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1.
Sci Adv ; 6(39)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32978154

RESUMO

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , DNA de Cadeia Simples/genética , Eletroforese em Gel de Ágar/métodos , Pneumonia Viral/diagnóstico , RNA Viral/genética , Análise de Sequência de RNA/métodos , Sequência de Bases , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/genética , Eletroforese em Gel de Ágar/economia , Humanos , Limite de Detecção , Pandemias , Pneumonia Viral/virologia , Saliva/virologia , Análise de Sequência de RNA/economia , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia
2.
Nat Protoc ; 15(8): 2279-2300, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612278

RESUMO

It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.


Assuntos
DNA Antigo , DNA de Cadeia Simples/genética , Biblioteca Gênica , Análise de Sequência de DNA/métodos , Automação , Sequência de Bases
3.
Nucleic Acids Res ; 48(15): 8796-8807, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652019

RESUMO

5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure-as witnessed from the ∼2°C decrease in the melting temperature and 5-10 kJ mol-1 decrease in ΔG°-and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van't Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.


Assuntos
Citosina/análogos & derivados , DNA/efeitos dos fármacos , Conformação Molecular/efeitos dos fármacos , Termodinâmica , Pareamento de Bases/genética , Ilhas de CpG/genética , Citosina/química , Citosina/farmacologia , DNA/química , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , Cinética , Espectroscopia de Ressonância Magnética , Temperatura de Transição
4.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
5.
Nat Commun ; 11(1): 2981, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532979

RESUMO

The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.


Assuntos
Bacteriófago T7/genética , DNA/genética , Regulação Viral da Expressão Gênica , Código Genético , Regiões Promotoras Genéticas/genética , Algoritmos , DNA de Cadeia Simples/genética , Modelos Genéticos , Transcrição Genética
6.
Mol Cell ; 79(1): 115-126.e6, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497497

RESUMO

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.


Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
7.
Nucleic Acids Res ; 48(15): e87, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32573728

RESUMO

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/isolamento & purificação , Oligonucleotídeos/genética , Animais , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos/genética , Camundongos , Inibidores da Síntese de Ácido Nucleico/química , Oligonucleotídeos/síntese química , Ribonuclease H/genética
8.
Nucleic Acids Res ; 48(12): 6624-6639, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463460

RESUMO

Repair of DNA double-strand breaks (DSBs) with homologous chromosomes is a hallmark of meiosis that is mediated by recombination 'bridges' between homolog axes. This process requires cooperation of DMC1 and RAD51 to promote homology search and strand exchange. The mechanism(s) regulating DMC1/RAD51-ssDNA nucleoprotein filament and the components of 'bridges' remain to be investigated. Here we show that MEIOK21 is a newly identified component of meiotic recombination bridges and is required for efficient formation of DMC1/RAD51 foci. MEIOK21 dynamically localizes on chromosomes from on-axis foci to 'hanging foci', then to 'bridges', and finally to 'fused foci' between homolog axes. Its chromosome localization depends on DSBs. Knockout of Meiok21 decreases the numbers of HSF2BP and DMC1/RAD51 foci, disrupting DSB repair, synapsis and crossover recombination and finally causing male infertility. Therefore, MEIOK21 is a novel recombination factor and probably mediates DMC1/RAD51 recruitment to ssDNA or their stability on chromosomes through physical interaction with HSF2BP.


Assuntos
Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Técnicas de Inativação de Genes , Proteínas de Choque Térmico/genética , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética
9.
Nat Methods ; 17(5): 515-523, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251394

RESUMO

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.


Assuntos
Aldeídos/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Animais , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Transcrição Genética
10.
Proc Natl Acad Sci U S A ; 117(11): 5801-5809, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32127479

RESUMO

All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.


Assuntos
Proteínas de Escherichia coli/química , Fator sigma/química , Iniciação da Transcrição Genética , Sítios de Ligação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Ligação Proteica , RNA/química , RNA/genética , RNA/metabolismo , Fator sigma/metabolismo
11.
Nature ; 579(7797): 141-145, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32076262

RESUMO

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA de Cadeia Simples/genética , Edição de Genes/métodos , Pareamento de Bases , DNA de Cadeia Simples/metabolismo , Exodesoxirribonuclease V/metabolismo , Exonucleases/metabolismo , Fluorescência , Cinética , Recombinação Genética/genética , Fatores de Tempo
12.
Cardiovasc Ther ; 2020: 6869856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042311

RESUMO

Objectives: To observe the effect of avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Background: Percutaneous transluminal coronary angioplasty (PTCA) is currently the preferred method for the treatment of coronary heart disease. However, vascular restenosis still occurs after PTCA treatment, severely affecting the clinical efficacy of PTCA. Integrin avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Methods: In this experiment, we used systematic evolution of ligands by exponential enrichment (SELEX) to screen out avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Results: In the present study, we found that avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). Avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). AvP < 0.05). Av. Conclusions: The findings suggest that avß3 ssDNA inhibited the proliferation and migration of VSMCs by suppressing the activation of Ras-PI3K/MAPK signaling.ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Movimento Celular , Proliferação de Células , DNA de Cadeia Simples/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/genética , Células Cultivadas , DNA de Cadeia Simples/genética , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Integrina alfaVbeta3/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteopontina/genética , Osteopontina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas ras/genética
13.
Arch Virol ; 165(3): 771-774, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31960157

RESUMO

Long-distance migratory insects carry microorganisms that can potentially play a crucial role in the life cycles of their hosts. Here, we used Illumina and Sanger sequencing to determine the complete genome sequence of a novel circular Rep-encoding single-stranded (ss) DNA virus from an important migratory pest, Agrotis ipsilon (Hufnagel). The full genome of this new virus is about 2, 242 nt in length and shares 55-75% genome-wide pairwise sequence identity with members of the family Genomoviridae but 91% nucleotide sequence identity with finch-associated genomovirus 3 isolate S30P_D, which is tentatively abbreviated "FaGmV-3". Viral infection rates in A. ipsilon from Yantai, Langfang and Xinxiang were 4.5% (n = 88), 11.8% (n = 85) and 0% (n = 35), respectively. Phylogenetic analysis based on the deduced amino acid sequence of Rep indicated that the Agrotis ipsilon-associated virus is closely related to members of the genus Gemykibivirus, and we propose it to be a new member of this genus. Hence, it is tentatively named "Agrotis ipsilon-associated genomovirus" (AiGmV).


Assuntos
Vírus de DNA , Vírus de Insetos/classificação , Vírus de Insetos/genética , Mariposas/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , DNA de Cadeia Simples/genética , Vírus de Insetos/isolamento & purificação , Análise de Sequência de DNA
14.
Viruses ; 12(2)2020 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-31991902

RESUMO

The Sonoran Desert tortoise Gopherus morafkai is adapted to the desert, and plays an important ecological role in this environment. There is limited information on the viral diversity associated with tortoises (family Testudinidae), and to date no DNA virus has been identified associated with these animals. This study aimed to assess the diversity of DNA viruses associated with the Sonoran Desert tortoise by sampling their fecal matter. A viral metagenomics approach was used to identify the DNA viruses in fecal samples from wild Sonoran Desert tortoises in Arizona, USA. In total, 156 novel single-stranded DNA viruses were identified from 40 fecal samples. Those belonged to two known viral families, the Genomoviridae (n = 27) and Microviridae (n = 119). In addition, 10 genomes were recovered that belong to the unclassified group of circular-replication associated protein encoding single-stranded (CRESS) DNA virus and five circular molecules encoding viral-like proteins.


Assuntos
Vírus de DNA/isolamento & purificação , Fezes/virologia , Tartarugas/virologia , Animais , Arizona , Vírus de DNA/classificação , Vírus de DNA/genética , DNA Circular , DNA de Cadeia Simples/genética , Genoma Viral , Microviridae/classificação , Microviridae/genética , Microviridae/isolamento & purificação , Microvirus/classificação , Microvirus/genética , Microvirus/isolamento & purificação , Filogenia , Recombinação Genética , Proteínas Virais/genética
15.
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
16.
Nucleic Acids Res ; 48(4): 1701-1714, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31919510

RESUMO

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving single-stranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA-ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA-RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1-7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteína de Replicação A/genética , Estruturas Cromossômicas/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Humanos , Ligação Proteica/genética , Proteína de Replicação A/química
17.
Nucleic Acids Res ; 48(5): e30, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31956898

RESUMO

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , DNA de Cadeia Simples/genética , Endonucleases/genética , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas de Bactérias/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/metabolismo , Endonucleases/metabolismo , Edição de Genes/métodos , RNA Guia/genética , RNA Guia/metabolismo , Padrões de Referência
18.
Nucleic Acids Res ; 48(4): 2173-2188, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31925419

RESUMO

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.


Assuntos
Reparo do DNA/genética , Modelos Moleculares , Proteína de Replicação A/química , Proteína de Xeroderma Pigmentoso Grupo A/química , DNA/química , DNA/genética , Dano ao DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica/genética , Proteína de Replicação A/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética
19.
Anal Chim Acta ; 1100: 258-266, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987149

RESUMO

MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all 'A' sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , DNA de Cadeia Simples/genética , MicroRNAs/análise , Animais , Bioensaio , Calibragem , Bovinos , Técnicas de Cultura de Células , Humanos , Limite de Detecção , Células MCF-7 , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico
20.
J Gen Virol ; 101(3): 240-241, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31961791

RESUMO

The family Spiraviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped, hollow, cylindrical virions are formed from a coiling fibre that consists of two intertwining halves of a single circular nucleoprotein filament. A short appendage protrudes from each end of the cylindrical virion. The genome is circular, positive-sense, single-stranded DNA of 24 893 nucleotides. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Spiraviridae, which is available at ictv.global/report/spiraviridae.


Assuntos
Aeropyrum/virologia , Vírus de DNA/classificação , Vírus de DNA/genética , Vírion/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Genoma Viral , Nucleoproteínas , Fases de Leitura Aberta , Replicação Viral
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