Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2019 Jul 10;28(9):1552–1566. doi: 10.1002/pro.3670

APOBEC3s: DNA‐editing human cytidine deaminases

Tania V Silvas 1,†, Celia A Schiffer 1,✉
PMCID: PMC6699113  PMID: 31241202

Abstract

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.

Keywords: APOBEC, crystal structure, cytidine deaminase, DNA binding, gene editing, substrate specificity

1. INTRODUCTION

1.1. APOBEC superfamily

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) super family of human cytidine deaminases consists of APOBEC1 (A1), APOBEC2 (A2), subfamily APOBEC3 (A3), APOBEC4 (A4), and activation‐induced cytidine deaminase (AID). APOBEC enzymes are gene editors that can deaminate cytidines into uridines in DNA and, in certain cases, RNA. The A3 subfamily of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. A3 enzymes deaminate cytidines in the viral single stranded (ss) DNA intermediate during reverse transcription (Fig. 1). During second‐strand DNA synthesis, adenosines are transcribed across from uridines, resulting in G to A hypermutations. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes APOBECs a double‐edged sword. When APOBECs are over expressed, the resulting misregulated deaminase activity can contribute to genomic instability and cancer.

Figure 1.

Figure 1

Open in a new tab

APOBEC3s deaminate cytidines in ssDNA. A schematic of G to A mutation catalyzed by APOBEC3s. Gray ribbons represent DNA. Green nucleotides represent normal progression of complementary strand synthesis in the absence of APOBEC3. Red nucleotides represent nucleotide change in the presence of A3 protein

Initially, A1 and AID were the most studied within the APOBEC superfamily. A1 modulates lipid metabolism and transport in the small intestine by editing the mRNA of apoB protein.1, 2 A1 deaminates specifically one cytidine in apoB mRNA, C6666, creating a premature stop codon yielding a truncated apoB protein. This truncated apoB protein is essential for proper transport of dietary lipids from the intestines to other locations in the body.3, 4, 5 AID plays an essential role in adaptive immunity, regulating antibody maturation and diversification in activated B cells.6 AID catalyzes the diversification in the sequence of immunoglobulin genes through three pathways: somatic hypermutation, gene conversion, and class switch recombination.7, 8 All three of these pathways are the consequences of AID's cytidine deamination activity in ssDNA of antibody and immunoglobulin genes.9 The physiological function for A2 and A4 has remained elusive, as neither enzyme has yet been shown to be catalytically active.10, 11, 12 A2 is primarily expressed in heart and skeletal muscle, while A4 expression in humans has not been detected.10 The A3 family of human enzymes was first discovered through database studies as APOBEC1‐like genes, but the function of the gene products had not yet been elucidated.13

Soon thereafter, a specific A3, the APOBEC3G (A3G) gene, was found to be expressed in primary human T cells, the target cell type for HIV, and other permissive cells. When A3G was overexpressed in permissive cells, A3G rendered vif‐deficient HIV‐1 noninfectious. Thus, APOBEC3G gene was first identified as an anti‐HIV factor.14 With further HIV‐focused studies, A3G was shown to deaminate cytidines in ssDNA intermediate of replicating HIV genomes.15, 16 Eventually, all seven members of the A3 family of cytidine deaminases were found to restrict replication of retroviruses and retrotransposons by inducing hypermutations in the viral genome (Fig. 1).14, 15, 17, 18, 19, 20, 21 Proteins and proviruses produced from this hypermutated viral genome will be defective, thus preventing further viral replication.22

Besides inhibiting retroviruses and retroelements, as described above, A3s also restrict DNA viruses. A3s can restrict nuclear‐replicating ssDNA viruses such as adeno‐associated virus,23 as well as nuclear and dsDNA viruses such as hepatitis B virus, herpes viruses, and HPV.24, 25, 26, 27 A3s mediate the clearance of foreign DNA from monocytes and macrophages. In particular, A3A, which is primarily expressed in phagocytic cells, was found to deaminate exogenous DNA, creating a substrate for UNG2‐mediated excision while managing to leave genomic DNA intact.28, 29 A3A is also much more efficient at deaminating methylated cytidine than other deaminases such as A3G or AID, and hence has the unique ability to clear methylated foreign DNA from the cell.30, 31, 32

1.2. Consequences of misregulated DNA cytidine deamination

The ability of those APOBECs that deaminate cytidines in DNA, such as the APOBEC3 subfamily and AID, has made them a double‐edged sword, because if misregulated, DNA‐editing APOBECs may promote cancers (Fig. 2). In the APOBEC3 family, A3B and A3H localize to the nucleus, while A3A and A3G are transiently recruited to the nucleus.33, 34, 35, 36 When overexpressed, A3B and A3H have been described as a major endogenous source for mutations in various types of human cancer, such as breast, bladder, head and neck, cervical, and lung cancer.36, 37, 38 Like A3B and A3H, AID localizes to the nucleus and when overexpressed can lead to cancer, as seen in non‐Hodgkin B cell lymphomas.39, 40

Figure 2.

Figure 2

Open in a new tab

Functions and malfunctions of the ssDNA deaminating APOBECs. Schematic of the effects of deaminating deoxycytidine into deoxyuridine in ssDNA by APOBECs. Green arrows represent functions in the cell that the APOBEC family helps promote or inhibit. Red arrow represents the consequence of overexpression of A3/AID

Poor survival rates for patients undergoing chemotherapy for lymphoma and glioblastoma were found to correlate with having cancer cells that overexpressed the A3B or A3G gene.41, 42, 43 In these cells, A3G was discovered to promote double‐strand break (DSB) repair. In lymphoma cells, A3G is transiently recruited to the nucleus and accumulates at DSB sites after irradiation. A3G can directly promote DSB repair through A3G's enzymatic activity by deaminating cytidines in resected ssDNA, which may lead to recruitment of repair factors such as base‐excision repair proteins. A3G can also contribute to DSB repair through A3G's ability to simultaneously bind two 3’ ssDNA ends, promoting ssDNA end joining.34

1.3. Structure of catalytically active ssDNA‐deaminating APOBEC3 domains

Each of the seven proteins in the A3 subfamily contains either one or two zinc‐coordinating (Z) domains [Fig. 3(a)].44 These A3 Z domains are separated into three distinct phylogenetic groups (Z1, Z2, and Z3) [Fig. 3(b)].45 A3A (Z1), A3C (Z2), and A3H (Z3) comprise a single cytidine deaminase Z domain, while A3B, A3D, A3F, and A3G contain both an N‐terminal pseudocatalytic (inactive) Z domain (all Z2) and a catalytically active C‐terminal Z domain (Z1, Z2, Z2, and Z1, respectively).46 Adding to the complexity, A3H has seven haplotypes with varying stability and enzymatic efficiency.47 AID consists of one catalytically active zinc‐coordinating domain.48 All these enzymes share a conserved overall fold (Fig. 4).

Figure 3.

Figure 3

Open in a new tab

APOBEC3 family of cytidine deaminases. (a) Schematic of the seven human APOBEC3s. N‐terminal and C‐terminal domains are shown as ovals. (b) Phylogenic tree of A3 Z domains. Active domains are marked with an orange star. Z1 domains are in red, Z2 domains are in blue, and Z3 domain is in green

Figure 4.

Figure 4

Open in a new tab

Structures of all human ssDNA deaminating APOBECs domains determined to date. Cartoon representation of human apo ssDNA deaminating APOBEC domain crystal or NMR structures. Z1 domains are shown in red, Z2 are in blue, and AID is in purple. Zinc is depicted as orange spheres

While enzymology and biological consequences of APOBEC function have been extensively studied, many studies focus on double‐domain A3s49, 50, 51 for which the mechanism by which APOBEC3s recognize and edit DNA remains largely elusive. The N‐terminal domain of these double‐domain A3s is insoluble in aqueous buffer, and thus, they are very difficult to study in vitro.52 Isolated C‐terminal domain of double‐domain A3s binds substrates only weakly53 compared with full length, and the N‐terminal domain can influence the activity of double‐domain A3s.54, 55, 56, 57 Single‐domain A3s can bind substrate ssDNA with as low as 100 nanomolar Kd values58, 59 and enable in vitro studies to elucidate specificity and structures of A3 complexes.

Sequence alignment of all catalytically active domains of ssDNA deaminating APOBECs (7 A3s and AID) reveals that residues necessary for catalysis, such as the catalytic glutamate and zinc coordinating residues, are 100% identical in all domains [yellow star and green diamonds; Figs. 5 and 6(a,b)]. Sequence conservation of residues among APOBEC enzymes displayed on the crystal structure of A3A reveals that the most highly conserved residues are located in the core of the enzyme [Fig. 6(a)], likely stabilizing the overall fold of all APOBEC domains (Fig. 4). The strictly conserved surface‐exposed residues include those that coordinate zinc [Fig. 6(b)]. There are also localized patches of residues with high conservation on the surface of the protein structure [Fig. 6(c)]. These patches may be important for biological function and DNA binding. In the active site, the cytidine‐binding pocket is lined with mostly 100% conserved residues [Fig. 6(d)]. Certain nonidentical pocket residues, located in Loop1 T31 (teal; T/S/A), Loop 3 A71 (marine; A/V), Loop 5 I96 (teal; (T/I/L), and Loop 7 Y130 (marine; Y/F), are highly conserved. The minor differences in amino acid side chains within the cytidine‐binding pocket may play a role in the differences in catalytic efficiency between APOBEC enzymes, possibly by modulating the stability of APOBEC–DNA interactions to affect binding or catalysis. Loops that line the cytidine‐binding pocket are the least conserved regions within the APOBEC active site [Fig. 6(d)]. Variations in loop length and sequence likely underlie differences in APOBEC substrate sequence specificity.

Figure 5.

Figure 5

Open in a new tab

Sequence alignment of ssDNA cytidine deaminase proteins. Sequence alignment of human AID and the seven members of the APOBEC3 subfamily. Catalytic glutamate is denoted by orange star. Zinc coordinating residues are denoted with green diamonds. Identical residues are highlighted in blue, residues 80–100% identical in light blue, and 60–80% in teal. Active‐site loops are denoted by horizontal red lines. Residues that make up the active‐site pocket are highlighted with red dashed boxes

Figure 6.

Figure 6

Open in a new tab

Tertiary structure analysis highlight similarities in the active sites of ssDNA deaminating APOBECs. (a) Cartoon view of A3A crystal structure highlighting the conservation of active domains of ssDNA deaminating APOBECs shown in Figure 5. (b) Example in which highly conserved residues are important for structural and function components of APOBEC proteins. Residues responsible for zinc coordination necessary for catalysis are 100% conserved and shown as sticks. (c) Surface view of A3A illustrating location of conserved residues on protein surface. (d) 180° rotation of (c) reveals the active site of A3A highlighted with a light orange box. The cytidine binding pocket is highly conserved between APOBEC proteins. Loops proposed to be responsible for ssDNA binding, labeled with red circles, are not conserved. Zinc is shown as light orange sphere. Identical residues are highlighted in blue, residues 80–100% identical in marine, and 60–80% in teal

Although human cytidine deaminases play different roles in the cell, the mechanism of deaminating cytidine is conserved.60, 61 These enzymes deaminate cytidine into uridine by catalyzing the exchange of the NH2 group to oxygen on cytidine bases [Fig. 7(a)]. Deamination of cytidine is carried out by a glutamate in the active‐site pocket, along with a coordinated zinc and water molecule [Figs. 6(b) and 7(b)]. The coordination of zinc atom and water molecule in the active site results in one zinc hydroxide. When a cytidine is coordinated properly within the active site, the oxygen of the zinc hydroxide molecule can make a nucleophilic attack on the C6 carbon of the cytidine base. The N3 nitrogen of the base may then be protonated by the OE1 oxygen of the catalytic glutamate. The hydrogen from newly activated zinc hydroxide group is then passed to the OE2 oxygen of the catalytic glutamate, which gets transferred to the NH2 group of the cytidine base, creating the leaving NH3 group. When ammonia is released from the cytidine base, the active site can be reset with the introduction of a new water molecule. This new water molecule allows for deprotonation by the carboxylate group of the catalytic glutamate to form the conserved zinc–water coordination necessary for another round of catalysis [Fig. 7(b)]. The highly conserved active sites of all catalytically active APOBEC domains, as described above, allow for the conserved mechanism in this protein family for deaminating cytidines in ssDNA.

Figure 7.

Figure 7

Open in a new tab

Deamination reaction mechanism. (a) Schematic of deoxycytidine conversion to deoxyuridine by ssDNA cytidine deaminases. Leaving NH2 group of cytidine base is in green and added oxygen is in red. Cytidine deaminase is depicted as gray cartoon. (b) Schematic of the steps of deamination by cytidine deaminases

1.4. Nucleic acid‐bound crystal structures of APOBECs

The recent crystal structures of A3A and A3G in complex with ssDNA not only visualize the active site poised for catalysis, but also pinpoint the residues that confer specificity toward CC/TC motifs.62, 63 The A3A–ssDNA structure was the first complex structure to be determined, which defined the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases [Fig. 8(a)]. The target cytidine was positioned compatible with catalysis as expected, while the flanking nucleic acid residues adopted rather tightly bent U shape [Fig. 9(a)]. While this overall ssDNA shape is likely shared with A3B,64 the recently determined A3G structure indicated a more open DNA conformation.63 Figure 8 shows a consolidation of poly nucleic acid bound structures of APOBEC3s determined [Fig. 8(d–f)]. Comparison of APOBECs bound to poly nucleic acids with a substrate cytosine [Fig. 9(a,b,f)] to those without [Fig. 9(c–f)] reveal the potential for alternative poly nucleic acid binding sites outside of the canonical active site of these proteins. Nucleic acid binding by inactive NTD of human APOBECs remains elusive.

Figure 8.

Figure 8

Open in a new tab

Solved structures of APOBEC‐poly nucleic acid complexes. (a) Our crystal structure of human A3A bound to the PolyT‐1C ssDNA sequence 5′‐TTTTTTTTCTTTTTT‐3′ (PDB ID: 5KEG). (b) Human A3B with active site A3A chimera bound to ssDNA 5′‐TTTTCAT‐3′ (PDB ID: 5TD5). (c) Primate A3G‐NTD bound to Poly T ssDNA sequence 5′‐TTTTTTTTTT‐3′ (PDB ID: 5K83). (d) Human A3F‐CTD bound to Poly T ssDNA sequence 5′‐TTTTTTTTTT‐3′ (PDB ID: 5W2M). Note ssDNA and zinc binding site located outside active site. (e) Macaque A3H bound to dsRNA of unknown sequence (PDB ID: 5W3V). (f) MBP‐fused human AID bound to dsDNA sequence 5′‐GTTCAAGGCCAG‐3′, 5′‐CTGGCCTTGAAC‐3′ and deoxycytidine monophosphate (PDB ID: 5W0U). All protein structures are shown in gray transparent surface view and cartoon. Zinc is shown as marine sphere. Nucleic acid is shown as orange sticks

Figure 9.

Figure 9

Open in a new tab

Active‐site view of APOBEC–poly nucleic acid complexes. (a) PolyT‐1C ssDNA with substrate deoxycytidine bound to the active site of human A3A (PDB ID: 5KEG). (b) ssDNA with substrate deoxycytidine bound to active site of human A3B‐active site A3A chimera (PDB ID: 5TD5). (c) Poly T ssDNA with only substrate deoxycytidine visible and bound to active site the Primate A3G‐NTD (PDB ID: 5K83). (d) Poly T ssDNA bound to “back side” of Human A3F‐CTD (PDB ID: 5W2M). (e) dsRNA of unknown sequence bound to the “top” of Macaque A3H (PDB ID: 5W3V). (f) Deoxycytidine monophosphate bound to active site of MBP fused human AID. dsDNA also bound to “top” of protein (PDB ID: 5W0U). All protein structures are shown in gray cartoon. Zinc is shown as marine sphere. Nucleic acid is shown as orange sticks

A human A3F‐CTD co‐crystal structure was solved using nonsubstrate Poly_T ssDNA [Fig. 8(d)].65 This structure revealed a unique zinc‐coordinated dimer interface. The crystal structure of apo A3A homodimer crystal structure was also coordinated by zinc.59 Further studies elucidating the role of zinc in modulating APOBEC oligomerization is necessary to determine the role of zinc beyond its function in the active site of APOBECs.

The first ever A3H structure was recently published of a macaque A3H (macA3H) bound to dsRNA [Fig. 8(e)].66 This was also the first structure of an APOBEC bound to RNA, albeit of unknown sequence (resolution was not high enough to identify the nucleotides of copurified RNA). Sequence alignment with other active APOBEC domains reveals that the residues involved in macA3H–RNA interactions are not conserved, other than in human A3H (Fig. 10), suggesting this RNA binding mode may be A3H specific.

Figure 10.

Figure 10

Open in a new tab

Sequence alignment of human AID, the seven members of the APOBEC3 subfamily active domains, and macA3H. Orange dots represent macA3H residues with side‐chain interactions with dsRNA and corresponding orange boxes to highlight the residue identity of other APOBEC3 at this position. Gray dots represent macA3H residues with backbone interactions with dsRNA and corresponding gray boxes to highlight the residue identity of other APOBEC3 at this position. Catalytic glutamate is denoted by orange star. Zinc coordinating residues are denoted with green diamond. Identical residues are highlighted in blue, residues 80–100% identical in light blue, and 60–80% in teal. Active site loops are denoted by red line. Residues that make up the active site pocket are highlighted with red dashed boxes

The first AID structure was published as MBP‐human AID fusion protein bound to dsDNA and deoxycytidine monophosphate [Figs. 8(f) and 9(f)].48 However, AID does not bind to dsDNA in solution and interactions in the crystal structure are solely interactions with PO4 backbone. Authors suggested dsDNA–AID interaction is a crystallization artifact; dsDNA is stacking in the crystal lattice and likely playing the role of neutralizing repulsion between the highly positively charged AID monomers. Despite efforts, solving a structure of biologically relevant AID bound to substrate DNA remains elusive and is still necessary to elucidate the molecular mechanism for AID substrate recognition.

To summarize, the nucleic acid bound structures of APOBEC superfamily members reveal both specific and nonspecific modes of binding, some being protein‐specific, while others may be shared within the family.

1.5. Models for cooperative DNA binding by A3A homodimers

Cooperative oligomerization of A3A may regulate the specific binding of A3A to ssDNA. Additionally, A3A forms multiple oligomeric states in solution. The apo A3A crystal structure revealed a homodimer with symmetric domain swapping of N‐terminal residues.59 Mutating the homodimer interface found in this structure resulted in a decrease in affinity for substrate as well as a decrease in Hill coefficient value relative to wildtype A3A. These results suggested that the homodimer interface seen in apo A3A crystal structure may mediate cooperative protein–protein interactions that affect A3A activity. However, cocrystal structures of A3A bound to ssDNA62 revealed a monomer in the asymmetric unit, with a 1:1 stoichiometry with ssDNA.

The apparent discrepancy between the results in the apo structures and the cocomplexes may be explained by a new hypothesis we propose for the mechanism of substrate binding by A3A, in light of a third structure of an A3–substrate complex reported recently,62 where human A3F‐CTD was bound to a nonsubstrate Poly_T ssDNA. This complex identified a second nonspecific polynucleotide binding site away from the active site [Fig. 8(d)].65 This binding site is located at the “backside” of APOBEC proteins relative to the substrate‐binding site seen in A3A–ssDNA structure [Fig. 9(d)]. More rigorous structural analysis combined with homology between other active APOBEC domains suggests that this second binding site may in fact be conserved throughout APOBEC proteins (Fig. 11). The ssDNA used to obtain this crystal structure, Poly_T 10‐mer oligo, likely did not bind to the active site of A3F‐CTD, because the sequence did not contain a substrate cytidine. Fluorescence anisotropy‐based binding assays with Poly_T ssDNA as a background sequence also indicated nonspecific binding,59, 67 which may be due to this second nonspecific binding site. This second site can also explain the effect of ssDNA length on A3A binding affinity to DNA, with longer oligonucleotide binding stronger, as described for A3A and A3G substrate length dependence.68, 69, 70

Figure 11.

Figure 11

Open in a new tab

A3F‐CTD poly T ssDNA binding residues. (a) A3F‐CTD backbone interactions with backbone PO4s. (b) Sequence alignment of residues involved in backbone–backbone protein–nucleic acid interactions with active APOBEC domains. (c) Y333 residue side‐chain base stacking with deoxythymine base. (d) Sequence alignment of residues involved in aromatic stacking protein–nucleic acid interactions with active APOBEC domains. For (a) and (c), residues 4 å away from ssDNA are in green sticks. Hydrogen bonds are represented as gray dashes. ssDNA is represented as orange sticks. Oxygen and nitrogen are colored as red and blue, respectively. In (b) and (d), sequence alignment is shown for human AID, the seven members of the APOBEC3 subfamily active domains, and macA3H. Orange dots represent A3F‐CTD residues with side‐chain interactions with ssDNA, and corresponding orange boxes to highlight the residue identity of other APOBEC3 at this position. Gray dots represent A3F‐CTD residues with backbone interactions with ssDNA, and corresponding gray boxes to highlight the residue identity of other APOBEC3 at this position. Identical residues are highlighted in blue, residues 80–100% identical in light blue, and 60–80% in teal

Considering crystal structures of A3A‐homodimer, A3A–ssDNA, and A3F‐CTD–ssDNA as well as sequence homology and biochemical data, we propose a new model for A3A binding to ssDNA (Figs. 12 and 13). Figure 12 illustrates the potential for an A3A homodimer binding to ssDNA in the active site as well as the distal nonspecific nucleic acid binding site. Figure 13 illustrates the potential for an A3A homodimer binding to two strands of single‐stranded nucleic acid. Note the antiparallel directionality that results from this model [Fig. 13(a)]. Interestingly, this model captures many highly conserved APOBEC residues within the path of nucleic acid binding [Fig. 13(b–d)]. Additionally, this model requires two A3A proteins to bind one strand of single‐stranded nucleic acid simultaneously [Fig. 13(d)] and would elucidate the structural mechanism for cooperative binding observed for A3A, A3F, and AID.48, 65

Figure 12.

Figure 12

Open in a new tab

Compilation of A3 apo and bound structures. (a) Our cocrystal structure of A3A‐ssDNA complex aligned with A3F‐CTD ssDNA structure. A3A is shown as gray cartoon and ssDNA bound to A3A in green sticks. PolyT ssDNA from A3F‐CTD‐ssDNA structure is depicted as orange sticks. Zinc is shown as marine sphere. (b) Compiled structure of (a) aligned to homodimer in our apo A3A crystal structure. A3A from complex structure is shown as gray surface. DNA is depicted as described for (a)

Figure 13.

Figure 13

Open in a new tab

Proposed model of A3 homodimer cooperatively binding to ssDNA. (a) Compiled structure as in Figure 12. ssDNA from solved crystal structures is depicted in light green sticks. Model of directionality of ssDNA binding is shown with green arrow. (b) Compiled structures seen in (a), with homologous residues highlighted in A3A surface. (c) 90° rotation of (b) with poly T ssDNA from A3F‐CTD structure shown in orange sticks. (d) Figure 13(c) with Model of directionality of ssDNA binding shown with green arrow. Model of directionality of ssDNA binding is shown with green arrow. A3A from complex structure shown as gray surface. Identical residues are highlighted in blue, residues 80–100% identical in light blue, and 60–80% in teal

1.6. Substrate specificity and enzymatic activity of APOBECs

All A3 and AID enzymes deaminate the second cytidine in either CC or TC dinucleotide motifs28, 71, 72, 73, 74, 75 (Table 1). However, sequence analysis has determined that not every cognate dinucleotide motif is deaminated, which suggests that the nucleotides flanking the dinucleotide motifs play a role in substrate recognition.36, 76, 77, 78, 79 Structures of A3s alone have not led to a clear explanation of the different preferred sequence context for the dinucleotide motifs among A3s and AID. Since A3 and AID active domains share an overall fold, major structural features cannot be responsible for differences in preference. The catalytic‐site residues of these enzymes are also highly conserved (Fig. 6) but the loops surrounding the catalytic site are more variable and may be responsible for the differences in sequence specificity and possibly also catalytic activity.

Table 1.

Consensus Sequence for Deamination Activity of Human AID and APOBEC Proteins

Consensus Sequence
AID (A/T)(A/G)C(A/C/T)
A3A TTCA
A3B ATCA
A3C TC
A3D TC
A3F TTC(A/T)
A3G (C/T)CC(A/C/T)
A3H TCA
Open in a new tab

The target C is bold and underlined, and the minimum motif is in bold.

Since the single‐domain A3A is soluble in vitro, catalytically efficient, and involved in viral restriction and cancer, A3A can be used as a benchmark to understand the structural basis for substrate recognition in ssDNA deaminating APOBECs. A3A has the highest catalytic activity among the deaminases in the APOBEC superfamily,80 and restricts HPV and retroelement LINE‐1.81, 82 When A3A is overexpressed or misregulated, it can also contribute to carcinogenesis.83 Our structure of A3A bound to ssDNA along with another A3 co‐crystal structure62, 84 elucidated the structural basic of substrate specificity for the TC motif. However, the molecular mechanism underlying substrate sequence specificity flanking the TC dinucleotide sequence was not evident from the crystal structures. Biochemical amenability of A3A enabled quantitatively characterizing substrate preferences and pH dependence of binding affinity/catalytic activity. Systematic assays with various substrate sequences enabled expanding the dinucleotide motif TC, which had been used as A3A signature sequence,28, 84, 85 to the 4‐mer motif (T/C)TC(A/G).67

1.7. pH dependence of APOBEC activity

A systematic measurement of A3A affinity for signature sequence in a broad range of pH values was also determined to verify and quantify the pH dependence of A3A affinity for substrate ssDNA.83, 85 This pH dependency may relate to the cellular compartmentalization of maximal activity. A3A was found to have an increase in ssDNA binding affinity with a decrease in pH value. The structural basis for the pH dependence of A3A was elucidated with analysis of our A3A–DNA cocrystal structure. The bound A3A structure shows that the active‐site His29 in Loop 1 of A3A can hydrogen‐bond with the ssDNA backbone and sugar of −1 and 0 nucleotides. This hydrogen bond network could only occur at pH values of 6.5 and below, when the histidine is protonated. Thus, the protonation state of histidine may be responsible for change in affinity seen at different pH values, and the maximum catalytic activity seen at pH 6.0 in previous studies.67, 83

The activity of A3G was also been shown to be pH‐dependent. Activity experiments with A3G‐CTD identified an increase in cytidine deamination with decreasing pH.50 These authors suggested that pH dependency is due to His216, also located in Loop 1, of the active site of A3G‐CTD.50 Thus, active‐site histidines in A3A and A3G may be an intraprotein regulation mechanism of enzymatic activity. Other A3 members lack an active‐site histidine at this position and may coordinate ssDNA via arginine side chains instead. Furthermore, A3A and A3G maximum activity occurring in the acidic pH range warrants further investigation of their role in endosomal‐related functions, such as foreign DNA sensing and potential exosomal‐related functions including cell–cell signaling, embryonic morphogenesis, regulation of host–pathogen interactions, as well as in the progression of neurodegenerative pathologies and cancer.

A3H is the only other APOBEC enzyme that has a histidine located close to its active site [Fig. 14(a)]. Unlike A3G and A3A, which have a histidine in Loop 1 of their active sites, the histidine of A3H is located in Loop 7. Homology model of A3H shows that the active‐site histidine is located close to the histidine in A3A and A3G, although in this model, A3H histidine is flipped away from the active site [Fig. 14(b)]. Upon substrate binding, it is possible that A3H active‐site histidine could flip back toward the active site to make hydrogen bonds with the substrate backbone, similar to what was seen for A3A. A3H is proposed to be the least stable and least active of the APOBECs, thus elucidating A3H activity and substrate specificity has remained elusive. Studying A3H activity and specificity in a quantitative and systematic way, as described for A3A, may reveal conditions in which A3H is more active than previously reported. Further studies testing A3H activity and specificity are warranted to determine if A3H activity is also regulated by pH.

Figure 14.

Figure 14

Open in a new tab

Active‐site histidines in APOBEC enzymes. (a) Sequence alignment of active site loops of all members of the APOBEC super family. (b) Solved structures of A3A and A3G‐CTD and homology model of A3H. Surface is depicted in gray, Z1 domains as red cartoon (A3A and A3G‐CTD), and Z3 domain as green cartoon (A3H). Active‐site histidine is shown as orange stick. Nitrogen and oxygen of the histidine base are colored blue and red, respectively. Active site Zinc is shown as gray spheres

1.8. RNA deamination

A3A is able to bind RNA in a highly specific and structural context‐dependent manner. Previous reports suggested that A3A binds only weakly to RNA and is not an RNA deaminase; however, these experiments were performed with unstructured RNA.70 A3A binding to hairpin RNA and not linear ssRNA, along with a recent study describing A3A preference for deaminating cytidines in the loop region of stable RNA hairpins, demonstrates that A3A's RNA deamination activity is highly dependent on sequence context and secondary structure.86

Analysis of the A3A–ssDNA structures illustrates a potential structural basis for A3A binding and deaminating RNA. A3A can conceivably bind RNA cytidine in the similar manner as substrate DNA. The extra oxygen of the cytidine sugar could be easily accommodated by the highly conserved residue Tyr130 and the zinc coordinating His70 [Fig. 15(a,b)]. Other RNA ribose oxygens in close proximity to A3A are at the −1 and − 2 position and could also be accommodated for by residues Tyr132, Gly27, and His29 [Fig. 15(c,d)]. A crystal structure of A3A bound to RNA is essential for determining the mechanism of A3A deamination of RNA as well as providing a foundation for using structural analysis to identify other A3s with the potential to deaminate RNA. A3A's ability to edit RNA opens up a new dimension of potential substrates that would augment the biological role of this enzyme.

Figure 15.

Figure 15

Open in a new tab

Potential structural mechanism for A3A binding RNA. (a) Our A3A‐PolyT‐1C co crystal structure (PDB ID: 5KEG). (b) A3A‐ATCG structure (PDB ID: 5SWW) illustrating substrate cytidine and A3A residues near where the extra oxygen would be in an RNA cytidine substrate. (c) Residues near −1 T and (d) near −2A oxygen in PDB 5SWW. A3A residues in proximity to extra oxygen on RNA ribose are shown as purple sticks. DNA is shown in orange. Location of extra oxygen is depicted in green, catalytic zinc in gray‐blue spheres, and active site oxygen in red sphere. Oxygen and nitrogen are colored red and blue, respectively

1.9. Determining sequence preferences of APOBECs

The comprehensive identification of A3 signature sequences enables a more accurate evaluation of A3 activity based on sequence analysis. For instance, many studies used only a single A3A signature sequence to implicate A3A's role in viral restriction or cancer progression. Systematic and quantitative analysis identified four almost equivalent substrate signature A3A sequences, TTCA, TTCG, CTCA, and CTCG,67 suggesting the use of a set of sequences rather than just one as a more accurate method to identify A3A's involvement in mutagenesis.

The quantitative and systematic approach to determine A3A's signature sequence can also be applied to identifying the signature sequences of other APOBECs. To date, substrate preference sequences of other APOBECs have not been determined in a comprehensive and quantitative manner. Additionally, the signature sequences of many other APOBECs are unknown outside their dinucleotide sequence motif. Further determination of the sequence specificity of other APOBECs will be a strong foundation for a more accurate identification of APOBECs' role in cancer, viral restriction, and other functions in the cell that rely on or are affected by modifications in ssDNA or RNA.

2. ACKNOWLEDGMENTS

We thank Ms. S. Hou and Dr. N. Kurt Yilmaz for critical reading of the manuscript and editorial help. Our research on APOBEC3 proteins is supported by National Institute of General Medical Sciences grant R01 GM118474 and TVS was supported by F31 GM119931.

Silvas TV, Schiffer CA. APOBEC3s: DNA‐editing human cytidine deaminases. Protein Science. 2019;28:1552–1566. 10.1002/pro.3670

Funding information National Institute of General Medical Sciences, Grant/Award Numbers: F31 GM119931 , R01 GM118474

REFERENCES

  • 1. Glickman RM, Rogers M, Glickman JN (1986) Apolipoprotein B synthesis by human liver and intestine in vitro. Proc Natl Acad Sci U S A 83:5296–5300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Greeve J, Altkemper I, Dieterich JH, Greten H, Windler E (1993) Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB‐containing plasma lipoproteins. J Lipid Res 34:1367–1383. [PubMed] [Google Scholar]
  • 3. Kane JP, Hardman DA, Paulus HE (1980) Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci U S A 77:2465–2469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Chen S‐H, Habib G, Yang CY, Gu ZW, Lee BR, Weng SA, Silberman SR, Cai SJ, Deslypere JP, Rosseneu M, Gotto AM Jr, Li W‐H, Chan L (1987) Apolipoprotein B‐48 is the product of a messenger RNA with an organ‐specific in‐frame stop codon. Science 238:363–366. [DOI] [PubMed] [Google Scholar]
  • 5. Innerarity TL, Boren J, Yamanaka S, Olofsson SO (1996) Biosynthesis of apolipoprotein B48‐containing lipoproteins. Regulation by novel post‐transcriptional mechanisms. J Biol Chem 271:2353–2356. [DOI] [PubMed] [Google Scholar]
  • 6. Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K, Davidson NO, Honjo T (1999) 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 274:18470–18476. [DOI] [PubMed] [Google Scholar]
  • 7. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T (2000) Class switch recombination and hypermutation require activation‐induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553–563. [DOI] [PubMed] [Google Scholar]
  • 8. Arakawa H, Hauschild J, Buerstedde JM (2002) Requirement of the activation‐induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295:1301–1306. [DOI] [PubMed] [Google Scholar]
  • 9. Schrader CE, Linehan EK, Mochegova SN, Woodland RT, Stavnezer J (2005) Inducible DNA breaks in Ig S regions are dependent on AID and UNG. J Exp Med 202:561–568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Liao W, Hong SH, Chan BH, Rudolph FB, Clark SC, Chan L (1999) APOBEC‐2, a cardiac‐ and skeletal muscle‐specific member of the cytidine deaminase supergene family. Biochem Biophys Res Commun 260:398–404. [DOI] [PubMed] [Google Scholar]
  • 11. Harris RS, Petersen‐Mahrt SK, Neuberger MS (2002) RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol Cell 10:1247–1253. [DOI] [PubMed] [Google Scholar]
  • 12. Lada AG, Krick CF, Kozmin SG, Mayorov VI, Karpova TS, Rogozin IB, Pavlov YI (2011) Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast. Biochemistry 76:131–146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Jarmuz A, Chester A, Bayliss J, Gisbourne J, Dunham I, Scott J, Navaratnam N (2002) An anthropoid‐specific locus of orphan C to U RNA‐editing enzymes on chromosome 22. Genomics 79:285–296. [DOI] [PubMed] [Google Scholar]
  • 14. Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that inhibits HIV‐1 infection and is suppressed by the viral Vif protein. Nature 418:646–650. [DOI] [PubMed] [Google Scholar]
  • 15. Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, Trono D (2003) Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424:99–103. [DOI] [PubMed] [Google Scholar]
  • 16. Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, Gao L (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV‐1 DNA. Nature 424:94–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Zheng YH, Irwin D, Kurosu T, Tokunaga K, Sata T, Peterlin BM (2004) Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication. J Virol 78:6073–6076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Dang Y, Siew LM, Wang X, Han Y, Lampen R, Zheng YH (2008) Human cytidine deaminase APOBEC3H restricts HIV‐1 replication. J Biol Chem 283:11606–11614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Dang Y, Wang X, Esselman WJ, Zheng YH (2006) Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family. J Virol 80:10522–10533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Bogerd HP, Wiegand HL, Doehle BP, Lueders KK, Cullen BR (2006) APOBEC3A and APOBEC3B are potent inhibitors of LTR‐retrotransposon function in human cells. Nucleic Acids Res 34:89–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Muckenfuss H, Hamdorf M, Held U, Perkovic M, Lower J, Cichutek K, Flory E, Schumann GG, Munk C (2006) APOBEC3 proteins inhibit human LINE‐1 retrotransposition. J Biol Chem 281:22161–22172. [DOI] [PubMed] [Google Scholar]
  • 22. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen‐Mahrt SK, Watt IN, Neuberger MS, Malim MH (2003) DNA deamination mediates innate immunity to retroviral infection. Cell 113:803–809. [DOI] [PubMed] [Google Scholar]
  • 23. Chen H, Lilley CE, Yu Q, Lee DV, Chou J, Narvaiza I, Landau NR, Weitzman MD (2006) APOBEC3A is a potent inhibitor of adeno‐associated virus and retrotransposons. Curr Biol 16:480–485. [DOI] [PubMed] [Google Scholar]
  • 24. Janahi EM, McGarvey MJ (2013) The inhibition of hepatitis B virus by APOBEC cytidine deaminases. J Viral Hepat 20:821–828. [DOI] [PubMed] [Google Scholar]
  • 25. Vieira VC, Leonard B, White EA, Starrett GJ, Temiz NA, Lorenz LD, Lee D, Soares MA, Lambert PF, Howley PM, Harris RS (2014) Human papillomavirus E6 triggers upregulation of the antiviral and cancer genomic DNA deaminase APOBEC3B. MBio 5:e02234–e02214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Wang Z, Wakae K, Kitamura K, Aoyama S, Liu G, Koura M, Monjurul AM, Kukimoto I, Muramatsu M (2014) APOBEC3 deaminases induce hypermutation in human papillomavirus 16 DNA upon beta interferon stimulation. J Virol 88:1308–1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Suspene R, Aynaud MM, Koch S, Pasdeloup D, Labetoulle M, Gaertner B, Vartanian JP, Meyerhans A, Wain‐Hobson S (2011) Genetic editing of herpes simplex virus 1 and Epstein‐Barr herpesvirus genomes by human APOBEC3 cytidine deaminases in culture and in vivo. J Virol 85:7594–7602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Stenglein MD, Burns MB, Li M, Lengyel J, Harris RS (2010) APOBEC3 proteins mediate the clearance of foreign DNA from human cells. Nat Struct Mol Biol 17:222–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Land AM, Law EK, Carpenter MA, Lackey L, Brown WL, Harris RS (2013) Endogenous APOBEC3A DNA cytosine deaminase is cytoplasmic and nongenotoxic. J Biol Chem 288:17253–17260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Wijesinghe P, Bhagwat AS (2012) Efficient deamination of 5‐methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G. Nucleic Acids Res 40:9206–9217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Suspene R, Aynaud MM, Vartanian JP, Wain‐Hobson S (2013) Efficient deamination of 5‐methylcytidine and 5‐substituted cytidine residues in DNA by human APOBEC3A cytidine deaminase. PLoS One 8:e63461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Carpenter MA, Li M, Rathore A, Lackey L, Law EK, Land AM, Leonard B, Shandilya SM, Bohn MF, Schiffer CA, Brown WL, Harris RS (2012) Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A. J Biol Chem 287:34801–34808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Koito A, Ikeda T (2013) Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases. Front Microbiol 4:28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Nowarski R, Wilner OI, Cheshin O, Shahar OD, Kenig E, Baraz L, Britan‐Rosich E, Nagler A, Harris RS, Goldberg M, Willner I, Kotler M (2012) APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase‐dependent DNA repair. Blood 120:366–375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Suspene R, Mussil B, Laude H, Caval V, Berry N, Bouzidi MS, Thiers V, Wain‐Hobson S, Vartanian JP (2017) Self‐cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage. Nucleic Acids Res 45:3231–3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Starrett GJ, Luengas EM, McCann JL, Ebrahimi D, Temiz NA, Love RP, Feng Y, Adolph MB, Chelico L, Law EK, Carpenter MA, Harris RS (2016) The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis. Nat Commun 7:12918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Burns MB, Temiz NA, Harris RS (2013) Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat Genet 45:977–983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P, Kiezun A, Kryukov GV, Carter SL, Saksena G, Harris S, Shah RR, Resnick MA, Getz G, Gordenin DA (2013) An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat Genet 45:970–976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Greeve J, Philipsen A, Krause K, Klapper W, Heidorn K, Castle BE, Janda J, Marcu KB, Parwaresch R (2003) Expression of activation‐induced cytidine deaminase in human B‐cell non‐Hodgkin lymphomas. Blood 101:3574–3580. [DOI] [PubMed] [Google Scholar]
  • 40. Hardianti MS, Tatsumi E, Syampurnawati M, Furuta K, Saigo K, Nakamachi Y, Kumagai S, Ohno H, Tanabe S, Uchida M, Yasuda N (2004) Activation‐induced cytidine deaminase expression in follicular lymphoma: association between AID expression and ongoing mutation in FL. Leukemia 18:826–831. [DOI] [PubMed] [Google Scholar]
  • 41. Jais JP, Haioun C, Molina TJ, Rickman DS, de Reynies A, Berger F, Gisselbrecht C, Briere J, Reyes F, Gaulard P, Feugier P, Labouyrie E, Tilly H, Bastard C, Coiffier B, Salles G, Leroy K, Groupe d'Etude des Lymphomes de lA (2008) The expression of 16 genes related to the cell of origin and immune response predicts survival in elderly patients with diffuse large B‐cell lymphoma treated with CHOP and rituximab. Leukemia 22:1917–1924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Prabhu P, Shandilya SM, Britan‐Rosich E, Nagler A, Schiffer CA, Kotler M (2016) Inhibition of APOBEC3G activity impedes double‐stranded DNA repair. FEBS J 283:112–129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Wang Y, Wu S, Zheng S, Wang S, Wali A, Ezhilarasan R, Sulman EP, Koul D, Alfred Yung WK (2017) APOBEC3G acts as a therapeutic target in mesenchymal gliomas by sensitizing cells to radiation‐induced cell death. Oncotarget 8:54285–54296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Conticello SG, Thomas CJ, Petersen‐Mahrt SK, Neuberger MS (2005) Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol Biol Evol 22:367–377. [DOI] [PubMed] [Google Scholar]
  • 45. LaRue RS, Andresdottir V, Blanchard Y, Conticello SG, Derse D, Emerman M, Greene WC, Jonsson SR, Landau NR, Lochelt M, Malik HS, Malim MH, Munk C, O'Brien SJ, Pathak VK, Strebel K, Wain‐Hobson S, Yu XF, Yuhki N, Harris RS (2009) Guidelines for naming nonprimate APOBEC3 genes and proteins. J Virol 83:494–497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Munk C, Willemsen A, Bravo IG (2012) An ancient history of gene duplications, fusions and losses in the evolution of APOBEC3 mutators in mammals. BMC Evol Biol 12:71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Wang X, Abudu A, Son S, Dang Y, Venta PJ, Zheng YH (2011) Analysis of human APOBEC3H haplotypes and anti‐human immunodeficiency virus type 1 activity. J Virol 85:3142–3152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Qiao Q, Wang L, Meng FL, Hwang JK, Alt FW, Wu H (2017) AID recognizes structured DNA for class switch recombination. Mol Cell 67:361–373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Harjes E, Gross PJ, Chen KM, Lu Y, Shindo K, Nowarski R, Gross JD, Kotler M, Harris RS, Matsuo H (2009) An extended structure of the APOBEC3G catalytic domain suggests a unique holoenzyme model. J Mol Biol 389:819–832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Harjes S, Solomon WC, Li M, Chen KM, Harjes E, Harris RS, Matsuo H (2013) Impact of H216 on the DNA binding and catalytic activities of the HIV restriction factor APOBEC3G. J Virol 87:7008–7014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Furukawa A, Nagata T, Matsugami A, Habu Y, Sugiyama R, Hayashi F, Kobayashi N, Yokoyama S, Takaku H, Katahira M (2009) Structure, interaction and real‐time monitoring of the enzymatic reaction of wild‐type APOBEC3G. EMBO J 28:440–451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Kouno T, Luengas EM, Shigematsu M, Shandilya SM, Zhang J, Chen L, Hara M, Schiffer CA, Harris RS, Matsuo H (2015) Structure of the Vif‐binding domain of the antiviral enzyme APOBEC3G. Nat Struct Mol Biol 22:485–491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53. Ara A, Love RP, Chelico L (2014) Different mutagenic potential of HIV‐1 restriction factors APOBEC3G and APOBEC3F is determined by distinct single‐stranded DNA scanning mechanisms. PLoS Pathog 10:e1004024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Feng Y, Chelico L (2011) Intensity of deoxycytidine deamination of HIV‐1 proviral DNA by the retroviral restriction factor APOBEC3G is mediated by the noncatalytic domain. J Biol Chem 286:11415–11426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55. Bulliard Y, Turelli P, Rohrig UF, Zoete V, Mangeat B, Michielin O, Trono D (2009) Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition. J Virol 83:12611–12621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56. Song C, Sutton L, Johnson ME, D'Aquila RT, Donahue JP (2012) Signals in APOBEC3F N‐terminal and C‐terminal deaminase domains each contribute to encapsidation in HIV‐1 virions and are both required for HIV‐1 restriction. J Biol Chem 287:16965–16974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57. Pak V, Heidecker G, Pathak VK, Derse D (2011) The role of amino‐terminal sequences in cellular localization and antiviral activity of APOBEC3B. J Virol 85:8538–8547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58. Love RP, Xu H, Chelico L (2012) Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A. J Biol Chem 287:30812–30822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59. Bohn MF, Shandilya SM, Silvas TV, Nalivaika EA, Kouno T, Kelch BA, Ryder SP, Kurt‐Yilmaz N, Somasundaran M, Schiffer CA (2015) The ssDNA mutator APOBEC3A is regulated by cooperative dimerization. Structure 23:903–911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60. Betts L, Xiang S, Short SA, Wolfenden R, Carter CW Jr (1994) Cytidine deaminase. The 2.3 a crystal structure of an enzyme: transition‐state analog complex. J Mol Biol 235:635–656. [DOI] [PubMed] [Google Scholar]
  • 61. Davidson NO, Anant S, MacGinnitie AJ (1995) Apolipoprotein B messenger RNA editing: insights into the molecular regulation of post‐transcriptional cytidine deamination. Curr Opin Lipidol 6:70–74. [DOI] [PubMed] [Google Scholar]
  • 62. Kouno T, Silvas TV, Hilbert BJ, Shandilya SMD, Bohn MF, Kelch BA, Royer WE, Somasundaran M, Kurt Yilmaz N, Matsuo H, Schiffer CA (2017) Crystal structure of APOBEC3A bound to single‐stranded DNA reveals structural basis for cytidine deamination and specificity. Nat Commun 8:15024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63. Maiti A, Myint W, Kanai T, Delviks‐Frankenberry K, Sierra Rodriguez C, Pathak VK, Schiffer CA, Matsuo H (2018) Crystal structure of the catalytic domain of HIV‐1 restriction factor APOBEC3G in complex with ssDNA. Nat Commun 9:2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64. Hou S, Silvas T, Leidner F, Nalivaika EA, Matsuo H, Kurt Yilmaz N, Schiffer CA (2019) Structural analysis of the active site and DNA binding of human cytidine deaminase APOBEC3B. J Chem Theory Comput 15:637–647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65. Fang Y, Xiao X, Li SX, Wolfe A, Chen XS (2018) Molecular interactions of a DNA modifying enzyme APOBEC3F catalytic domain with a single‐stranded DNA. J Mol Biol 430:87–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66. Bohn JA, Thummar K, York A, Raymond A, Brown WC, Bieniasz PD, Hatziioannou T, Smith JL (2017) APOBEC3H structure reveals an unusual mechanism of interaction with duplex RNA. Nat Commun 8:1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Silvas TV, Hou S, Myint W, Nalivaika E, Somasundaran M, Kelch BA, Matsuo H, Kurt Yilmaz N, Schiffer CA (2018) Substrate sequence selectivity of APOBEC3A implicates intra‐DNA interactions. Sci Rep 8:7511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68. Chelico L, Pham P, Calabrese P, Goodman MF (2006) APOBEC3G DNA deaminase acts processively 3′ ‐‐> 5′ on single‐stranded DNA. Nat Struct Mol Biol 13:392–399. [DOI] [PubMed] [Google Scholar]
  • 69. Byeon IJ, Ahn J, Mitra M, Byeon CH, Hercik K, Hritz J, Charlton LM, Levin JG, Gronenborn AM (2013) NMR structure of human restriction factor APOBEC3A reveals substrate binding and enzyme specificity. Nat Commun 4:1890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70. Mitra M, Hercik K, Byeon IJ, Ahn J, Hill S, Hinchee‐Rodriguez K, Singer D, Byeon CH, Charlton LM, Nam G, Heidecker G, Gronenborn AM, Levin JG (2014) Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties. Nucleic Acids Res 42:1095–1110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71. Pham P, Bransteitter R, Petruska J, Goodman MF (2003) Processive AID‐catalysed cytosine deamination on single‐stranded DNA simulates somatic hypermutation. Nature 424:103–107. [DOI] [PubMed] [Google Scholar]
  • 72. Beale RC, Petersen‐Mahrt SK, Watt IN, Harris RS, Rada C, Neuberger MS (2004) Comparison of the differential context‐dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J Mol Biol 337:585–596. [DOI] [PubMed] [Google Scholar]
  • 73. Esnault C, Heidmann O, Delebecque F, Dewannieux M, Ribet D, Hance AJ, Heidmann T, Schwartz O (2005) APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses. Nature 433:430–433. [DOI] [PubMed] [Google Scholar]
  • 74. Hultquist JF, Lengyel JA, Refsland EW, LaRue RS, Lackey L, Brown WL, Harris RS (2011) Human and rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity to restrict Vif‐deficient HIV‐1. J Virol 85:11220–11234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B, Refsland EW, Kotandeniya D, Tretyakova N, Nikas JB, Yee D, Temiz NA, Donohue DE, McDougle RM, Brown WL, Law EK, Harris RS (2013) APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494:366–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76. Larijani M, Petrov AP, Kolenchenko O, Berru M, Krylov SN, Martin A (2007) AID associates with single‐stranded DNA with high affinity and a long complex half‐life in a sequence‐independent manner. Mol Cell Biol 27:20–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77. Rogozin IB, Diaz M (2004) 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 172:3382–3384. [DOI] [PubMed] [Google Scholar]
  • 78. Liddament MT, Brown WL, Schumacher AJ, Harris RS (2004) APOBEC3F properties and hypermutation preferences indicate activity against HIV‐1 in vivo. Curr Biol 14:1385–1391. [DOI] [PubMed] [Google Scholar]
  • 79. Chan K, Roberts SA, Klimczak LJ, Sterling JF, Saini N, Malc EP, Kim J, Kwiatkowski DJ, Fargo DC, Mieczkowski PA, Getz G, Gordenin DA (2015) An APOBEC3A hypermutation signature is distinguishable from the signature of background mutagenesis by APOBEC3B in human cancers. Nat Genet 47:1067–1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80. Wu CH, Fallini C, Ticozzi N, Keagle PJ, Sapp PC, Piotrowska K, Lowe P, Koppers M, McKenna‐Yasek D, Baron DM, Kost JE, Gonzalez‐Perez P, Fox AD, Adams J, Taroni F, Tiloca C, Leclerc AL, Chafe SC, Mangroo D, Moore MJ, Zitzewitz JA, Xu ZS, van den Berg LH, Glass JD, Siciliano G, Cirulli ET, Goldstein DB, Salachas F, Meininger V, Rossoll W, Ratti A, Gellera C, Bosco DA, Bassell GJ, Silani V, Drory VE, Brown RH Jr, Landers JE (2012) Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature 488:499–503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81. Bogerd HP, Wiegand HL, Hulme AE, Garcia‐Perez JL, O'Shea KS, Moran JV, Cullen BR (2006) Cellular inhibitors of long interspersed element 1 and Alu retrotransposition. Proc Natl Acad Sci U S A 103:8780–8785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82. Vartanian JP, Guetard D, Henry M, Wain‐Hobson S (2008) Evidence for editing of human papillomavirus DNA by APOBEC3 in benign and precancerous lesions. Science 320:230–233. [DOI] [PubMed] [Google Scholar]
  • 83. Pham P, Landolph A, Mendez C, Li N, Goodman MF (2013) A biochemical analysis linking APOBEC3A to disparate HIV‐1 restriction and skin cancer. J Biol Chem 288:29294–29304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84. Shi K, Carpenter MA, Banerjee S, Shaban NM, Kurahashi K, Salamango DJ, McCann JL, Starrett GJ, Duffy JV, Demir O, Amaro RE, Harki DA, Harris RS, Aihara H (2017) Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B. Nat Struct Mol Biol 24:131–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85. Byeon IJ, Byeon CH, Wu T, Mitra M, Singer D, Levin JG, Gronenborn AM (2016) Nuclear magnetic resonance structure of the APOBEC3B catalytic domain: structural basis for substrate binding and DNA deaminase activity. Biochemistry 55:2944–2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86. Sharma S, Baysal BE (2017) Stem‐loop structure preference for site‐specific RNA editing by APOBEC3A and APOBEC3G. PeerJ 5:e4136. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES