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Molecular Therapy. Nucleic Acids Dec 2023Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at... (Review)
Review
Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.
PubMed: 38028200
DOI: 10.1016/j.omtn.2023.102062 -
BioRxiv : the Preprint Server For... Nov 2023Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We...
Replication of the complex retrovirus mouse mammary tumor virus (MMTV) is antagonized by murine Apobec3 (mA3), a member of the Apobec family of cytidine deaminases. We have shown that MMTV-encoded Rem protein inhibits proviral mutagenesis by the Apobec enzyme, activation-induced cytidine deaminase (AID) during viral replication in BALB/c mice. To further study the role of Rem , we have infected C57BL/6 (B6) mice with a superantigen-independent lymphomagenic strain of MMTV (TBLV-WT) or a mutant strain (TBLV-SD) that is defective in Rem and its cleavage product Rem-CT. Unlike MMTV, TBLV induced T-cell tumors in µMT mice, indicating that mature B cells, which express the highest AID levels, are not required for TBLV replication. Compared to BALB/c, B6 mice were more susceptible to TBLV infection and tumorigenesis. The lack of Rem expression accelerated B6 tumorigenesis at limiting doses compared to TBLV-WT in either wild-type B6 or AID-deficient mice. However, unlike proviruses from BALB/c mice, high-throughput sequencing indicated that proviral G-to-A or C-to-T changes did not significantly differ in the presence and absence of Rem expression. stimulation showed higher levels of mA3 relative to AID in B6 compared to BALB/c splenocytes, but effects of agonists differed in the two strains. RNA-Seq revealed increased transcripts related to growth factor and cytokine signaling in TBLV-SD-induced tumors relative to those from TBLV-WT, consistent with a third Rem function. Thus, Rem-mediated effects on tumorigenesis in B6 mice are independent of Apobec-mediated proviral hypermutation.
PubMed: 37961113
DOI: 10.1101/2023.11.02.565355 -
The Journal of Antimicrobial... Dec 2023Hypermutated viruses induced by APOBEC3 (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3) proteins comprise some of the defective viruses in the HIV... (Clinical Trial)
Clinical Trial
BACKGROUND
Hypermutated viruses induced by APOBEC3 (apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3) proteins comprise some of the defective viruses in the HIV reservoir. Here, we assessed the proportion of APOBEC3-induced defective proviruses in HIV-positive patients before and after receiving dolutegravir + lamivudine dual therapy.
METHODS
PBMCs of virologically suppressed patients enrolled in the ANRS 167 LAMIDOL trial, evaluating a switch from triple therapy to dolutegravir + lamivudine, were collected 8 weeks before (W-8) and 48 weeks after (W48) dual-therapy initiation. The Vif and RT regions were subject to next-generation sequencing. Bioinformatic algorithms were developed to identify APOBEC3-defective sequences and APOBEC3-related drug resistance mutations (APOMuts). All hypermutated sequences and those containing at least one stop codon were considered as defective.
RESULTS
One hundred and four patients were enrolled (median virological suppression duration: 4.2 years; IQR: 2.0-9.1). Proviral defective reads at W-8 and W48 were detected in Vif in 22% and 29% of patients, respectively, and in RT in 38% and 42% of patients, respectively. At least one APOMut was present in proviruses of 27% and 38% of patients at W-8 and W48, respectively. The ratio of APOMuts/number of potential APOMut sites was significantly higher at W48 (16.5%) than at W-8 (9.8%, P = 0.007). The presence of APOBEC3-defective viruses at W-8 was not associated with HIV total DNA level, nor with the third drug class received prior to switching to dolutegravir + lamivudine, nor with the duration of virological suppression.
CONCLUSIONS
Whereas no significant change in the proportion of patients with APOBEC3-defective proviruses was evidenced after 1 year of dolutegravir + lamivudine maintenance, enrichment in APOMuts was observed. Further longer-term studies are needed to assess the other forms of defective viruses with dual-therapy.
Topics: Humans; Anti-HIV Agents; APOBEC Deaminases; DNA; Heterocyclic Compounds, 3-Ring; HIV Infections; Lamivudine; Pyridones; Viral Load
PubMed: 37930812
DOI: 10.1093/jac/dkad344 -
Science Advances Nov 2023Mutational signatures represent a genomic footprint of endogenous and exogenous mutational processes through tumor evolution. However, their functional impact on the...
Mutational signatures represent a genomic footprint of endogenous and exogenous mutational processes through tumor evolution. However, their functional impact on the proteome remains incompletely understood. We analyzed the protein-coding impact of single-base substitution (SBS) signatures in 12,341 cancer genomes from 18 cancer types. Stop-gain mutations (SGMs) (i.e., nonsense mutations) were strongly enriched in SBS signatures of tobacco smoking, APOBEC cytidine deaminases, and reactive oxygen species. These mutational processes alter specific trinucleotide contexts and thereby substitute serines and glutamic acids with stop codons. SGMs frequently affect cancer hallmark pathways and tumor suppressors such as , , and . Tobacco-driven SGMs in lung cancer correlate with smoking history and highlight a preventable determinant of these harmful mutations. APOBEC-driven SGMs are enriched in YTCA motifs and associate with expression. Our study exposes SGM expansion as a genetic mechanism by which endogenous and carcinogenic mutational processes directly contribute to protein loss of function, oncogenesis, and tumor heterogeneity.
Topics: Humans; Mutation; Neoplasms; Cytidine Deaminase; APOBEC Deaminases; Tobacco Smoking
PubMed: 37922356
DOI: 10.1126/sciadv.adh3083 -
Science (New York, N.Y.) Nov 2023Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa....
Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.
Topics: Animals; Humans; Africa, Central; Africa, Western; APOBEC Deaminases; Disease Outbreaks; Mpox (monkeypox); Monkeypox virus; Mutation; Phylogeny; Viral Zoonoses; RNA Editing
PubMed: 37917680
DOI: 10.1126/science.adg8116 -
Nature Communications Oct 2023Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper...
Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper replication, especially at head-on transcription-replication conflicts (TRCs). Here we show that TRCs trigger a DNA Damage Response (DDR) involving the chromatin network to prevent genome instability. Depletion of the key chromatin factors INO80, SMARCA5 and MTA2 results in TRCs, fork stalling and R-loop-mediated DNA damage which mostly accumulates at S/G2, while histone H3 Ser10 phosphorylation, a mark of chromatin compaction, is enriched at TRCs. Strikingly, TRC regions show increased mutagenesis in cancer cells with signatures of homologous recombination deficiency, transcription-coupled nucleotide excision repair (TC-NER) and of the AID/APOBEC cytidine deaminases, being predominant at head-on collisions. Thus, our results support that the chromatin network prevents R-loops and TRCs from genomic instability and mutagenic signatures frequently associated with cancer.
Topics: Humans; Chromatin; DNA Replication; Transcription, Genetic; Mutagenesis; DNA Damage; Genomic Instability; Neoplasms; Histone Deacetylases; Repressor Proteins
PubMed: 37898641
DOI: 10.1038/s41467-023-42653-0 -
BMC Bioinformatics Oct 2023Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites....
BACKGROUND
Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.
RESULTS
We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.
CONCLUSIONS
FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE .
Topics: RNA Editing; Workflow; RNA; RNA-Seq; Binding Sites; APOBEC-1 Deaminase
PubMed: 37784060
DOI: 10.1186/s12859-023-05452-4 -
Science Advances Sep 2023CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes. Here, we develop optimized C-to-T base editing systems for the...
CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes. Here, we develop optimized C-to-T base editing systems for the generation of precise loss- or gain-of-function alleles in and identify temperature as a crucial parameter for efficiency. We find that a variant of the widely used APOBEC1 deaminase has attenuated activity at 18° to 29°C and shows considerable dose-dependent toxicity. In contrast, the temperature-tolerant evoCDA1 domain mediates editing of typically more than 90% of alleles and is substantially better tolerated. Furthermore, formation of undesired mutations is exceptionally rare in compared to other species. The predictable editing outcome, high efficiency, and product purity enables near homogeneous induction of STOP codons or alleles encoding protein variants in vivo. Last, we demonstrate how optimized expression enables conditional base editing in marked cell populations. This work substantially facilitates creation of precise alleles in and provides key design parameters for developing efficient base editing systems in other ectothermic species.
Topics: Drosophila; Gene Editing; Animals; APOBEC-1 Deaminase; Cytidine Deaminase; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Silencing
PubMed: 37647411
DOI: 10.1126/sciadv.adj1568 -
MBio Aug 2023HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4 T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B...
HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4 T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4 T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4 T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of -, -, and -to--null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and -null THP-1 cells, and a more modest decrease in infectivity in -null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted 3 to 3 genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.
Topics: Humans; HIV-1; Cytidine Deaminase; vif Gene Products, Human Immunodeficiency Virus; Protein Binding; APOBEC-3G Deaminase; Cytosine Deaminase; Cell Line; HIV Infections; Myeloid Cells; Virion; APOBEC Deaminases
PubMed: 37555667
DOI: 10.1128/mbio.00782-23 -
Genome Research Sep 2023The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced...
The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we used a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. We also determined whether each deletion was epistatic with Ung1 loss, which indicated whether the encoded factors participate in the homologous recombination (HR)-dependent bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single-stranded DNA (ssDNA). We found that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics, we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, (2) defective CTF18-RFC complex function, and (3) defective HR-mediated bypass of APOBEC-induced lesions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancers display three- to fourfold more APOBEC-induced mutations. Mirroring our results in yeast, Rev1-mediated C-to-G substitutions are mainly responsible for increased APOBEC-signature mutations in BRCA1/2-deficient tumors, and these mutations associate with lagging strand synthesis during replication. These results identify important factors that influence DNA replication dynamics and likely the abundance of APOBEC-induced mutation during tumor progression. They also highlight a novel role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.
Topics: Humans; Female; BRCA1 Protein; Saccharomyces cerevisiae; BRCA2 Protein; Mutagenesis; Mutation; Cytidine Deaminase; Breast Neoplasms; Minor Histocompatibility Antigens
PubMed: 37532520
DOI: 10.1101/gr.277430.122