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.     

Murine APOBEC1 is a powerful mutator of retroviral and cellular RNA in vitro and in vivo

Mammalian APOBEC molecules comprise a large family of cytidine deaminases with specificity for RNA and single-stranded DNA (ssDNA). APOBEC1s are invariably highly specific and edit a single residue in a cellular mRNA, while the cellular targets for APOBEC3s are not clearly established, although they may curtail the transposition of some retrotransposons. Two of the seven member human APOBEC3 enzymes strongly restrict human immunodeficiency virus type 1 in vitro and in vivo. We show here that ssDNA hyperediting of an infectious exogenous gammaretrovirus, the Friend-murine leukemia virus, by murine APOBEC1 and APOBEC3 deaminases occurs in vitro. Murine APOBEC1 was able to hyperdeaminate cytidine residues in murine leukemia virus genomic RNA as well. Analysis of the edited sites shows that the deamination in vivo was due to mouse APOBEC1 rather than APOBEC3. Furthermore, murine APOBEC1 is able to hyperedit its primary substrate in vivo, the apolipoprotein B mRNA, and a variety of heterologous RNAs. In short, murine APOBEC1 is a hypermutator of both RNA and ssDNA in vivo, which could exert occasional side effects upon overexpression.     

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.     

APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

Many APOBEC cytidine deaminase members are known to induce ‘off-target’ cytidine deaminations in 5′TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.     

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.     

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.     

Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single-stranded DNA binding

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.     

APOBEC3A damages the cellular genome during DNA replication

The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.     

An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA,which is affected by the concentrations of APOBEC3F and single-stranded DNA

BackgroundAPOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation.MethodsThe real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions.ResultsA3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect.ConclusionsThe concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA.General significanceThe discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.     

Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family

A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.     

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of human immunodeficiency virus-1, other retroviruses, and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1-helix (residues 201-206) that was not apparent in the shorter protein, and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5, and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different from the continuous β2 strand of another family member, APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain, including the interdomain linker and some of the last α-helix. These structured residues (residues 191-196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from each other to participate in this interaction.     

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.     

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.     

Efficient Deamination of 5-Methylcytidine and 5-Substituted Cytidine Residues in DNA by Human APOBEC3A Cytidine Deaminase

Deamination of 5-methylcytidine (5MeC) in DNA results in a G:T mismatch unlike cytidine (C) deamination which gives rise to a G:U pair. Deamination of C was generally considered to arise spontaneously. It is now clear that human APOBEC3A (A3A), a polynucleotide cytidine deaminase (PCD) with specificity for single stranded DNA, can extensively deaminate human nuclear DNA. It is shown here that A3A among all human PCDs can deaminate 5-methylcytidine in a variety of single stranded DNA substrates both in vitro and in transfected cells almost as efficiently as cytidine itself. This ability of A3A to accommodate 5-methyl moiety extends to other small and physiologically relevant substituted cytidine bases such as 5-hydroxy and 5-bromocytidine. As 5MeCpG deamination hotspots characterize many genes associated with cancer it is plausible that A3A is a major player in the onset of cancer.     

Genetic Editing of HBV DNA by Monodomain Human APOBEC3 Cytidine Deaminases and the Recombinant Nature of APOBEC3G

Hepatitis B virus (HBV) DNA is vulnerable to editing by human cytidine deaminases of the APOBEC3 (A3A-H) family albeit to much lower levels than HIV cDNA. We have analyzed and compared HBV editing by all seven enzymes in a quail cell line that does not produce any endogenous DNA cytidine deaminase activity. Using 3DPCR it was possible to show that all but A3DE were able to deaminate HBV DNA at levels from 10⁻² to 10⁻⁵ in vitro, with A3A proving to be the most efficient editor. The amino terminal domain of A3G alone was completely devoid of deaminase activity to within the sensitivity of 3DPCR (∼10⁻⁴ to 10⁻⁵). Detailed analysis of the dinucleotide editing context showed that only A3G and A3H have strong preferences, notably CpC and TpC. A phylogenic analysis of A3 exons revealed that A3G is in fact a chimera with the first two exons being derived from the A3F gene. This might allow co-expression of the two genes that are able to restrict HIV-1Δvif efficiently.     

A single amino acid in human APOBEC3F alters susceptibility to HIV-1 Vif

Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu(324) as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu(324) is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.     

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off‐target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin‐containing TCP‐1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A‐induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.