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1.
Mol Cell ; 79(5): 703-704, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888434

RESUMO

Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , Citosina , DNA de Cadeia Simples , Desaminação
2.
Science ; 369(6503): 566-571, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732424

RESUMO

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.


Assuntos
Adenina/química , Adenosina Desaminase/química , Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , Proteínas de Escherichia coli/química , Edição de Genes , Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , Microscopia Crioeletrônica , Desaminação , Proteínas de Escherichia coli/genética
3.
Mol Cell Biol ; 40(16)2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32513818

RESUMO

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.


Assuntos
Linfócitos B/citologia , Citosina/metabolismo , Switching de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Citidina Desaminase/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Desaminação/imunologia , Recombinação Genética/genética
4.
PLoS One ; 15(5): e0233467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437404

RESUMO

The enzymatic reactions leading to the deamination of ß-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards ß-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that ß-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that ß-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.


Assuntos
Amônia-Liases/metabolismo , Modelos Moleculares , Sítios de Ligação , Desaminação , Simulação de Acoplamento Molecular , Conformação Proteica , Relação Estrutura-Atividade
5.
Nat Commun ; 11(1): 2052, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345976

RESUMO

Cytosine base editors (CBEs) enable efficient, programmable reversion of T•A to C•G point mutations in the human genome. Recently, cytosine base editors with rAPOBEC1 were reported to induce unguided cytosine deamination in genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 with either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B [wt, R54Q], or PpAPOBEC1 [wt, H122A, R33A]) that display comparable DNA on-target editing frequencies, whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the transcriptome, and up to a 45-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Further, no enrichment of genome-wide C•G to T•A edits are observed in mammalian cells following transfection of mRNA encoding five of these next-generation editors. Taken together, these next-generation CBEs represent a collection of base editing tools for applications in which minimized off-target and high on-target activity are required.


Assuntos
Citosina/metabolismo , DNA/genética , Edição de Genes , RNA/genética , Desaminase APOBEC-1/metabolismo , Citosina Desaminase/metabolismo , Replicação do DNA/genética , Desaminação , Genoma , Células HEK293 , Humanos , Mutagênese/genética , Transcrição Genética , Transcriptoma/genética
6.
Nucleic Acids Res ; 48(3): 1353-1371, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31943071

RESUMO

The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.


Assuntos
Citidina Desaminase/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Desaminação/genética , Sítios de Ligação/genética , Linhagem Celular , Citidina Desaminase/química , Citosina Desaminase/química , Citosina Desaminase/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , HIV-1/genética , HIV-1/patogenicidade , Vírus da Hepatite B/genética , Humanos , Mutagênese/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Retroelementos/genética
7.
Biochim Biophys Acta Gen Subj ; 1864(2): 129346, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30986508

RESUMO

BACKGROUND: APOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation. METHODS: The real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions. RESULTS: A3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect. CONCLUSIONS: The concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA. GENERAL SIGNIFICANCE: The discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.


Assuntos
Citosina Desaminase/química , DNA de Cadeia Simples/química , Citidina Desaminase/química , Desaminação , Genoma Viral , HIV-1/genética , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Mutação , Oligonucleotídeos/química , Ligação Proteica
8.
Bioanalysis ; 11(21): 1955-1965, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31829055

RESUMO

Aim: A novel single-stranded deaminated oligonucleotide metabolite resulting from a REVERSIR™ oligonucleotide was discovered and identified in monkey liver after subcutaneous administration. Results & methodology: REVERSIR-A and its metabolites were extracted from biological matrices by solid phase extraction and analyzed using LC coupled with high-resolution MS under negative ionization mode. A novel 9-mer metabolite of REVERSIR-A, resulting from deamination of the 3' terminal 2'-O-methyl-adenosine nucleotide to 2'-O-methyl-inosine, was discovered at significant levels in monkey liver. The metabolite's identity was confirmed by LC-MS/MS. Conclusion: This report describes the first observation of a long-chain deaminated metabolite of a single-stranded REVERSIR oligonucleotide in vivo in monkey liver.


Assuntos
Espectrometria de Massas/métodos , Oligonucleotídeos/metabolismo , Animais , Desaminação , Inosina/metabolismo , Fígado/metabolismo , Macaca fascicularis
9.
Nucleic Acids Res ; 47(20): 10815-10829, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31566237

RESUMO

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.


Assuntos
Citidina Desaminase/metabolismo , DNA/genética , Região Variável de Imunoglobulina/genética , RNA Polimerase II/metabolismo , Transcrição Genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desaminação , Células HeLa , Humanos , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Especificidade por Substrato , Fatores de Elongação da Transcrição/metabolismo , Proteínas Virais/metabolismo
10.
Chemistry ; 25(66): 15164-15172, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31538684

RESUMO

UV irradiation induces DNA lesions particularly at dipyrimidine sites. Using time-resolved UV pump (250 nm) and mid-IR probe spectroscopy the triplet pathway of cyclobutane pyrimidine dimer (CPD) formation within TpC and CpT sequences was studied. The triplet state is initially localized at the thymine base but decays with 30 ns under formation of a biradical state extending over both bases of the dipyrimidine. Subsequently this state either decays back to the electronic ground state on the 100 ns time scale or forms a cyclobutane pyrimidine dimer lesion (CPD). Stationary IR spectroscopy and triplet sensitization via 2'-methoxyacetophenone (2-M) in the UVA range shows that the lesions are formed with an efficiency of approximately 1.5 %. Deamination converts the cytosine moiety of the CPD lesions on the time scale of 10 hours into uracil which gives CPD(UpT) and CPD(TpU) lesions in which the coding potential of the initial cytosine base is vanished.


Assuntos
Citosina/química , DNA/química , Timina/química , Sequência de Bases , Dano ao DNA/efeitos da radiação , Desaminação , Dímeros de Pirimidina/química , Teoria Quântica , Espectroscopia de Infravermelho com Transformada de Fourier , Raios Ultravioleta
11.
Nat Methods ; 16(12): 1275-1280, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548708

RESUMO

N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.


Assuntos
Adenosina/análogos & derivados , Sequência de Bases , Desaminase APOBEC-1/genética , Adenosina/análise , Desaminação , Células HEK293 , Humanos , Transcriptoma
12.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
13.
Amino Acids ; 51(9): 1353-1363, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31446487

RESUMO

Interferons are signaling proteins that belong to the large class of cytokines and human interferons which are classified based on the type of receptor interactions: type I, II and III. IFNα2b belongs to the type I interferon class with a major therapeutic application for the treatment of hepatitis B and C infections. A recombinant form of IFNα2b expressed in E. coli, known as IntronA, has been approved by US Food and Drug Administration (FDA). IFN γ, also known as type II interferon, plays a significant role in the inhibition of viral replication. Actimmune® is a US Food and Drug Administration (FDA) approved version of IFN γ for the indication of reducing infections associated with chronic granulomatous disease and severe malignant osteopetrosis. In this study we have applied advanced analytical methods for the characterization of IFNα2b and IFN γ produced from Pichia pastoris. The multi-enzyme digestion approach has been developed to allow measurement of 100% sequence coverage and detailed analysis of post-translational variants and degradation products. In this manner, we identified the following variants in IFN α2b: N-terminal residual leader sequence, an amino acid substitution, oxidation of methionine residues and two sites of high mannose N-glycosylation. In the Pichia IFN γ produced material, our approach detected variants resulting from glycosylation, C-terminal proteolysis, oxidation of methionine residues and deamidation. In this manner, the analytical program was able to support rapid process development as well as identify product variants and degradation products in the resulting product.


Assuntos
Antivirais/química , Interferon-alfa/química , Interferon gama/química , Pichia/metabolismo , Sequência de Aminoácidos , Antivirais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Desaminação , Glicosilação , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Espectrometria de Massas/métodos , Oxirredução , Sinais Direcionadores de Proteínas , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
14.
Biochim Biophys Acta Gen Subj ; 1863(11): 129415, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31404619

RESUMO

BACKGROUND: AID/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. METHODS: Substrates 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. RESULTS: All 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. CONCLUSIONS: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged. GENERAL SIGNIFICANCE: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.


Assuntos
Citidina Desaminase/química , Dano ao DNA , Desoxicitidina/química , Antígenos de Histocompatibilidade Menor/química , Proteínas/química , Citidina Desaminase/metabolismo , Desaminação , Desoxicitidina/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Oxirredução , Proteínas/metabolismo
15.
Nat Commun ; 10(1): 3717, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420547

RESUMO

Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.


Assuntos
Aminoácido Oxirredutases/síntese química , Geobacillus stearothermophilus/enzimologia , Cetonas/metabolismo , Aminação , Aminas , Amônia/metabolismo , Biocatálise , Desaminação , Estereoisomerismo
16.
Dev Comp Immunol ; 101: 103437, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31288046

RESUMO

The complement system is a critical part of teleost immune defences, with complement component C4 forming part of the classical and lectin pathways. Cod C4-like protein was isolated from plasma, specific antibodies generated and C4-like protein was assessed in cod sera, mucus and in extracellular vesicles (EVs) from serum and mucus. Higher levels of C4-like protein were detected in serum- than mucus-derived EVs. Post-translational deimination, caused by conversion of arginine into citrulline, can affect protein structure and function. Here we detected deiminated forms of C4-like protein in cod serum and at lower levels in mucus. C4-like protein was also found in deiminated form at low levels in EVs from both serum and mucus. C4-like protein was assessed by immunohistochemistry in cod larvae and detected in a range of organs including in liver, kidney, gut, muscle, skin and mucus, as well as in neuronal tissues of the brain, spinal cord and eye. This abundance of C4-like protein during early development may indicate roles in tissue remodelling, in addition to immune defences. The presence of deiminated C4-like protein in serum and mucosa, as well as in EVs, may suggest C4 protein moonlighting via post-translational deimination.


Assuntos
Complemento C4/metabolismo , Proteínas de Peixes/metabolismo , Gadus morhua/imunologia , Gadus morhua/metabolismo , Animais , Complemento C4/imunologia , Desaminação , Vesículas Extracelulares/metabolismo , Proteínas de Peixes/imunologia , Processamento de Proteína Pós-Traducional/imunologia
17.
Naturwissenschaften ; 106(7-8): 44, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267209

RESUMO

Deaminations (A->G, C->T) increase with DNA singlestrandedness during replication, presumably creating spontaneous genomic mutational and nucleotide frequency gradients. Alternatively, genes are positioned to avoid deaminations. Deamination gradients affect directly mitogene third codon positions; conserved vertebrate mitochondrial tRNA and protein coding gene arrangements minimize deaminations in anticodons, and first and second codon positions in mitogenes. Here we describe deamination gradients across theoretical minimal RNA rings, 22 nucleotide-long RNAs designed to simulate prebiotic RNAs. These RNA rings code for a start/stop codon and a single codon for each amino acid, and form stem-loop hairpins slowing degradation. They resemble consensus tRNAs, defining potential anticodons and cognate amino acids. Theoretical minimal RNA rings are not designed to include deamination gradients, yet deamination gradients occur in RNA rings. tRNA homology produces stronger evidence for deamination gradients than RNA ring homology defined by coding properties. Deamination gradients start at predicted RNA ring anticodons, corresponding to known homologies between mitochondrial tRNAs and replication origins, and between bacterial tRNA synthetases and mitochondrial DNA polymerase gamma. Deamination gradients are strongest for RNA rings with predicted anticodons matching cognate amino acids that integrated early the genetic code. Presumably protections against deaminations evolved while amino acids integrated the genetic code. Results confirm tRNA-RNA ring homologies. Coding constraints defining RNA rings presumably produce deamination gradients starting at predicted anticodons. Hence, the universal genetic code determines nucleotide deamination gradients in theoretical minimal RNA rings, suggesting adaptation to prevent consequences of deamination mutations. Results also indicate that the genetic code's structure determined evolution of tRNAs, their cognates, tRNA synthetases, and polymerases.


Assuntos
Evolução Molecular , Modelos Químicos , RNA/química , Códon/química , Simulação por Computador , Desaminação , RNA de Transferência/química
18.
Molecules ; 24(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181774

RESUMO

Hydrodeaminated and monodeuterated aromatics were obtained via a visible-light driven reaction of arylazo sulfones. Deuteration occurs efficiently in deuterated media such as isopropanol-d8 or in THF-d8/water mixtures and exhibits a high tolerance to the nature and the position of the aromatic substituents.


Assuntos
Deutério/química , Hidrogênio/química , Luz , Metais/química , Sulfonas/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Catálise , Desaminação , Espectroscopia de Prótons por Ressonância Magnética
19.
Chem Asian J ; 14(13): 2235-2241, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116511

RESUMO

Human APOBEC3G (A3G) inhibits the replication of human immunodeficiency virus-1 by deaminating cytidine at the 3'-end in the target motif 5'-CCC-3' in viral cDNA during reverse transcription. It in vitro deaminates two consecutive cytidines in a 3'->5' order. Although a crystal structure of the A3G catalytic domain (A3G-CD2) with DNA was reported, it is unknown why residues involved in enzymatic reaction are distributed widely. Here, we introduced an iodine atom into the C-5 position of cytidine (dC6 I ) in DNA 5'-ATTC4 C5 C6 I A7 ATT-3' (TCCC6 I ). It switches the deamination sequence preference from CCC to TCC, although small dC6 I deamination was observed. Solution structures of A3G-CD2 in complexes with products DNA TCUC6 I and TCUU6 I indicate that the substrate DNA binds A3G-CD2 in TCC and CCC modes. The dC6 deamination correlates with the 4th base type. The CCC mode favours dC6 deamination, while the TCC mode results in dC5 deamination. These studies present an extensive basis to design inhibitors to impede viral evolvability.


Assuntos
Desaminase APOBEC-3G/metabolismo , Citidina/metabolismo , DNA/metabolismo , Desaminase APOBEC-3G/química , Sequência de Bases , Domínio Catalítico , Citidina/química , DNA/química , Desaminação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética
20.
J Pharm Biomed Anal ; 173: 56-61, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31121454

RESUMO

A novel analytical approach capable of measuring deaminated degradation products of oligonucleotide therapeutics is described. The method employs high-resolution mass spectrometry to assess the shift in isotopic distribution that accompanies deamination. Isotopic Distribution Factors (IDF), derived directly from the peak heights of the isotopic pattern, are employed to measure deamination levels of as little as 0.5%. Results obtained from application of the method to a phosphorothioate diester oligonucleotide exposed to various temperatures were used to determine deamination rates.


Assuntos
Espectrometria de Massas/métodos , Oligonucleotídeos Fosforotioatos/análise , Desaminação , Armazenamento de Medicamentos , Oligonucleotídeos Fosforotioatos/metabolismo , Temperatura
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