Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA

BACKGROUND: Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. PRINCIPAL FINDINGS/METHODOLOGY: We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. SIGNIFICANCE: Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.     

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.     

Phosphorylation directly regulates the intrinsic DNA cytidine deaminase activity of activation-induced deaminase and APOBEC3G protein

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.     

RecA protein filaments disassemble in the 5' to 3' direction on single-stranded DNA

RecA protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5' to 3' direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5' end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5' to 3' direction.     

The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain

The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus. These proteins have two putative deaminase domains, and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well characterized single domain deaminases. Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation. A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminase domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F. This 5'-TC-->TT mutational specificity was confirmed in a heterologous Escherichia coli-based mutation assay, in which the 5'-CC-->CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain. An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in an AID-like mutational signature. Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.     

APOBEC3G subunits self-associate via the C-terminal deaminase domain

Human APOBEC3G (hA3G) is a cytidine deaminase active on HIV single-stranded DNA. Small angle x-ray scattering and molecular envelope restorations predicted a C-terminal dimeric model for RNA-depleted hA3G in solution. Each subunit was elongated, suggesting that individual domains of hA3G are solvent-exposed and therefore may interact with other macromolecules even as isolated substructures. In this study, co-immunoprecipitation and in-cell quenched fluorescence resonance energy transfer assays reveal that hA3G forms RNA-independent oligomers through interactions within its C terminus. Residues 209-336 were necessary and sufficient for homoligomerization. N-terminal domains of hA3G were unable to multimerize but remained functional for Gag and viral infectivity factor (Vif) interactions when expressed apart from the C terminus. These findings corroborate the small angle x-ray scattering structural model and are instructive for development of high throughput screens that target specific domains and their functions to identify HIV/AIDS therapeutics.     

Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution

APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C → U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3′ → 5′ direction. A similar polarity observed in vivo is believed responsible for initiating localized C → T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5′ hot motif, a motif-dependent DNA contraction greatest for 5′ hot > 3′ hot > 5′ cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.     

Escherichia coli DbpA is a 3' --> 5' RNA helicase

Previous work has demonstrated that Escherichia coli DbpA is a nonprocessive RNA helicase that can disrupt short RNA helices on either the 5' side or 3' side of hairpin 92 of 23S rRNA. Here the directionality of the helicase activity of DbpA was determined by using substrates containing a short reporter helix in the presence of a second adjacent helix of varying stability placed either 5' or 3' of the reporter helix. When the second helix was on the 5' side of the reporter helix, it had no effect on the dissociation rate of the reporter helix. However, when the second helix was on the 3' side of the reporter helix, its dissociation rate determined the dissociation rate of the reporter helix. This defines DbpA as a 3' --> 5' helicase. Like other helicases, DbpA requires a single-stranded RNA loading site on the 3' side of the duplex for disruption to be observed. Since the loading site could be on either strand of the helix that was disrupted, hairpin 92 does not influence the directionality of the helicase but only aids in targeting RNA substrates.     

Extensive mutagenesis experiments corroborate a structural model for the DNA deaminase domain of APOBEC3G

APOBEC3G is a single-strand DNA cytosine deaminase capable of blocking retrovirus and retrotransposon replication. APOBEC3G has two conserved zinc-coordinating motifs but only one is required for catalysis. Here, deletion analyses revealed that the minimal catalytic domain consists of residues 198-384. Size exclusion assays indicated that this protein is monomeric. Many (31/69) alanine substitution derivatives of APOBEC3G198-384 retained significant to full levels of activity. These data corroborated an APOBEC2-based structural model for the catalytic domain of APOBEC3G indicating that most non-essential residues are solvent accessible and most essential residues cluster within the protein core.     

The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G

APOBEC3G is a human cellular enzyme that is incorporated into retroviral particles and acts to restrict retroviral replication in infected cells by deaminating dC to dU in the first (minus)-strand cDNA replication intermediate. HIV, however, encodes a protein (virion infectivity factor, Vif ), which overcomes APOBEC3G-mediated restriction but by an unknown mechanism. Here, we show that Vif triggers APOBEC3G degradation by a proteasome-dependent pathway and that an 80 amino acid region of APOBEC3G surrounding its first zinc coordination motif is sufficient to confer the ability to partake in an interaction involving Vif. Inhibitors of this interaction might therefore prove therapeutically useful in blocking Vif-mediated APOBEC3G destruction.     

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.     

T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.     

TWINKLE Has 5' -> 3' DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein

Mutations in TWINKLE cause autosomal dominant progressive external ophthalmoplegia, a human disorder associated with multiple deletions in the mitochondrial DNA. TWINKLE displays primary sequence similarity to the phage T7 gene 4 primase-helicase, but no specific enzyme activity has been assigned to the protein. We have purified recombinant TWINKLE to near homogeneity and demonstrate here that TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements. The protein needs a stretch of 10 nucleotides of single-stranded DNA on the 5'-side of the duplex to unwind duplex DNA. In addition, helicase activity is not observed unless a short single-stranded 3'-tail is present. The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate, with UTP being the optimal substrate. DNA unwinding by TWINKLE is specifically stimulated by the mitochondrial single-stranded DNA-binding protein. Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork and provides evidence for a close relationship of the DNA replication machinery in bacteriophages and mammalian mitochondria.     

The nuclear DNA deaminase AID functions distributively whereas cytoplasmic APOBEC3G has a processive mode of action

AID deaminates cytosine in the context of single stranded DNA to generate uracil, essential for effective class-switch recombination, somatic hypermutation and gene conversion at the B cell immunoglobulin locus. As a nuclear DNA mutator, AID activity must be tightly controlled and regulated, but the genetic analysis of AID and other DNA deaminases has left unstudied a number of important biochemical details. We have asked fundamental questions regarding AID's substrate recognition and processing, i.e. whether AID acts distributively or processively. We demonstrate that in vitro, human AID exhibits turnover, a prerequisite for our analysis, and show that it exhibits a distributive mode of action. Using a variety of different assays, we established that human AID is alone unable to act processively on any of a number of DNA substrates, i.e. one AID molecule is unable to carry out multiple, sequential deamination events on the same substrate. This is in contrast to the cytoplasmically expressed anti-viral DNA deaminase APOBEC3G, which acts in a processive manner, possibly suggesting that evolutionary pressure has altered the ability of DNA deaminases to act in a processive or distributive manner, depending on the physiological need.     

The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments

Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo.