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1.
Plant Mol Biol ; 103(1-2): 225-234, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32140819

RESUMO

DNA N6-methyladenine (6 mA) is one of the most vital epigenetic modifications and involved in controlling the various gene expression levels. With the avalanche of DNA sequences generated in numerous databases, the accurate identification of 6 mA plays an essential role for understanding molecular mechanisms. Because the experimental approaches are time-consuming and costly, it is desirable to develop a computation model for rapidly and accurately identifying 6 mA. To the best of our knowledge, we first proposed a computational model named i6mA-Fuse to predict 6 mA sites from the Rosaceae genomes, especially in Rosa chinensis and Fragaria vesca. We implemented the five encoding schemes, i.e., mononucleotide binary, dinucleotide binary, k-space spectral nucleotide, k-mer, and electron-ion interaction pseudo potential compositions, to build the five, single-encoding random forest (RF) models. The i6mA-Fuse uses a linear regression model to combine the predicted probability scores of the five, single encoding-based RF models. The resultant species-specific i6mA-Fuse achieved remarkably high performances with AUCs of 0.982 and 0.978 and with MCCs of 0.869 and 0.858 on the independent datasets of Rosa chinensis and Fragaria vesca, respectively. In the F. vesca-specific i6mA-Fuse, the MBE and EIIP contributed to 75% and 25% of the total prediction; in the R. chinensis-specific i6mA-Fuse, Kmer, MBE, and EIIP contribute to 15%, 65%, and 20% of the total prediction. To assist high-throughput prediction for DNA 6 mA identification, the i6mA-Fuse is publicly accessible at https://kurata14.bio.kyutech.ac.jp/i6mA-Fuse/.


Assuntos
Adenina/análogos & derivados , DNA de Plantas/metabolismo , Rosaceae/metabolismo , Adenina/metabolismo , Algoritmos , Sítios de Ligação , Biologia Computacional , Conjuntos de Dados como Assunto , Aprendizado de Máquina , Modelos Genéticos , Rosaceae/genética
2.
Nat Biomed Eng ; 4(1): 97-110, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31937940

RESUMO

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann-Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.


Assuntos
Adenina/metabolismo , Citosina/metabolismo , Dependovirus/fisiologia , Edição de Genes/métodos , Animais , Encéfalo/metabolismo , Vetores Genéticos/administração & dosagem , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Retina/metabolismo
3.
BMC Bioinformatics ; 21(1): 31, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996134

RESUMO

BACKGROUND: The analysis and comparison of RNA m6A methylation profiles have become increasingly important for understanding the post-transcriptional regulations of gene expression. However, current m6A profiles in public databases are not readily intercomparable, where heterogeneous profiles from the same experimental report but different cell types showed unwanted high correlations. RESULTS: Several normalizing or correcting methods were tested to remove such laboratory bias. And m6Acorr, an effective pipeline for correcting m6A profiles, was presented on the basis of quantile normalization and empirical Bayes batch regression method. m6Acorr could efficiently correct laboratory bias in the simulated dataset and real m6A profiles in public databases. The preservation of biological signals was examined after correction, and m6Acorr was found to better preserve differential methylation signals, m6A regulated targets, and m6A-related biological features than alternative methods. Finally, the m6Acorr server was established. This server could eliminate the potential laboratory bias in m6A methylation profiles and perform profile-profile comparisons and functional analysis of hyper- (hypo-) methylated genes based on corrected methylation profiles. CONCLUSION: m6Acorr was established to correct the existing laboratory bias in RNA m6A methylation profiles and perform profile comparisons on the corrected datasets. The m6Acorr server is available at http://www.rnanut.net/m6Acorr. A stand-alone version with the correction function is also available in GitHub at https://github.com/emersON106/m6Acorr.


Assuntos
Adenina/análogos & derivados , RNA/metabolismo , Software , Adenina/metabolismo , Animais , Teorema de Bayes , Humanos , Internet , Masculino , Metilação , Camundongos , RNA/química , Processamento Pós-Transcricional do RNA
4.
Phys Chem Chem Phys ; 22(5): 2999-3007, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957771

RESUMO

Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of the three protonated base-pair mismatches containing 9-ethylguanine (9eG) in the gas phase. Computational chemistry has been used to determine the relative energies and compute the infrared spectra of these complexes. By comparing the IRMPD spectra with the computed spectra, in all cases, it was possible to deduce that what was observed experimentally were the lowest energy computed structures. The protonated complex between 9eG and 1-methylthymine (1mT) is protonated at N7 of 9eG-the most basic site of all three bases in this study-and bound in a Hoogsteen type structure with two hydrogen bonds. The experimental IRMPD spectrum for the protonated complex between 9eG and 9-methyladenine (9mA) is described as arising from a combination of the two lowest energy structures, both which are protonated at N1 of adenine and each containing two hydrogen bonds with 9eG being the acceptor of both. The protonated dimer of 9eG is protonated at N7 with an N7-H+-N7 ionic hydrogen bond. It also contains an interaction between a C-H of protonated guanine and the O6 carbonyl of neutral guanine which is manifested in a slight red shift of that carbonyl stretch. The protonated 9eG/9mA structures have been previously identified by X-ray crystallography in RNA and are contained within the protein database.


Assuntos
Gases/química , Guanina/análogos & derivados , Espectrofotometria Infravermelho , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Pareamento Incorreto de Bases , Cristalografia por Raios X , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Fótons , Timina/análogos & derivados , Timina/química , Timina/metabolismo
5.
Nat Commun ; 10(1): 5636, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822664

RESUMO

Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.


Assuntos
Adenina/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Sequência de Bases , Reagentes para Ligações Cruzadas/química , Exonucleases/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo
6.
Phys Rev E ; 100(2-1): 022412, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31574664

RESUMO

Riboswitches are RNA-structured elements that modulate gene expression through changing their conformation in response to specific metabolite binding. However, the regulation mechanisms of riboswitches by ligand binding are still not well understood. At present two possible ligand-binding mechanisms have been proposed: conformational selection and induced fit. Based on explicit-solvent molecular dynamics (MD) simulations, we have studied the process of the binding of ligands (adenines) to add adenine riboswitch aptamer (AARA) in detail. Our results show that the relative high flexibility of the junction J23 of AARA allows the ligands to be captured by the binding pocket of AARA in a near-native state, which may be driven by hydrophobic and base-stacking interactions. In addition, the binding of a ligand makes the stem P1 and the junction J23 of AARA more stable than in the absence of the ligand. Generally, our analyses show that the ligand-binding process of the add adenine riboswitch may be partially embodied by a conformational selection mechanism.


Assuntos
Adenina/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Simulação de Dinâmica Molecular , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Ligantes , Conformação de Ácido Nucleico , Termodinâmica
7.
Nucleic Acids Res ; 47(15): 8154-8162, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31276580

RESUMO

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.


Assuntos
Adenina/metabolismo , DNA Catalítico/metabolismo , Guanina/metabolismo , Sódio/metabolismo , Algoritmos , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , DNA Catalítico/química , DNA Catalítico/genética , Cinética , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA/genética , RNA/metabolismo , Especificidade por Substrato
8.
Nature ; 571(7764): 275-278, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181567

RESUMO

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , Transfecção
9.
BMC Genomics ; 20(1): 445, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159718

RESUMO

BACKGROUND: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. RESULTS: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. CONCLUSIONS: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.


Assuntos
Adenina/análogos & derivados , Artefatos , Citosina/análogos & derivados , Metilação de DNA , DNA/genética , Eucariotos/genética , Genoma , Adenina/análise , Adenina/metabolismo , Animais , Células Cultivadas , Citosina/análise , Citosina/metabolismo , Genômica , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo
10.
Nucleic Acids Res ; 47(15): 7929-7941, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31216025

RESUMO

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.


Assuntos
Metilação de DNA , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Salmonella enterica/genética , Fatores de Transcrição/genética , Adenina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Salmonella enterica/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
11.
Saudi J Kidney Dis Transpl ; 30(3): 723-725, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31249241

RESUMO

Adenine phosphoribosyltransferase deficiency is an inherited condition presenting from infancy to late adulthood. The common features are recurrent kidney and urinary tract stones and obstructive symptoms. The stones are characteristically radiolucent. 2, 8-Dihydroxyadenine (2, 8-DHA) formation is blocked by xanthine oxidase blocker allopurinol. Here, we report the case of an eight-month-old baby girl who presented with obstructive acute kidney injury secondary to calculi which was treated with surgical removal of stone. The analysis of the calculi revealed 2, 8-DHA crystals.


Assuntos
Lesão Renal Aguda/etiologia , Adenina Fosforribosiltransferase/deficiência , Adenina/análogos & derivados , Cálculos Renais/etiologia , Erros Inatos do Metabolismo/complicações , Urolitíase/complicações , Lesão Renal Aguda/diagnóstico , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/cirurgia , Adenina/metabolismo , Alopurinol/uso terapêutico , Cristalização , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Lactente , Cálculos Renais/diagnóstico , Cálculos Renais/metabolismo , Cálculos Renais/cirurgia , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/tratamento farmacológico , Resultado do Tratamento , Urolitíase/diagnóstico , Urolitíase/tratamento farmacológico , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/metabolismo
12.
Mol Genet Genomics ; 294(5): 1173-1182, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31055655

RESUMO

DNA N6-methyladenine is a non-canonical DNA modification that occurs in different eukaryotes at low levels and it has been identified as an extremely important function of life. Moreover, about 0.2% of adenines are marked by DNA N6-methyladenine in the rice genome, higher than in most of the other species. Therefore, the identification of them has become a very important area of study, especially in biological research. Despite the few computational tools employed to address this problem, there still requires a lot of efforts to improve their performance results. In this study, we treat DNA sequences by the continuous bags of nucleobases, including sub-word information of its biological words, which then serve as features to be fed into a support vector machine algorithm to identify them. Our model which uses this hybrid approach could identify DNA N6-methyladenine sites with achieved a jackknife test sensitivity of 86.48%, specificity of 89.09%, accuracy of 87.78%, and MCC of 0.756. Compared to the state-of-the-art predictor as well as the other methods, our proposed model is able to yield superior performance in all the metrics. Moreover, this study provides a basis for further research that can enrich a field of applying natural language-processing techniques in biological sequences.


Assuntos
Adenina/análogos & derivados , DNA de Plantas/genética , Genoma de Planta/genética , Oryza/genética , Adenina/metabolismo , Algoritmos , Biologia Computacional/métodos , Máquina de Vetores de Suporte
13.
Cell Mol Life Sci ; 76(15): 2957-2966, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31143960

RESUMO

DNA modifications are a major form of epigenetic regulation that eukaryotic cells utilize in concert with histone modifications. While much work has been done elucidating the role of 5-methylcytosine over the past several decades, only recently has it been recognized that N(6)-methyladenine (N6-mA) is present in quantifiable and biologically active levels in the DNA of eukaryotic cells. Unlike prokaryotes which utilize N6-mA to recognize "self" from "foreign" DNA, eukaryotes have been found to use N6-mA in varying ways, from regulating transposable elements to gene regulation in response to hypoxia and stress. In this review, we examine the current state of the N6-mA in research field, and the current understanding of the biochemical mechanisms which deposit and remove N6-mA from the eukaryotic genome.


Assuntos
Adenina/análogos & derivados , Eucariotos/metabolismo , Adenina/metabolismo , Animais , Metilação de DNA , Enzimas Reparadoras do DNA/metabolismo , Epigenômica , Eucariotos/genética , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases N-Desmetilantes/metabolismo , Estresse Fisiológico
14.
Nat Commun ; 10(1): 2219, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101825

RESUMO

A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. We have previously shown that N6-methyladenine (6mA) DNA modification is highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. Here we report that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. We find that zelda encoding the pioneer factor Zelda is marked by 6mA. Our genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, our findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.


Assuntos
DNA/metabolismo , Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário/fisiologia , Fatores de Transcrição/metabolismo , Zigoto/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Embrião não Mamífero , Epigênese Genética/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Genoma de Inseto , Masculino , Proteínas Nucleares , Fatores de Transcrição/genética
15.
G3 (Bethesda) ; 9(6): 1893-1900, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988038

RESUMO

N6-methyladenine (6mA or m6dA) is a DNA modification that has long been known to play an important role in a variety of biological functions in prokaryotes. This modification has only recently been described in eukaryotes, where it seems to have evolved species-specific functions ranging from nucleosome positioning to transposon repression. In Drosophila, 6mA has been shown to be important for enforcing the tissue specificity of neuronal genes in the brain and suppressing transposable element expression in the ovaries. In this study, we have analyzed the raw signal data from nanopore sequencing to identify 6mA positions in the D. melanogaster genome at single-base resolution. We find that this modification is enriched upstream from transcription start sites, within the introns and 3' UTRs of genes, as well as in simple repeats. These 6mA positions are enriched for sequence motifs that are recognized by known transcriptional activators involved in development, such as Bicoid and Caudal, and the genes that carry this modification are enriched for functions involved in development, regulation of transcription, and neuronal activity. These genes show high expression specificity in a variety of tissues besides the brain, suggesting that this modification may play a more general role in enforcing the specificity of gene expression across many tissues, throughout development, and between the sexes.


Assuntos
Adenina/metabolismo , Metilação de DNA , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Animais , Biologia Computacional/métodos , Sequência Conservada , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Motivos de Nucleotídeos
16.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 299-306, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950831

RESUMO

Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Šresolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.


Assuntos
Adenina/metabolismo , Dissacarídeos/biossíntese , Enzimas/química , Enzimas/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Lactamas , Ligantes , Homologia Estrutural de Proteína , Especificidade por Substrato
17.
Chem Res Toxicol ; 32(5): 840-849, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-30938985

RESUMO

N6-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo-). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo-. The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo- reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency ( kpol/ Kd,dNTP) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate ( kpol), and Hyp increased the dissociation constant ( Kd,dNTP). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.


Assuntos
Adenina/metabolismo , Bacteriófagos/enzimologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Proteínas Virais/metabolismo , Adenina/análogos & derivados , DNA/química , Espectroscopia de Ressonância de Spin Eletrônica , Cinética
18.
DNA Repair (Amst) ; 78: 81-90, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991231

RESUMO

N6-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (kpol) and increased the dissociation constant (Kd,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.


Assuntos
Adenina/análogos & derivados , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Epigênese Genética , Adenina/metabolismo , Humanos , Cinética , Nucleotídeos/metabolismo
19.
Biosci Biotechnol Biochem ; 83(7): 1300-1305, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999827

RESUMO

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine - but not caffeine - transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium-proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.


Assuntos
Adenina/metabolismo , Coffea/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Arabidopsis/metabolismo , Cafeína/metabolismo , Coffea/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Nigericina/farmacologia , Oryza/metabolismo
20.
Mol Cell ; 74(6): 1138-1147.e6, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30982744

RESUMO

Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing.


Assuntos
Adenina/análogos & derivados , Homólogo AlkB 4 da Lisina Desmetilase/genética , Anormalidades Craniofaciais/genética , DNA/genética , Metiltransferases/genética , Proteínas Repressoras/genética , Adenina/metabolismo , Homólogo AlkB 4 da Lisina Desmetilase/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , DNA/metabolismo , Metilação de DNA , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Feminino , Inativação Gênica , Genes Letais , Histonas/genética , Histonas/metabolismo , Endogamia , Masculino , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Proteólise , Proteínas Repressoras/metabolismo , Transdução de Sinais , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Transcrição Genética , Ubiquitina/genética , Ubiquitina/metabolismo
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