RESUMEN
Activation-induced cytidine deaminase (AID) initiates antibody diversification by mutating immunoglobulin loci in B lymphocytes. AID and related APOBEC3 (A3) enzymes also induce genome-wide mutations and lesions implicated in tumorigenesis and tumor progression. The most prevalent mutation signatures across diverse tumor genomes are attributable to the mistargeted mutagenic activities of AID/A3s. Thus, inhibiting AID/A3s has been suggested to be of therapeutic benefit. We previously used a computational-biochemical approach to gain insight into the structure of AID's catalytic pocket, which resulted in the discovery of a novel type of regulatory catalytic pocket closure that regulates AID/A3s that we termed the "Schrodinger's CATalytic pocket". Our findings were subsequently confirmed by direct structural studies. Here, we describe our search for small molecules that target the catalytic pocket of AID. We identified small molecules that inhibit purified AID, AID in cell extracts, and endogenous AID of lymphoma cells. Analogue expansion yielded derivatives with improved potencies. These were found to also inhibit A3A and A3B, the two most tumorigenic siblings of AID. Two compounds exhibit low micromolar IC50 inhibition of AID and A3A, exhibiting the strongest potency for A3A. Docking suggests key interactions between their warheads and residues lining the catalytic pockets of AID, A3A, and A3B and between the tails and DNA-interacting residues on the surface proximal to the catalytic pocket opening. Accordingly, mutants of these residues decreased inhibition potency. The chemistry and abundance of key stabilizing interactions between the small molecules and residues within and immediately outside the catalytic pockets are promising for therapeutic development.
RESUMEN
Activation-induced cytidine deaminase (AID) mutates immunoglobulin genes and acts genome-wide. AID targets robustly transcribed genes, and purified AID acts on single-stranded (ss) but not double-stranded (ds) DNA oligonucleotides. Thus, it is believed that transcription is the generator of ssDNA for AID. Previous cell-free studies examining the relationship between transcription and AID targeting have employed a bacterial colony count assay wherein AID reverts an antibiotic resistance stop codon in plasmid substrates, leading to colony formation. Here, we established a novel assay where kb-long dsDNA of varying topologies is incubated with AID, with or without transcription, followed by direct sequencing. This assay allows for an unselected and in-depth comparison of mutation frequency and pattern of AID targeting in the absence of transcription or across a range of transcription dynamics. We found that without transcription, AID targets breathing ssDNA in supercoiled and, to a lesser extent, in relaxed dsDNA. The most optimal transcription only modestly enhanced AID action on supercoiled dsDNA in a manner dependent on RNA polymerase speed. These data suggest that the correlation between transcription and AID targeting may reflect transcription leading to AID-accessible breathing ssDNA patches naturally occurring in de-chromatinized dsDNA, as much as being due to transcription directly generating ssDNA.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN de Cadena Simple/química , ADN/química , Plásmidos/genética , Transcripción Genética , Citidina Desaminasa/genética , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Plásmidos/química , Plásmidos/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: AID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (Wâ¯=â¯A/T, Râ¯=â¯A/G) and YC (Yâ¯=â¯T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases. METHODS: Substrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs. RESULTS: All three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases. CONCLUSIONS: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged. GENERAL SIGNIFICANCE: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.
Asunto(s)
Citidina Desaminasa/química , Daño del ADN , Desoxicitidina/química , Antígenos de Histocompatibilidad Menor/química , Proteínas/química , Citidina Desaminasa/metabolismo , Desaminación , Desoxicitidina/metabolismo , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Oxidación-Reducción , Proteínas/metabolismoRESUMEN
Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.
Asunto(s)
Desaminasas APOBEC-1/genética , Citidina Desaminasa/clasificación , Citidina Desaminasa/genética , Variaciones en el Número de Copia de ADN , Lampreas/genética , Linfocitos/inmunología , Receptores de Antígenos/genética , Desaminasas APOBEC-1/química , Desaminasas APOBEC-1/inmunología , Secuencia de Aminoácidos , Animales , Citidina Desaminasa/química , Citidina Desaminasa/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Conformación Proteica , Receptores de Antígenos/clasificación , Homología de Secuencia , Secuenciación Completa del GenomaRESUMEN
Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.
Asunto(s)
Linfocitos B/metabolismo , Citidina Desaminasa/metabolismo , Cambio de Clase de Inmunoglobulina , Mutagénesis , Elongación de la Transcripción Genética , Animales , Arginina/química , Línea Celular Tumoral , Cromatina/química , ADN/química , Desaminación , Escherichia coli/metabolismo , Genes de Inmunoglobulinas , Humanos , Inmunoglobulinas/química , Lipopolisacáridos/química , Ratones , Microscopía Confocal , Mutación , Dominios Proteicos , Hipermutación Somática de Inmunoglobulina , Transcripción GenéticaRESUMEN
Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN/metabolismo , Región de Cambio de la Inmunoglobulina , Conformación de Ácido Nucleico , ARN/metabolismo , Composición de Base , Secuencia de Bases , Catálisis , Citidina Desaminasa/química , Citidina Desaminasa/aislamiento & purificación , ADN de Cadena Simple/metabolismo , Desaminación , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Hibridación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.
Asunto(s)
Citidina Desaminasa/metabolismo , Roturas del ADN , Reparación del ADN por Unión de Extremidades/fisiología , Cambio de Clase de Inmunoglobulina/genética , Análisis de Varianza , Animales , Linfocitos B/inmunología , Western Blotting , Línea Celular , Inmunoprecipitación de Cromatina , Reparación del ADN por Unión de Extremidades/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Translocación Genética/genética , Uracil-ADN Glicosidasa/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.
Asunto(s)
5-Metilcitosina/metabolismo , Citidina Desaminasa/metabolismo , Desoxicitidina/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Dominio Catalítico , Citidina Desaminasa/química , ADN de Cadena Simple/metabolismo , Humanos , Modelos Moleculares , Pez Cebra , Proteínas de Pez Cebra/químicaRESUMEN
Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.