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1.
Soft Matter ; 15(21): 4284-4293, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31094392

RESUMO

Despite their great promise as fluorescent biological probes and sensors, the structure and dynamics of Ag complexes derived from single stranded DNA (ssDNA) are less understood than their double stranded counterparts. In this work, we seek new insights into the structure of single AgNssDNA clusters using analytical ultracentrifugation (AUC), nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular dynamics simulations (MD) of a fluorescent (AgNssDNA)8+ nanocluster. The results suggest that the purified (AgNssDNA)8+ nanocluster is a mixture of predominantly Ag15 and Ag16 species that prefer two distinct long-lived conformational states: one extended, the other approaching spherical. However, the ssDNA strands within these clusters are highly mobile. Ag(i) interacts preferentially with the nucleobase rather than the phosphate backbone, causing a restructuring of the DNA strand relative to the bare DNA. Infrared spectroscopy and MD simulations of (AgNssDNA)8+ and model nucleic acid homopolymers suggest that Ag(i) has a higher affinity for cytosine over guanine bases, little interaction with adenine, and virtually none with thymine. Ag(i) shows a tendency to interact with cytosine N3 and O2 and guanine N7 and O6, opening the possibility for a Ag(i)-base bifurcated bond to act as a nanocluster nucleation and strand stabilizing site. This work provides valuable insight into nanocluster structure and dynamics which drive stability and optical properties, and additional studies using these types of characterization techniques are important for the rational design of single stranded AgDNA nanocluster sensors.


Assuntos
DNA de Cadeia Simples/química , Prata/química , Sequência de Bases , DNA de Cadeia Simples/genética , Conformação Molecular , Simulação de Dinâmica Molecular
2.
Arch Virol ; 164(7): 1883-1887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079213

RESUMO

Using next-generation sequencing to characterize agents associated with a severe stunting disease of parsley from Germany, we identified a hitherto undescribed virus. We sequenced total RNA and rolling-circle-amplified DNA from diseased plants. The genome sequence of the virus shows that it is a member of the genus Nanovirus, but it lacks DNA-U4. In addition to the seven genomic DNAs of the virus, we identified a second DNA-R and seven distinct alphasatellites associated with the disease. We propose the name "parsley severe stunt associated virus" (PSSaV) for this novel nanovirus.


Assuntos
DNA Viral/genética , Nanovirus/genética , Nanovirus/isolamento & purificação , Petroselinum/virologia , Doenças das Plantas/virologia , Sequência de Bases , DNA Circular/genética , DNA Satélite/genética , DNA de Cadeia Simples/genética , Genoma Viral/genética , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Nanovirus/classificação
3.
Analyst ; 144(10): 3216-3220, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-30984925

RESUMO

A DSN-RNAse-TdT-T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity-the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.


Assuntos
DNA Nucleotidilexotransferase/química , Exodesoxirribonucleases/química , MicroRNAs/sangue , Ribonucleases/química , Bacteriófago T7/enzimologia , Sondas de DNA/síntese química , Sondas de DNA/genética , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Mensageiro/genética
4.
Anal Bioanal Chem ; 411(13): 2925-2935, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30957202

RESUMO

We present an electrochemical DNA detection strategy based on self-assembled ferrocene-cored poly(amidoamine) dendrimers for the detection of a gene relevant to breast cancer. The chemisorption of three ferrocene-cored poly(amidoamine) generations and hybridization of single-stranded DNA on a Au electrode were studied by cyclic voltammetry and differential pulse voltammetry. The biosensor demonstrated high sensitivity of 0.13 µA/(ng/ml) in the detection of the target DNA with a linear range of 1.3-20 nM and a detection limit of 0.38 nM. The DNA biosensor also has high selectivity for the target DNA, showing a clear signal difference from a noncomplementary sequence and a single-base-mismatch sequence, which was used as a model of BRAC1 gene mutation. The results shown are highly motivating for exploring DNA biosensing technology in the diagnosis of breast cancer caused by mutation of the BRAC1 gene. Graphical abstract.


Assuntos
Técnicas Biossensoriais/métodos , Neoplasias da Mama/genética , DNA/genética , Técnicas Eletroquímicas/métodos , Genes BRCA1 , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Análise Mutacional de DNA/métodos , DNA de Cadeia Simples/genética , Feminino , Compostos Ferrosos/química , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/genética , Metalocenos/química , Mutação , Hibridização de Ácido Nucleico , Poliaminas/química
5.
Appl Microbiol Biotechnol ; 103(8): 3559-3570, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30879090

RESUMO

Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.


Assuntos
DNA Primase/genética , DNA de Cadeia Simples/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Engenharia Genética/métodos , Sistemas CRISPR-Cas , DNA Primase/metabolismo , DNA Bacteriano/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Edição de Genes , Genoma Bacteriano/genética , Recombinação Homóloga , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo
6.
Biosens Bioelectron ; 133: 24-31, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30903938

RESUMO

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn2O3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM - 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM - 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples/química , DNA/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/isolamento & purificação , DNA/química , DNA de Cadeia Simples/genética , Grafite/química , Humanos , Nuclease do Micrococo/genética , Nuclease do Micrococo/isolamento & purificação , Nanofibras/química , Hibridização de Ácido Nucleico , Proteínas de Ligação às Penicilinas/isolamento & purificação , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/isolamento & purificação , Staphylococcus aureus/genética
7.
RNA ; 25(6): 737-746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926754

RESUMO

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/química , Biomarcadores Tumorais/química , DNA de Cadeia Simples/química , Exorribonucleases/química , Magnésio/química , Proteínas Mitocondriais/química , Oligorribonucleotídeos/química , RNA/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sítios de Ligação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Magnésio/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Analyst ; 144(8): 2773-2779, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30869659

RESUMO

With the use of a double-cycle system involving two catalytic reactions by RNase H and DNAzyme, the signal of oligoDNAs has been specifically amplified in an isothermal mode. The precursor of DNAzyme was introduced to the system as a ring-structured and inactivated form, which involves the 6-nt RNA portion being complementary to target oligoDNA. In the presence of target oligoDNA, the RNA portion forms a DNA/RNA hetero-duplex and is cut by RNase H. This scission converts the precursor to catalytically active DNAzyme, which in turn disconnects the molecular beacon to produce the amplified signal. Because the covalent bonds were disconnected to provide discrete structural changes in both cycles, high sensitivity and specificity are obtained, indicating the strong potential of this double catalytic cycle method for versatile applications.


Assuntos
DNA Catalítico/química , DNA de Cadeia Simples/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Oligonucleotídeos/análise , Ribonuclease H/química , Antraquinonas/química , DNA Catalítico/genética , DNA de Cadeia Simples/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Perileno/química , Ribonuclease H/genética
9.
Analyst ; 144(8): 2755-2764, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30869681

RESUMO

A ratiometric and sensitive microfluidic chip based aptasensor was developed for antibiotic detection with kanamycin (Kana) as a model analyte. A novel stir bar assisted sorptive extraction and rolling circle amplification strategy was designed to largely amplify the signal and overcome complex matrix interference in food samples. The detection mechanism was as follows: firstly, many duplex DNA probes (a single-stranded DNA as a primer hybrid with an aptamer sequence) were modified on a stir bar. In the presence of Kana, the probes on the bar could specifically capture Kana and release the primer to trigger RCA in the presence of a circular DNA template (CDT). As the reaction proceeds, the amount of CDT decreased and the number of RCA products increased. It is worth mentioning that they can be efficiently separated and detected using a microfluidic chip. The signal ratio of RCA products and CDT (IR/IC) can be employed to qualify Kana in a wide linear range from 0.8 pg mL-1 to 10 ng mL-1 with a low detection limit of 0.3 pg mL-1. This method exhibited excellent sensitivity and selectivity and can obviously reduce the matrix interference through a ratiometric strategy combined with stir bar extraction. The aptasensor was successfully tested in milk and fish samples, confirming that it can be applied for on-site quantitation of antibiotic residues in foods.


Assuntos
Antibacterianos/análise , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Canamicina/análise , Técnicas Analíticas Microfluídicas/métodos , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Peixes , Ouro/química , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Leite/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Alimentos Marinhos/análise
10.
Proc Natl Acad Sci U S A ; 116(12): 5493-5498, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819888

RESUMO

The filamentous bacteriophage IKe infects Escherichia coli cells bearing IncN pili. We report the cryo-electron microscopy structure of the micrometer-long IKe viral particle at a resolution of 3.4 Å. The major coat protein [protein 8 (p8)] consists of 47 residues that fold into a ∼68-Å-long helix. An atomic model of the coat protein was built. Five p8 helices in a horizontal layer form a pentamer, and symmetrically neighboring p8 layers form a right-handed helical cylinder having a rise per pentamer of 16.77 Å and a twist of 38.52°. The inner surface of the capsid cylinder is positively charged and has direct interactions with the encapsulated circular single-stranded DNA genome, which has an electron density consistent with an unusual left-handed helix structure. Similar to capsid structures of other filamentous viruses, strong capsid packing in the IKe particle is maintained by hydrophobic residues. Despite having a different length and large sequence differences from other filamentous phages, π-π interactions were found between Tyr9 of one p8 and Trp29 of a neighboring p8 in IKe that are similar to interactions observed in phage M13, suggesting that, despite sequence divergence, overall structural features are maintained.


Assuntos
Bacteriófago IKe/ultraestrutura , Bacteriófago IKe/genética , Bacteriófago IKe/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/ultraestrutura , Modelos Moleculares , Alinhamento de Sequência , Montagem de Vírus
11.
Nat Commun ; 10(1): 1061, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837459

RESUMO

The self-assembly of a DNA origami structure, although mostly feasible, represents indeed a rather complex folding problem. Entropy-driven folding and nucleation seeds formation may provide possible solutions; however, until now, a unified view of the energetic factors in play is missing. Here, by analyzing the self-assembly of origami domains with identical structure but different nucleobase composition, in function of variable design and experimental parameters, we identify the role played by sequence-dependent forces at the edges of the structure, where topological constraint is higher. Our data show that the degree of mechanical stress experienced by these regions during initial folding reshapes the energy landscape profile, defining the ratio between two possible global conformations. We thus propose a dynamic model of DNA origami assembly that relies on the capability of the system to escape high structural frustration at nucleation sites, eventually resulting in the emergence of a more favorable but previously hidden state.


Assuntos
DNA de Cadeia Simples/química , Nanoestruturas/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Estresse Mecânico , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/ultraestrutura , Entropia , Transferência Ressonante de Energia de Fluorescência , Microscopia de Força Atômica , Nanotecnologia/métodos , Oligonucleotídeos/genética
12.
Methods Mol Biol ; 1963: 75-83, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875046

RESUMO

Genomic library preparation from highly degraded DNA is more efficient when library molecules are prepared separately from the complementary strands of DNA fragments. We describe a protocol in which libraries are constructed from single DNA strands in a three-step procedure: single-stranded ligation of the first adapter with T4 DNA ligase in the presence of a splinter oligonucleotide, copying of the DNA strand with a proofreading polymerase, and blunt-end ligation of the second double-stranded adapter with T4 DNA ligase.


Assuntos
DNA Antigo/análise , DNA de Cadeia Simples/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , DNA Antigo/química , DNA Antigo/isolamento & purificação
13.
Biosens Bioelectron ; 130: 1-19, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30716589

RESUMO

Mucin 1 protein (MUC1) is a membrane-associated glycoprotein overexpressed in the majority of human malignancies and considered as a predominant protein biomarker in cancers. Owing to the crucial role of MUC1 in cancer dissemination and metastasis, detection and quantification of this biomarker is of great importance in clinical diagnostics. Today, there exist a wide variety of strategies for the determination of various types of disease biomarkers, especially MUC1. In this regard, aptamers, as artificial single-stranded DNA or RNA oligonucleotides with catalytic and receptor properties, have drawn lots of attention for the development of biosensing platforms. So far, various sensitivity-enhancement techniques in combination with a broad range of smart nanomaterials have integrated into the design of novel aptamer-based biosensors (aptasensors) to improve detection limit and sensitivity of analyte determination. This review article provides a brief classification and description of the research progresses of aptamer-based biosensors and nanobiosensors for the detection and quantitative determination of MUC1 based on optical and electrochemical platforms.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Mucina-1/isolamento & purificação , Neoplasias/diagnóstico , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Mucina-1/química , Mucina-1/genética , Nanoestruturas/química
14.
Mol Cell ; 73(5): 900-914.e9, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733119

RESUMO

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Cromossomos Fúngicos , DNA Helicases/genética , DNA Fúngico/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mitose , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
15.
Molecules ; 24(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704053

RESUMO

The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein⁻DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.


Assuntos
DNA/química , Imagem Individual de Molécula , Fenômenos Biofísicos , DNA/genética , DNA/ultraestrutura , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência , Polímeros , Análise de Sequência de DNA , Imagem Individual de Molécula/métodos
16.
DNA Repair (Amst) ; 75: 18-28, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710866

RESUMO

A missense mutation in C. elegans RAD-54, a homolog of RAD54 that operates in the homologous recombination (HR) pathway, was found to decrease ATPase activity in vitro. The hypomorphic mutation caused hypersensitivity of C. elegans germ cells to double-strand DNA breaks (DSBs). Although the formation of RAD-51 foci at DSBs was normal in both the mutant and knockdown worms, their subsequent dissipation was slow. The rad-54-deficient phenotypes were greatly aggravated when combined with an xpf-1 mutation, suggesting a conservative role of single-strand annealing (SSA) for DSB repair in HR-defective worms. The phenotypes of doubly-deficient rad-54;xpf-1 worms were partially suppressed by a mutation of lig-4, a nonhomologous end-joining (NHEJ) factor. In summary, RAD-54 is required for the dissociation of RAD-51 from DSB sites in C. elegans germ cells. Also, NHEJ and SSA exert negative and positive effects, respectively, on genome stability when HR is defective.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Células Germinativas/metabolismo , Recombinação Homóloga , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA de Cadeia Simples/genética , Mutação
17.
Nat Commun ; 10(1): 944, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808869

RESUMO

The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal - and therefore the cell's DNA damage load - is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection to induce quantitatively different ssDNA signals at a site-specific double strand break in budding yeast and identify two distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is unresponsive to increased amounts of ssDNA, while the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. The global checkpoint signal critically depends on the 9-1-1 and its downstream acting signalling axis, suggesting that ssDNA quantification depends on at least two sensor complexes.


Assuntos
Dano ao DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Saccharomyces cerevisiae/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , DNA de Cadeia Simples/genética , Histonas/metabolismo , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
18.
J Hum Genet ; 64(5): 459-466, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30796324

RESUMO

Recent findings have highlighted the possibility that polymorphisms within the annexin A5 gene (ANXA5) promoter contribute to the etiology of various obstetric complications. However, the underlying mechanisms are unknown. The M2 haplotype of the ANXA5 shows lower activity and less expression of ANXA5 mRNA. This gene promoter region has a motif that potentially forms a G-quadruplex structure. In vitro G-quadruplex propensity estimated by circular dichroism indicated that the M2 haplotype oligonucleotide manifested a decreased potential for G-quadruplex formation. In addition, in vivo G-quadruplex formation of the promoter region was evidenced by the presence of single-stranded DNA shown by sodium bisulfite treatment of placental genomic DNA. Comparative analysis indicated less potential in the M2 allele than the major allele. Promoter activity of the two haplotypes determined by luciferase reporter analysis correlated with the estimated G-quadruplex propensity. Our data lend support to the developing paradigm that genomic variation affects gene expression levels via DNA secondary structures leading to the disease susceptibility.


Assuntos
Anexina A5 , Quadruplex G , Regulação da Expressão Gênica/fisiologia , Polimorfismo Genético , Complicações na Gravidez , Regiões Promotoras Genéticas , Anexina A5/biossíntese , Anexina A5/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Feminino , Haplótipos , Humanos , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia
19.
Mikrochim Acta ; 186(3): 176, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30771011

RESUMO

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.


Assuntos
Trifosfato de Adenosina/sangue , Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Catalítico/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Cor , Sondas de DNA/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Ouro/química , Humanos , Limite de Detecção , Magnésio/química , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico
20.
Appl Microbiol Biotechnol ; 103(6): 2783-2795, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30762073

RESUMO

Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redß and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.


Assuntos
Sistemas CRISPR-Cas , DNA de Cadeia Simples/genética , Edição de Genes/métodos , Genoma Bacteriano , Pseudomonas putida/genética , Biotecnologia , Proteína 9 Associada à CRISPR/metabolismo , Deleção de Genes , Plasmídeos/genética , Mutação Puntual
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