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1.
Anticancer Res ; 40(4): 2025-2032, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32234893

RESUMEN

BACKGROUND/AIM: The winemaking procedure results in the generation of stems, a by-product that is harmful to the environment. Concomitantly, stems are rich in polyphenols and, hence, they are putatively beneficial for human health. MATERIALS AND METHODS: In this study, the grape stem extracts derived from three native Greek vine varieties, namely Mavrodaphne, Muscat and Rhoditis were examined for their chemical composition and antioxidant and antimutagenic properties using a battery of in vitro biomarkers. RESULTS: All extracts are rich in polyphenols. Moreover, they exhibit potent antioxidant and antimutagenic properties with the extract of Mavrodaphne being the strongest in reducing the DPPH• and O2 -• radicals and the Fe3+ and in protecting plasmid DNA from peroxyl radical-induced oxidative modification. CONCLUSION: Therefore, although they are serious pollutants, grape stems contain phytochemicals with important biological properties and can be used as (ingredients of) bio-functional foods to improve certain aspects of human health.


Asunto(s)
Antioxidantes/farmacología , ADN/efectos de los fármacos , Polifenoles/farmacología , Vitis/química , ADN/metabolismo , Daño del ADN/efectos de los fármacos , Grecia , Humanos , Oxidación-Reducción , Extractos Vegetales/química , Extractos Vegetales/farmacología , Tallos de la Planta/química , Plásmidos/efectos de los fármacos , Plásmidos/metabolismo , Polifenoles/química , Especies Reactivas de Oxígeno/metabolismo
2.
Chem Biol Interact ; 323: 109030, 2020 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-32205154

RESUMEN

The review summarizes literature data on the DNA-binding, DNA-protecting and DNA-damaging activities of a range of natural human endogenous and exogenous compounds. Small natural organic molecules bind DNA in a site-specific mode, by arranging tight touch with the structure of the major and minor grooves, as well as individual bases in the local duplex DNA. Polyphenols are the best-studied exogenous compounds from this point of view. Many of them demonstrate hormetic effects, producing both beneficial and damaging effects. An attempt to establish the dependence of DNA damage or DNA protection on the concentration of the compound turned out to be successful for some polyphenols, daidzein, genistein and resveratrol, which were DNA protecting in low concentrations and DNA damaging in high concentrations. There was no evident dependence on concentration for quercetin and kaempferol. Probably, the DNA-protecting effect is associated with the affinity to DNA. Caffeine and theophylline are DNA binders; at the same time, they favor DNA repair. Although most alkaloids damage DNA, berberine can protect DNA against damage. Among the endogenous compounds, hormones belonging to the amine class, thyroid and steroid hormones appear to bind DNA and produce some DNA damage. Thus, natural compounds continue to reveal beneficial or adverse effects on genome integrity and provide a promising source of therapeutic activities.


Asunto(s)
Productos Biológicos/metabolismo , Reparación del ADN , ADN/metabolismo , Alimentos , Compuestos Orgánicos/metabolismo , Productos Biológicos/química , Hormonas/metabolismo , Compuestos Orgánicos/química
3.
Phys Chem Chem Phys ; 22(13): 6984-6992, 2020 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-32188961

RESUMEN

Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.


Asunto(s)
Aminoácidos Diaminos/metabolismo , Daño del ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Rayos Ultravioleta , ADN/química , Plásmidos/química , Plásmidos/metabolismo , Plásmidos/efectos de la radiación , Dispersión del Ángulo Pequeño , Resonancia por Plasmón de Superficie , Difracción de Rayos X
4.
BMC Bioinformatics ; 21(1): 96, 2020 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-32131723

RESUMEN

BACKGROUND: Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost-sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are thrown away. RESULTS: In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows "reuniting" these reads with their respective families increasing the output of the method and making it more cost effective. CONCLUSIONS: We combine an error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0. It is written in Python, C, AWK, and Bash. It is open source and readily available through Galaxy, Bioconda, and Github: https://github.com/galaxyproject/dunovo.


Asunto(s)
Interfaz Usuario-Computador , Algoritmos , ADN/química , ADN/metabolismo , Humanos , Alineación de Secuencia , Análisis de Secuencia de ADN
5.
Chem Commun (Camb) ; 56(16): 2379-2390, 2020 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-32022004

RESUMEN

DNA forms not only the canonical duplex structure but also non-canonical structures. Most potential sequences that induce the formation of non-canonical structures are present in disease-related genes. Interestingly, biological reactions are inhibited or dysregulated by non-canonical structure formation in disease-related genes. To control biological reactions, methods for inducing the formation of non-canonical structures have been developed using small molecules and oligonucleotides. In this feature article, we review biological reactions such as replication, transcription, and reverse transcription controlled by non-canonical DNA structures formed by disease-related genes. Furthermore, we discuss recent studies aimed at developing methods for regulating these biological reactions using drugs targeting the DNA structure.


Asunto(s)
ADN/antagonistas & inhibidores , Preparaciones Farmacéuticas/química , Animales , ADN/química , ADN/metabolismo , Humanos , Conformación de Ácido Nucleico
6.
Mutat Res ; 849: 503128, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32087849

RESUMEN

A physiological decrease in extracellular pH (pHe) alters the efficiency of DNA repair and increases formation of DNA double-strand breaks (DSBs). Whether this could translate into genetic instability and variations, was investigated using the TK6 cell model, in which positive selection of the TK1 gene loss-of-function mutations can be achieved from resistance to trifluorothymidine. Cell exposure to suboptimal pH (down to 6.9) for 3 weeks resulted in the 100 % frequency of a stronger frameshift mutation that has spread to both TK1 alleles, whereas weaker frameshift mutations within the 3'exon were eliminated during the selection. Suboptimal pHe values were also found to alter the proportion of the TK1 splicing variant expressed as percent spliced in index values and promote selection of truncated exons as well as intron retention. Although recovery at pH 7.4 did not reverse the selected frameshift mutation, reversal of splice variants and exon truncation towards control values were observed. Hence, suboptimal pHe can induce a combination of mutational events and splicing alterations within the same gene in the resistant clones. This model of positive selection for loss-of-function clearly demonstrates that suboptimal pHe may confer a similar growth advantage when such instability occurs within tumor suppressor genes.


Asunto(s)
Empalme Alternativo , Mutación del Sistema de Lectura , Linfocitos/metabolismo , Modelos Genéticos , Mutagénesis , Timidina Quinasa/genética , Antimetabolitos/farmacología , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , Exones , Espacio Extracelular/química , Espacio Extracelular/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Intrones , Linfocitos/citología , Linfocitos/efectos de los fármacos , Timidina Quinasa/metabolismo , Trifluridina/farmacología
7.
Mutat Res ; 849: 503129, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32087850

RESUMEN

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.


Asunto(s)
Linfocitos/efectos de los fármacos , Mutagénesis , Pruebas de Mutagenicidad/normas , Mutación , Timidina Quinasa/genética , 4-Nitroquinolina-1-Óxido/toxicidad , Benzo(a)pireno/toxicidad , Línea Celular Tumoral , Ciclofosfamida/toxicidad , ADN/genética , ADN/metabolismo , Daño del ADN , Diclofenaco/toxicidad , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Regulación de la Expresión Génica , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Metilmetanosulfonato/toxicidad , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Mitomicina/toxicidad , Timidina Quinasa/metabolismo , Vinblastina/toxicidad
8.
Mutat Res ; 849: 503141, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32087855

RESUMEN

The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G0 lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.


Asunto(s)
Radiación de Fondo/efectos adversos , Bioensayo , Reparación del ADN/efectos de los fármacos , Rayos gamma/efectos adversos , Linfocitos/efectos de la radiación , Adulto , Animales , Células CHO , Fusión Celular , Cromosomas Humanos/efectos de los fármacos , Cromosomas Humanos/efectos de la radiación , Cricetulus , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Masculino , Proyectos Piloto , Cultivo Primario de Células , Dosis de Radiación , Wortmanina/farmacología
9.
Nat Struct Mol Biol ; 27(2): 127-133, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32015553

RESUMEN

The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA-strand transfer complex of mouse RAG at 3.1-Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuates unwanted transposition. In contrast to strand transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Microscopía por Crioelectrón , ADN/química , División del ADN , Proteínas de Unión al ADN/química , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica
10.
Nat Struct Mol Biol ; 27(2): 119-126, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32015552

RESUMEN

A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Secuencia de Bases , Dominio Catalítico , Microscopía por Crioelectrón , ADN/química , Proteínas de Unión al ADN/química , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica
11.
Chem Commun (Camb) ; 56(19): 2901-2904, 2020 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-32037435

RESUMEN

The enzymatic-assisted signal amplification of DNA sensors is rarely applied in living cells due to the difficulties in protein delivery. In this study, we have proposed a biomineralization-based DNA nanoprobe to transport nucleases and DNA sensors for enzyme-assisted imaging of microRNA in living cells.


Asunto(s)
Biomineralización , Sondas de ADN/química , ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Nanopartículas/química , Humanos , Estructuras Metalorgánicas/química , MicroARNs/metabolismo
12.
Nat Struct Mol Biol ; 27(2): 109-118, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32042149

RESUMEN

Understanding how the genome is structurally organized as chromatin is essential for understanding its function. Here, we review recent developments that allowed the readdressing of old questions regarding the primary level of chromatin structure, the arrangement of nucleosomes along the DNA and the folding of the nucleosome fiber in nuclear space. In contrast to earlier views of nucleosome arrays as uniformly regular and folded, recent findings reveal heterogeneous array organization and diverse modes of folding. Local structure variations reflect a continuum of functional states characterized by differences in post-translational histone modifications, associated chromatin-interacting proteins and nucleosome-remodeling enzymes.


Asunto(s)
Cromatina/genética , ADN/genética , Nucleosomas/genética , Animales , Cromatina/metabolismo , ADN/metabolismo , Código de Histonas , Humanos , Nucleosomas/metabolismo , Regiones Promotoras Genéticas
13.
Nat Genet ; 52(2): 146-159, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32060489

RESUMEN

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.


Asunto(s)
Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Naftiridinas/farmacología , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Animales , Cuerpo Estriado/efectos de los fármacos , ADN/metabolismo , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Transgénicos , Inestabilidad de Microsatélites , Mutación , Ribonucleasas/metabolismo , Proteína de Unión a TATA-Box/genética , Transcripción Genética
14.
Top Curr Chem (Cham) ; 378(1): 20, 2020 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-32016608

RESUMEN

Nucleic acids are considered not only extraordinary carriers of genetic information but also are perceived as the perfect elemental materials of molecular recognition and signal transduction/amplification for assembling programmable artificial reaction networks or circuits, which are similar to conventional electronic logic devices. Among these sophisticated DNA-based reaction networks, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and DNAzyme represent the typical nonenzymatic amplification methods with high robustness and efficiency. Furthermore, their extensive hierarchically cascade integration into multi-layered autonomous DNA circuits establishes novel paradigms for constructing more different catalytic DNA nanostructures and for regenerating or replicating diverse molecular components with specific functions. Various DNA and inorganic nanoscaffolds have been used to realize the surface-confined DNA reaction networks with significant biomolecular sensing and signal-regulating functions in living cells. Especially, the specific aptamers and metal-ion-bridged duplex DNA nanostructures could extend their paradigms for detecting small molecules and proteins in even living entities. Herein, the varied enzyme-free DNA circuits are introduced in general with an extensive explanation of their underlying molecular reaction mechanisms. Challenges and outlook of the autonomous enzyme-free DNA circuits will also be discussed at the end of this chapter.


Asunto(s)
Técnicas Biosensibles/métodos , ADN/química , ADN/metabolismo , ADN Catalítico/química , ADN Catalítico/metabolismo , Entropía , Transferencia Resonante de Energía de Fluorescencia , Humanos , Microscopía Fluorescente , Nanoestructuras/química , Hibridación de Ácido Nucleico
15.
Nat Commun ; 11(1): 978, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-32080196

RESUMEN

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.


Asunto(s)
ADN/metabolismo , Transducción de Señal , Adenosina Trifosfato/metabolismo , Células Artificiales/química , Materiales Biomiméticos/química , Biomimética/métodos , Homeostasis , Nanoestructuras/química , Biología Sintética/métodos
16.
Biochemistry (Mosc) ; 85(2): 192-204, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32093595

RESUMEN

8-Oxoguanine-DNA N-glycosylase (OGG1) is a eukaryotic DNA repair enzyme responsible for the removal of 8-oxoguanine (oxoG), one of the most abundant oxidative DNA lesions. OGG1 catalyzes two successive reactions - N-glycosidic bond hydrolysis (glycosylase activity) and DNA strand cleavage on the 3'-side of the lesion by ß-elimination (lyase activity). The enzyme also exhibits lyase activity with substrates containing apurinic/apyrimidinic (AP) sites (deoxyribose moieties lacking the nucleobase). OGG1 is highly specific for the base opposite the lesion, efficiently excising oxoG and cleaving AP sites located opposite to C, but not opposite to A. The activity is also profoundly decreased by amino acid changes that sterically interfere with oxoG binding in the active site of the enzyme after the lesion is everted from the DNA duplex. Earlier, the molecular dynamics approach was used to study the conformational dynamics of such human OGG1 mutants in complexes with the oxoG:C-containing substrate DNA, and the population density of certain conformers of two OGG1 catalytic residues, Lys249 and Asp268, was suggested to determine the enzyme activity. Here, we report the study of molecular dynamics of human OGG1 bound to the oxoG:A-containing DNA and OGG1 mutants bound to the AP:C-containing DNA. We showed that the enzyme low activity is associated with a decrease in the populations of Lys249 and Asp268 properly configured for catalysis. The experimentally measured rate constants for the OGG1 mutants show a good agreement with the models. We conclude that the enzymatic activity of OGG1 is determined majorly by the population density of the catalytically competent conformations of the active site residues Lys249 and Asp268.


Asunto(s)
Biocatálisis , ADN Glicosilasas/metabolismo , Dominio Catalítico , ADN/química , ADN/metabolismo , ADN Glicosilasas/química , ADN Glicosilasas/genética , Reparación del ADN , Humanos , Simulación de Dinámica Molecular , Conformación Proteica
17.
Nat Struct Mol Biol ; 27(3): 240-248, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32066963

RESUMEN

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.


Asunto(s)
Reparación del ADN , ADN/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitina/química , Animales , Sitios de Unión , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Daño del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinación
18.
Biochemistry (Mosc) ; 85(2): 147-166, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32093592

RESUMEN

Recently, there has been a rapid progress in the development of techniques for isothermal amplification of nucleic acids as an alternative to polymerase chain reaction (PCR). The advantage of these methods is that the nucleic acids amplification can be carried out at constant temperature, unlike PCR, which requires cyclic temperature changes. Moreover, isothermal amplification can be conducted directly in living cells. This review describes the principles of isothermal amplification techniques and demonstrates their high efficiency in designing new highly sensitive detection methods of nucleic acids and enzymes involved in their modifications. The data on successful application of isothermal amplification methods for the analysis of cells and biomolecules with the use of DNA/RNA aptamers are presented.


Asunto(s)
ADN/análisis , ADN/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/análisis , ARN/genética , Temperatura , Animales , ADN/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , ARN/metabolismo
19.
Nat Commun ; 11(1): 908, 2020 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-32075966

RESUMEN

Cyclic cGMP-AMP synthase (cGAS) is a pattern recognition cytosolic DNA sensor that is essential for cellular senescence. cGAS promotes inflammatory senescence-associated secretory phenotype (SASP) through recognizing cytoplasmic chromatin during senescence. cGAS-mediated inflammation is essential for the antitumor effects of immune checkpoint blockade. However, the mechanism by which cGAS recognizes cytoplasmic chromatin is unknown. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP1cc) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence. TOP1cc localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.


Asunto(s)
Senescencia Celular/inmunología , ADN-Topoisomerasas de Tipo I/metabolismo , Proteína HMGB2/metabolismo , Nucleotidiltransferasas/metabolismo , Animales , Antígeno B7-H1/inmunología , Línea Celular , Cromatina/inmunología , Cromatina/metabolismo , Citosol/inmunología , Citosol/metabolismo , ADN/inmunología , ADN/metabolismo , Daño del ADN/inmunología , ADN-Topoisomerasas de Tipo I/genética , Técnicas de Silenciamiento del Gen , Proteína HMGB2/genética , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BL , Mutación , Neoplasias/inmunología , Nucleotidiltransferasas/genética , Unión Proteica , Ensayos Antitumor por Modelo de Xenoinjerto
20.
Nat Protoc ; 15(3): 840-876, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31969721

RESUMEN

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.


Asunto(s)
Cromosomas , Regulación del Desarrollo de la Expresión Génica/fisiología , Procesamiento de Imagen Asistido por Computador , Transcripción Genética/fisiología , Animales , Cromatina , ADN/química , ADN/genética , ADN/metabolismo , Drosophila/embriología , Colorantes Fluorescentes , Hibridación Fluorescente in Situ/métodos , ARN/química , ARN/genética , ARN/metabolismo
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