Human Tribbles 3 Protects Nuclear DNA from Cytidine Deamination by APOBEC3A

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.     

Methylcytosine and Normal Cytosine Deamination by the Foreign DNA Restriction Enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

APOBEC3A通过脱氨基作用抑制HBV复制

目的:研究APOBEC3A抑制HBV复制的分子机制。方法:首先在肝癌细胞HuH7中过表达APOBEC3A,通过MTT法检测了APOBEC3A对细胞毒性的影响;通过免疫荧光检测了APOBEC3A在细胞中的定位,通过IP试验进一步证实了APOBEC3A与病毒颗粒的相互作用;并通过ELISA,特异性荧光定量PCR检测了HBV复制的参数包括上清中HBsAg,病毒核心颗粒中HBV DNA以及核内的cccDNA的表达水平;最后通过3D PCR方法分析了核心颗粒中HBV DNA脱氨基作用。结果:过表达APOBEC3A对HuH7细胞毒性没有显著性差异;APOBEC3A主要位于细胞核,但APOBEC3A可以与病毒颗粒结合,在逆转录环节对HBV复制发生抑制作用;共转染HBV复制质粒和APOBEC3A表达质粒后,细胞上清中HBsAg,核心颗粒中的HBV DNA以及核内的cccDNA均显著下降;最后通过3D PCR和克隆测序表明核心颗粒中的HBV DNA负链发生了大量的G-A突变,同时正链也发生了较多的C-T突变。结论:APOBEC3A可与病毒颗粒结合,在HBV复制的逆转录环节可作用于HBV单链,发生脱氨基作用,从而抑制HBV的复制。     

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.     

The restriction of zoonotic PERV transmission by human APOBEC3G

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.     

Molecular basis of the attenuated phenotype of human APOBEC3B DNA mutator enzyme

The human APOBEC3A and APOBEC3B genes (A3A and A3B) encode DNA mutator enzymes that deaminate cytidine and 5-methylcytidine residues in single-stranded DNA (ssDNA). They are important sources of mutations in many cancer genomes which show a preponderance of CG->TA transitions. Although both enzymes can hypermutate chromosomal DNA in an experimental setting, only A3A can induce double strand DNA breaks, even though the catalytic domains of A3B and A3A differ by only 9% at the protein level. Accordingly we sought the molecular basis underlying A3B attenuation through the generation of A3A-A3B chimeras and mutants. It transpires that the N-terminal domain facilitates A3B activity while a handful of substitutions in the catalytic C-terminal domain impacting ssDNA binding serve to attenuate A3B compared to A3A. Interestingly, functional attenuation is also observed for the rhesus monkey rhA3B enzyme compared to rhA3A indicating that this genotoxic dichotomy has been selected for and maintained for some 38 million years. Expression of all human ssDNA cytidine deaminase genes is absent in mature sperm indicating they contribute to somatic mutation and cancer but not human diversity.     

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3′ and 5′ of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.     

Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G

The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC’s only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.     

AID,APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation

BackgroundAID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases.MethodsSubstrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs.ResultsAll three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases.ConclusionsAID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged.General significanceBeyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.     

APOBEC3B and APOBEC3F inhibit L1 retrotransposition by a DNA deamination-independent mechanism

The most common transposable genetic element in humans, long interspersed element 1 (L1), constitutes about 20% of the genome. The activity of L1 and related transposons such as Alu elements causes disease and contributes to speciation. Little is known about the cellular mechanisms that control their spread. We show that expression of human APOBEC3B or APOBEC3F decreased the rate of L1 retrotransposition by 5-10-fold. Expression of two related proteins, APOBEC3D or APOBEC3G, had little effect. The mechanism of L1 inhibition did not correlate with an obvious subcellular protein distribution as APOBEC3B appeared predominantly nuclear and APOBEC3F was mostly cytosolic. Two lines of evidence indicated that these APOBEC3 proteins use a deamination-independent mechanism to inhibit L1. First, a catalytically inactive APOBEC3B mutant maintained L1 inhibition activity. Second, cDNA strand-specific C --> T hypermutations were not detected among L1 elements that had replicated in the presence of APOBEC3B or APOBEC3F. In addition, lower levels of retrotransposed L1 DNA accumulated in the presence of APOBEC3B and APOBEC3F. Together, these data combined to suggest a model in which APOBEC3B or APOBEC3F provide a preintegration barrier to L1 retrotransposition. A particularly high level of APOBEC3F protein in human testes and an inverse correlation between L1 activity and APOBEC3 gene number suggest the relevance of this mechanism to mammals.     

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.     

Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C-U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.     

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.