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2.
Nat Commun ; 11(1): 4714, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948754

RESUMO

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.


Assuntos
DNA/química , Fenômenos Magnéticos , Magnetismo/métodos , Nanotecnologia , Análise Espectral/métodos , Ciprofloxacino/farmacologia , DNA/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Conformação de Ácido Nucleico , Pinças Ópticas , Fenômenos Físicos , Software
3.
Int J Mol Sci ; 21(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867123

RESUMO

A considerable amount of rapid-paced research is underway to combat the SARS-CoV-2 pandemic. In this work, we assess the 3D structure of the 5' untranslated region of its RNA, in the hopes that stable secondary structures can be targeted, interrupted, or otherwise measured. To this end, we have combined molecular dynamics simulations with previous Nuclear Magnetic Resonance measurements for stem loop 2 of SARS-CoV-1 to refine 3D structure predictions of that stem loop. We find that relatively short sampling times allow for loop rearrangement from predicted structures determined in absence of water or ions, to structures better aligned with experimental data. We then use molecular dynamics to predict the refined structure of the transcription regulatory leader sequence (TRS-L) region which includes stem loop 3, and show that arrangement of the loop around exchangeable monovalent potassium can interpret the conformational equilibrium determined by in-cell dimethyl sulfate (DMS) data.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Regiões 5' não Traduzidas/genética , Humanos , Sequências Repetidas Invertidas/genética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Pandemias , RNA Viral/genética
4.
PLoS Pathog ; 16(9): e1008825, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886709

RESUMO

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.


Assuntos
Alphavirus , Genoma Viral , Conformação de Ácido Nucleico , RNA Replicase , RNA Viral , Proteínas Virais , Alphavirus/química , Alphavirus/genética , Alphavirus/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Nat Commun ; 11(1): 4531, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913225

RESUMO

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.


Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição Genética
6.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
7.
Mol Cell ; 79(6): 917-933.e9, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32755595

RESUMO

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/ultraestrutura , Troca de Cromátide Irmã/genética , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Microscopia Crioeletrônica , DNA/genética , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
8.
PLoS One ; 15(8): e0237747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822369

RESUMO

With the great significance of biomolecular flexibility in biomolecular dynamics and functional analysis, various experimental and theoretical models are developed. Experimentally, Debye-Waller factor, also known as B-factor, measures atomic mean-square displacement and is usually considered as an important measurement for flexibility. Theoretically, elastic network models, Gaussian network model, flexibility-rigidity model, and other computational models have been proposed for flexibility analysis by shedding light on the biomolecular inner topological structures. Recently, a topology-based machine learning model has been proposed. By using the features from persistent homology, this model achieves a remarkable high Pearson correlation coefficient (PCC) in protein B-factor prediction. Motivated by its success, we propose weighted-persistent-homology (WPH)-based machine learning (WPHML) models for RNA flexibility analysis. Our WPH is a newly-proposed model, which incorporate physical, chemical and biological information into topological measurements using a weight function. In particular, we use local persistent homology (LPH) to focus on the topological information of local regions. Our WPHML model is validated on a well-established RNA dataset, and numerical experiments show that our model can achieve a PCC of up to 0.5822. The comparison with the previous sequence-information-based learning models shows that a consistent improvement in performance by at least 10% is achieved in our current model.


Assuntos
RNA/química , Algoritmos , Elasticidade , Aprendizado de Máquina , Distribuição Normal , Conformação de Ácido Nucleico
9.
Nucleic Acids Res ; 48(15): 8529-8544, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738045

RESUMO

Myocyte enhancer factor-2B (MEF2B) has the unique capability of binding to its DNA target sites with a degenerate motif, while still functioning as a gene-specific transcriptional regulator. Identifying its DNA targets is crucial given regulatory roles exerted by members of the MEF2 family and MEF2B's involvement in B-cell lymphoma. Analyzing structural data and SELEX-seq experimental results, we deduced the DNA sequence and shape determinants of MEF2B target sites on a high-throughput basis in vitro for wild-type and mutant proteins. Quantitative modeling of MEF2B binding affinities and computational simulations exposed the DNA readout mechanisms of MEF2B. The resulting binding signature of MEF2B revealed distinct intricacies of DNA recognition compared to other transcription factors. MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif, where A-tract polarity dictates nuances of binding. The predominant role of shape readout at the center of the core motif, with most contacts formed in the minor groove, differs from previously observed protein-DNA readout modes. MEF2B, therefore, represents a unique protein for studies of the role of DNA shape in achieving binding specificity. MEF2B-DNA recognition mechanisms are likely representative for other members of the MEF2 family.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/ultraestrutura , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/ultraestrutura , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica/genética
10.
Proc Natl Acad Sci U S A ; 117(33): 19661-19663, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747537

RESUMO

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A-H2B heterodimers and one histone (H3-H4)2 tetramer, wrapped around by ∼146 bp of DNA. The N-terminal flexible histone tails stick out from the histone core and have extensive posttranslational modifications, causing epigenetic changes of chromatin. Although crystal and cryogenic electron microscopy structures of nucleosomes are available, the flexible tail structures remain elusive. Using NMR, we have examined the dynamics of histone H3 tails in nucleosomes containing unmodified and tetra-acetylated H4 tails. In unmodified nucleosome, the H3 tail adopts a dynamic equilibrium structure between DNA-contact and reduced-contact states. In acetylated H4 nucleosome, however, the H3 tail equilibrium shifts to a mainly DNA-contact state with a minor reduced-contact state. The acetylated H4 tail is dynamically released from its own DNA-contact state to a reduced-contact state, while the H3 tail DNA-contact state becomes major. Notably, H3 K14 in the acetylated H4 nucleosome is much more accessible to acetyltransferase Gcn5 relative to unmodified nucleosome, possibly due to the formation of a favorable H3 tail conformation for Gcn5. In summary, each histone tail adopts a characteristic dynamic state but regulates one other, probably creating a histone tail network even on a nucleosome.


Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Motivos de Aminoácidos , DNA/genética , DNA/metabolismo , Histonas/genética , Humanos , Conformação de Ácido Nucleico , Nucleossomos/genética
11.
Sci Rep ; 10(1): 12331, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704018

RESUMO

The outbreak of COVID-19 has brought great threat to human health. Its causative agent is a severe acute respiratory syndrome-related coronavirus which has been officially named SARS-CoV-2. Here we report the discovery of extremely low CG abundance in its open reading frames. We found that CG reduction in SARS-CoV-2 is achieved mainly through mutating C/G into A/T, and CG is the best target for mutation. Meanwhile, 5'-untranslated region of SARS-CoV-2 has high CG content and is capable of forming an internal ribosome entry site (IRES) to recruit host ribosome for translating its RNA. These features allow SARS-CoV-2 to reproduce efficiently in host cells, because less energy is consumed in disrupting the stem-loops formed by its genomic RNA. Notably, genomes of cellular organisms also have very low CG abundance, suggesting that mutating C/G into A/T occurs universally in all life forms. Moreover, CG is the dinucleotide related to CpG island, mutational hotspot and single nucleotide polymorphism in cellular organisms. The relationship between these features is worthy of further investigations.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Fosfatos de Dinucleosídeos/metabolismo , Fases de Leitura Aberta/genética , Pneumonia Viral/virologia , Regiões 5' não Traduzidas , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Ilhas de CpG , Humanos , Conformação de Ácido Nucleico , Razão de Chances , Pandemias , Pneumonia Viral/patologia , Polimorfismo de Nucleotídeo Único , Replicação Viral
12.
Nat Commun ; 11(1): 3422, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647223

RESUMO

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Autoantígeno Ku/química , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Homologia Estrutural de Proteína
13.
Nat Commun ; 11(1): 3557, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678091

RESUMO

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.


Assuntos
Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Pequeno RNA não Traduzido/genética , Transcriptoma , Acetilglucosamina/metabolismo , Proteínas de Bactérias/genética , Bacteroides thetaiotaomicron/crescimento & desenvolvimento , Bacteroides thetaiotaomicron/metabolismo , Meios de Cultura/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Navegador
14.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
15.
PLoS One ; 15(7): e0235655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628740

RESUMO

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.


Assuntos
RNA Nuclear Pequeno/metabolismo , Spliceossomos/metabolismo , Criança , Bases de Dados Genéticas , Nanismo/genética , Nanismo/patologia , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Humanos , Microcefalia/genética , Microcefalia/patologia , Conformação de Ácido Nucleico , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Processamento de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética
16.
Nat Commun ; 11(1): 3599, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680990

RESUMO

Notwithstanding the central biological role of the (6-4) photoadduct in the induction of skin cancer by sunlight, crucial mechanistic details about its formation have evaded characterization despite efforts spanning more than half a century. 4-Thiothymidine (4tT) has been widely used as an important model system to study its mechanism of formation, but the excited-state precursor, the intermediate species, and the time scale leading to the formation of the (6-4) photoadduct have remained elusive. Herein, steady-state and time-resolved spectroscopic techniques are combined with new and reported quantum-chemical calculations to demonstrate the excited state leading to the formation of the thietane intermediate, its rate, and the formation of the (6-4) photoadduct using the 5'-TT(4tT)T(4tT)TT-3' DNA oligonucleotide. Efficient, sub-1 ps intersystem crossing leads to the population of a triplet minimum of the thietane intermediate in as short as 3 ps, which intersystem crosses to its ground state and rearranges to form the (6-4) photoadduct.


Assuntos
Adutos de DNA/genética , DNA/genética , Neoplasias Cutâneas/genética , Tionucleosídeos/química , Timidina/análogos & derivados , Raios Ultravioleta/efeitos adversos , DNA/química , Adutos de DNA/efeitos da radiação , Humanos , Estrutura Molecular , Conformação de Ácido Nucleico/efeitos da radiação , Neoplasias Cutâneas/etiologia , Timidina/química
17.
Nat Commun ; 11(1): 3474, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651392

RESUMO

RNase MRP is an essential eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle. RNase MRP is related to the ribozyme-based RNase P, but it has evolved to have distinct cellular roles. We report a cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme solved to 3.0 Å. We describe the structure of this 450 kDa complex, interactions between its components, and the organization of its catalytic RNA. We show that some of the RNase MRP proteins shared with RNase P undergo an unexpected RNA-driven remodeling that allows them to bind to divergent RNAs. Further, we reveal how this RNA-driven protein remodeling, acting together with the introduction of new auxiliary elements, results in the functional diversification of RNase MRP and its progenitor, RNase P, and demonstrate structural underpinnings of the acquisition of new functions by catalytic RNPs.


Assuntos
Microscopia Crioeletrônica , Endorribonucleases/ultraestrutura , Ribonucleoproteínas/ultraestrutura , Carbono/química , Catálise , Domínio Catalítico , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Catalítico/química , RNA Fúngico/química , Ribonuclease P/química , Saccharomyces cerevisiae/enzimologia
18.
Nat Commun ; 11(1): 3723, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709850

RESUMO

DNA methylation maintenance by DNMT1 is an essential process in mammals but molecular mechanisms connecting DNA methylation patterns and enzyme activity remain elusive. Here, we systematically analyzed the specificity of DNMT1, revealing a pronounced influence of the DNA sequences flanking the target CpG site on DNMT1 activity. We determined DNMT1 structures in complex with preferred DNA substrates revealing that DNMT1 employs flanking sequence-dependent base flipping mechanisms, with large structural rearrangements of the DNA correlating with low catalytic activity. Moreover, flanking sequences influence the conformational dynamics of the active site and cofactor binding pocket. Importantly, we show that the flanking sequence preferences of DNMT1 highly correlate with genomic methylation in human and mouse cells, and 5-azacytidine triggered DNA demethylation is more pronounced at CpG sites with flanks disfavored by DNMT1. Overall, our findings uncover the intricate interplay between CpG-flanking sequence, DNMT1-mediated base flipping and the dynamic landscape of DNA methylation.


Assuntos
Sequência de Bases , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , DNA/química , DNA/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Cinética , Camundongos Knockout , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos , Conformação Proteica , Especificidade por Substrato
19.
Nat Commun ; 11(1): 3749, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719310

RESUMO

xrRNAs from flaviviruses survive in host cells because of their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, [Formula: see text] and [Formula: see text], are driven through a pore. Pulling the [Formula: see text] end, as done by replicases, elicits a progressive unfolding; pulling the [Formula: see text] end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the [Formula: see text] end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials.


Assuntos
RNA Viral/metabolismo , Zika virus/genética , Sequência de Bases , Conformação de Ácido Nucleico , Transporte de RNA , RNA Viral/química , RNA Viral/genética , Estresse Mecânico , Termodinâmica
20.
Nat Commun ; 11(1): 3613, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680994

RESUMO

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.


Assuntos
Sítios Frágeis do Cromossomo/genética , Período de Replicação do DNA/genética , Genoma Humano , Instabilidade Genômica , Afidicolina/farmacologia , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Mapeamento Cromossômico/métodos , DNA/química , Período de Replicação do DNA/efeitos dos fármacos , Fibroblastos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genética , Conformação de Ácido Nucleico , Sensibilidade e Especificidade , Transcrição Genética/efeitos dos fármacos
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