Incarnation of classical pro- and eukaryotic mechanisms of mutagenesis in hypermutagenesis and immunity of vertebrates

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.     

APOBEC4, a New Member of the AID/APOBEC Family of Polynucleotide (Deoxy)Cytidine Deaminases Predicted by Computational Analysis

Using iterative database searches, we identified a new subfamily of the AID/APOBEC family of RNA/DNA editing cytidine deaminases. The new subfamily, which is represented by readily identifiable orthologs in mammals, chicken, and frog, but not fishes, was designated APOBEC4. The zinc-coordinating motifs involved in catalysis and the secondary structure of the APOBEC4 deaminase domain are evolutionarily conserved, suggesting that APOBEC4 proteins are active polynucleotide (deoxy)cytidine deaminases. In reconstructed maximum likelihood phylogenetic trees, APOBEC4 forms distinct clade with a high statistical support. APOBEC4 and APOBEC1 are joined in a moderately supported cluster clearly separated from AID, APOBEC2 and APOBEC3 subfamilies. In mammals, APOBEC4 is expressed primarily in testis which suggests the possibility that it is an editing enzyme for mRNAs involved in spermatogenesis.     

Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This cannot be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.     

Creative deaminases, self-inflicted damage, and genome evolution

Organisms minimize genetic damage through complex pathways of DNA repair. Yet a gene family-the AID/APOBECs-has evolved in vertebrates with the sole purpose of producing targeted damage in DNA/RNA molecules through cytosine deamination. They likely originated from deaminases involved in A>I editing in tRNAs. AID, the archetypal AID/APOBEC, is the trigger of the somatic diversification processes of the antibody genes. Its homologs may have been associated with the immune system even before the evolution of the antibody genes. The APOBEC3s, arising from duplication of AID, are involved in the restriction of exogenous/endogenous threats such as retroviruses and mobile elements. Another family member, APOBEC1, has (re)acquired the ability to target RNA while maintaining its ability to act on DNA. The AID/APOBECs have shaped the evolution of vertebrate genomes, but their ability to mutate nucleic acids is a double-edged sword: AID is a key player in lymphoproliferative diseases by triggering mutations and chromosomal translocations in B cells, and there is increasing evidence suggesting that other AID/APOBECs could be involved in cancer development as well.     

Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases

The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing). The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast. Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family. We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals. The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish. Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile. The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation. By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci). We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.     

PrimPol prevents APOBEC/AID family mediated DNA mutagenesis

PrimPol is a DNA damage tolerant polymerase displaying both translesion synthesis (TLS) and (re)-priming properties. This led us to study the consequences of a PrimPol deficiency in tolerating mutagenic lesions induced by members of the APOBEC/AID family of cytosine deaminases. Interestingly, during somatic hypermutation, PrimPol counteracts the generation of C>G transversions on the leading strand. Independently, mutation analyses in human invasive breast cancer confirmed a pro-mutagenic activity of APOBEC3B and revealed a genome-wide anti-mutagenic activity of PRIMPOL as well as most Y-family TLS polymerases. PRIMPOL especially prevents APOBEC3B targeted cytosine mutations within TpC dinucleotides. As C transversions induced by APOBEC/AID family members depend on the formation of AP-sites, we propose that PrimPol reprimes preferentially downstream of AP-sites on the leading strand, to prohibit error-prone TLS and simultaneously stimulate error-free homology directed repair. These in vivo studies are the first demonstrating a critical anti-mutagenic activity of PrimPol in genome maintenance.     

Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo

To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.     

APOBEC3 proteins and genomic stability: The high cost of a good defense

The human APOBEC3 family of cytidine deaminases constitutes a cellular intrinsic defense mechanism that is effective against a range of viruses and retro-elements. While it is well-established that these enzymes are powerful mutators of viral DNA, the possibility that their activity could threaten the integrity of the host genome has only recently begun to be investigated. Here, we discuss the implications of new evidence suggesting that APOBEC3 proteins can mediate the deamination of cellular DNA. The maintenance of genomic integrity in the face of this potential off-target activity must require high-fidelity DNA repair and strict regulation of APOBEC3 gene expression and enzyme activity. Conversely, the ability of specific members of the APOBEC3 family to activate DNA damage signaling pathways might also reflect another way that these proteins contribute to the host immune response.Key words: APOBEC3, cytidine deaminase, AID, uracil-DNA glycosylase, DNA damage, hypermutation, genomic instability, cancer     

The AID/APOBEC family of nucleic acid mutators

The AID/APOBECs, a group of cytidine deaminases, represent a somewhat unusual protein family that can insert mutations in DNA and RNA as a result of their ability to deaminate cytidine to uridine. The ancestral AID/APOBECs originated from a branch of the zinc-dependent deaminase superfamily at the beginning of the vertebrate radiation. Other members of the family have arisen in mammals and present a history of complex gene duplications and positive selection. All AID/APOBECs have a characteristic zinc-coordination motif, which forms the core of the catalytic site. The crystal structure of human APOBEC2 shows remarkable similarities to that of the bacterial tRNA-editing enzyme TadA, which suggests a conserved mechanism by which polynucleotides are recognized and deaminated. The AID/APOBECs seem to have diverse roles. AID and the APOBEC3s are DNA mutators, acting in antigen-driven antibody diversification processes and in an innate defense system against retroviruses, respectively. APOBEC1 edits the mRNA for apolipoprotein B, a protein involved in lipid transport. A detailed understanding of the biological roles of the family is still some way off, however, and the functions of some members of the family are completely unknown. Given their ability to mutate DNA, a role for the AID/APOBECs in the onset of cancer has been proposed.     

APOBEC deaminases-mutases with defensive roles for immunity

In recent years, tremendous progress has been made in the elucidation of the biological roles and molecular mechanisms of the apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of enzymes. The APOBEC family of cytidine deaminases has important functional roles within the adaptive and innate immune system. Activation induced cytidine deaminase (AID) plays a central role in the biochemical steps of somatic hypermutation and class switch recombination during antibody maturation, and the APOBEC 3 enzymes are able to inhibit the mobility of retroelements and the replication of retroviruses and DNA viruses, such as the human immunodeficiency virus type-1 and hepatitis B virus. Recent advances in structural and functional studies of the APOBEC enzymes provide new biochemical insights for how these enzymes carry out their biological roles. In this review, we provide an overview of these recent advances in the APOBEC field with a special emphasis on AID and APOBEC3G.     

Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.     

RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators

APOBEC1 is the catalytic component of an RNA editing complex but shows homology to activation-induced cytidine deaminase (AID), a protein whose function is to potentiate diversification of immunoglobulin gene DNA. Here, we show that APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit potent DNA mutator activity in an E. coli assay. Indeed, like AID, these proteins appear to trigger DNA mutation through dC deamination. However, each protein exhibits a distinct local target sequence specificity. The results reveal the existence of a family of potential active dC/dG mutators, with possible implications for cancer.     

APOBEC-mediated viral restriction: not simply editing?

The APOBEC family of cytidine deaminases inhibit the mobility of diverse retroviruses, retrotransposons and other viruses. Initial reports proposed that these effects were due to the DNA editing capabilities of these enzymes; however, many recent studies have provided evidence suggesting that APOBEC proteins can inhibit these elements by several mechanisms, including editing-dependent and editing-independent processes. Investigating these modes of action and the potential contribution that each one makes to the antiviral activities of various APOBEC proteins is vital if we are to understand how APOBEC proteins protect host genomes from invading nucleic acids.     

APOBEC3A can activate the DNA damage response and cause cell-cycle arrest

Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins constitute a family of cytidine deaminases that mediate restriction of retroviruses, endogenous retro-elements and DNA viruses. It is well established that these enzymes are potent mutators of viral DNA, but it is unclear whether their editing activity is a threat to the integrity of the cellular genome. We show that expression of APOBEC3A can lead to induction of DNA breaks and activation of damage responses in a deaminase-dependent manner. Consistent with these observations, APOBEC3A expression induces cell-cycle arrest. These results indicate that cellular DNA is vulnerable to APOBEC3 activity and deregulated expression of APOBEC3A could threaten genomic integrity.     

An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22

The cytidine (C) to uridine (U) editing of apolipoprotein (apo) B mRNA is mediated by tissue-specific, RNA-binding cytidine deaminase APOBEC1. APOBEC1 is structurally homologous to Escherichia coli cytidine deaminase (ECCDA), but has evolved specific features required for RNA substrate binding and editing. A signature sequence for APOBEC1 has been used to identify other members of this family. One of these genes, designated APOBEC2, is found on chromosome 6. Another gene corresponds to the activation-induced deaminase (AID) gene, which is located adjacent to APOBEC1 on chromosome 12. Seven additional genes, or pseudogenes (designated APOBEC3A to 3G), are arrayed in tandem on chromosome 22. Not present in rodents, this locus is apparently an anthropoid-specific expansion of the APOBEC family. The conclusion that these new genes encode orphan C to U RNA-editing enzymes of the APOBEC family comes from similarity in amino acid sequence with APOBEC1, conserved intron/exon organization, tissue-specific expression, homodimerization, and zinc and RNA binding similar to APOBEC1. Tissue-specific expression of these genes in a variety of cell lines, along with other evidence, suggests a role for these enzymes in growth or cell cycle control.     

Mutagenic Activity of AID/APOBEC Deaminases in Antiviral Defense and Carcinogenesis

Molecular Biology - Proteins of the AID/APOBEC family are capable of cytidine deamination in nucleic acids forming uracil. These enzymes are involved in mRNA editing, protection against viruses,...     

APOBEC3s: DNA‐editing human cytidine deaminases

Nucleic acid editing enzymes are essential components of the human immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins. Among these enzymes are cytidine deaminases of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) super family, each with unique target sequence specificity and subcellular localization. We focus on the DNA‐editing APOBEC3 enzymes that have recently attracted attention because of their involvement in cancer and potential in gene‐editing applications. We review and compare the crystal structures of APOBEC3 (A3) domains, binding interactions with DNA, substrate specificity, and activity. Recent crystal structures of A3A and A3G bound to ssDNA have provided insights into substrate binding and specificity determinants of these enzymes. Still many unknowns remain regarding potential cooperativity, nucleic acid interactions, and systematic quantification of substrate preference of many APOBEC3s, which are needed to better characterize the biological functions and consequences of misregulation of these gene editors.