the most robust catalytic activity (APOBEC3A) has the lowest surface charge and most acidic pH preference, while the paralogue with the most lethargic catalytic rate (AID) has the most positive surface charge and highest optimal pH. We suggest one possible mechanism is through surface charge dictating an overall optimal pH that is different from the optimal pH of the catalytic pocket microenvironment. These findings illuminate an additional structural mechanism that regulates AID/APOBEC3 mutagenesis.

Two different kinds of interaction modes of deaminase APOBEC3A with single-stranded DNA in solution detected by nuclear magnetic resonance.

APOBEC3A (A3A) deaminates deoxycytidine in target motif TC in a single-stranded DNA (we termed it as TC DNA), which mortally mutates viral pathogens and immunoglobulins, and leads to the diversification and lethality of cancers. The crystal structure of A3A-DNA revealed a unique U-shaped recognition mode of target base dC0 . However, when TC DNA was titrated into 15 N-labeled A3A solution, we observed two sets of 1 H-15 N cross-peaks of A3A in HSQC spectra, and two sets of 1 H-1 H cross-peaks of DNA in two-dimensional 13 C,15 N-filtered TOCSY spectra, indicating two different kinds of conformers of either A3A or TC DNA existing in solution. Here, mainly by NMR, we demonstrated that one DNA conformer interacted with one A3A conformer, forming a specific complex A3AS -DNAS in a way almost similar to that observed in the reported crystal A3A-DNA structure, where dC0 inserted into zinc ion binding center. While the other DNA conformer bound with another A3A conformer, but dC0 did not extend into the zinc-binding pocket, forming a nonspecific A3ANS -DNANS complex. The NMR solution structure implied three sites Asn61 , His182 and Arg189 were necessary to DNA recognition. These observations indicate a distinctive way from that reported in X-ray crystal structure, suggesting an unexpected mode of deaminase APOBEC3A to identify target motif TC in DNA in solution.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

Divergence in Dimerization and Activity of Primate APOBEC3C.

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and we determined that Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity, 44, 45, and 144, also promoted dimerization and changes to the dynamics of loop 1, near the enzyme active site. Although forced evolution of rhA3C resulted in a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C and establish the amino acid network for dimerization and increased activity. Based on identification of the key amino acids determining Old World Monkey antiviral activity we predict that other Old World Monkey A3Cs did not impart anti-lentiviral activity, despite fixation of a key residue needed for hominid A3C activity. Overall, the coevolutionary analysis of the A3C dimerization interface presented also provides a basis from which to analyze dimerization interfaces of other A3 family members.

Creating RNA Specific C-to-U Editase from APOBEC3A by Separation of Its Activities on DNA and RNA Substrates.

APOBEC3A (A3A) is a cytidine deaminase involved in innate immune response and is able to catalyze deamination on both DNA and RNA substrates. It was used in creating the CRISPR-mediated base editor, but has since been held back due to its dual activities. On the other hand, it has been a challenge to separate A3A's dual activities in order to enable it for single-base RNA editors. Here we developed the reporter system for C-to-U RNA editing and employed rational design for mutagenesis to differentiate deaminase activities on RNA and DNA substrates to obtain an RNA-specific editase. Generation and examination of 23 previous A3A mutants showed their deamination activity on RNA was mostly abolished when their activity on DNA was impaired, with the exception of mutant N57Q that displayed an inverse change. We designed new mutations on Loops 1 and 7 based on A3A's crystal structure and found mutants H29R and Y132G had differential effects on catalytic activity on RNA and DNA substrates. In order to engineer an A3A with RNA-specific deaminase activity, we combined Y132G with mutations in Loop 1 or helix 6 by rational design. Two multipoint mutants, Y132G/K30R and Y132G/G188A/R189A/L190A, were successful in retaining high deaminase activity on RNA substrate while eliminating deaminase activity on DNA. We, for the first time, created novel human A3A variants with RNA-specific cytidine deaminase activity, providing insight into A3A's mechanism on substrate recognition and a new addition of a toolset to the creation of a RNA-specific C-to-U base editor.

Elucidation of the Complicated Scenario of Primate APOBEC3 Gene Evolution.

APOBEC3 proteins play pivotal roles in defenses against retroviruses, including HIV-1, as well as retrotransposons. Presumably due to the evolutionary arms race between the hosts and retroelements, APOBEC3 genes have rapidly evolved in primate lineages through sequence diversification, gene amplification and loss, and gene fusion. Consequently, modern primates possess a unique set or "repertoire" of APOBEC3 genes. The APOBEC3 gene repertoire of humans has been well investigated. There are three types of catalytic domains (Z domain; A3Z1, A3Z2, and A3Z3), 11 Z domains, and 7 independent genes, including 4 genes encoding double Z domains. However, the APOBEC3 gene repertoires of nonhuman primates remain largely unclear. Here, we characterize APOBEC3 gene repertoires among primates and investigated the evolutionary scenario of primate APOBEC3 genes using phylogenetic and comparative genomics approaches. In the 21 primate species investigated, we identified 145 APOBEC3 genes, including 69 double-domain type APOBEC3 genes. We further estimated the ages of the respective APOBEC3 genes and revealed that APOBEC3B, APOBEC3D, and APOBEC3F are the youngest in humans and were generated in the common ancestor of Catarrhini. Notably, invasion of the LINE1 retrotransposon peaked during the same period as the generation of these youngest APOBEC3 genes, implying that LINE1 invasion was one of the driving forces of the generation of these genes. Moreover, we found evidence suggesting that sequence diversification by gene conversions among APOBEC3 paralogs occurred in multiple primate lineages. Together, our analyses reveal the hidden diversity and the complicated evolutionary scenario of APOBEC3 genes in primates.IMPORTANCE In terms of virus-host interactions and coevolution, the APOBEC3 gene family is one of the most important subjects in the field of retrovirology. APOBEC3 genes are composed of a repertoire of subclasses based on sequence similarity, and a paper by LaRue et al. provides the standard guideline for the nomenclature and genomic architecture of APOBEC3 genes. However, it has been more than 10 years since this publication, and new information, including RefSeq, which we used in this study, is accumulating. Based on accumulating knowledge, APOBEC3 genes, particularly those of primates, should be refined and reannotated. This study updates knowledge of primate APOBEC3 genes and their genomic architectures. We further inferred the evolutionary scenario of primate APOBEC3 genes and the potential driving forces of APOBEC3 gene evolution. This study will be a landmark for the elucidation of the multiple aspects of APOBEC3 family genes in the future.

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

Structural plasticity of substrate selection by activation-induced cytidine deaminase as a regulator of its genome-wide mutagenic activity.

Activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class-switch recombination of antibodies. Computational-biochemical and crystallography analyses of AID have identified three surface grooves for binding single-stranded DNA (ssDNA). Functional studies have also found evidence for RNA-binding motifs on AID. Although AID and the related apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes share a conserved core, AID uniquely features multiple substrate-binding motifs on its surface. Here we suggest that combinatorial deployment of AID's multiple ssDNA- or RNA-binding motifs yields many substrate-binding modes that can accommodate ssDNA, RNA, or DNA/RNA substrates of diverse structures. We also suggest that AID oligomerization generates yet additional novel substrate-binding modes. We propose that this plasticity in substrate choice is an evolved aspect of AID's structure that contributes to the regulation of its differential mutagenic activity at various loci.