RESUMEN
Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.
Asunto(s)
Proteínas Argonautas , Proteínas Bacterianas , Plásmidos , Vibrio cholerae , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteínas Argonautas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/ultraestructura , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Modelos Moleculares , Plásmidos/genética , Plásmidos/inmunología , Plásmidos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Vibrio cholerae/genética , Vibrio cholerae/inmunología , Vibrio cholerae/patogenicidadRESUMEN
Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.
Asunto(s)
Proteínas Argonautas , Proteínas Bacterianas , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/química , Plásmidos/genética , Plásmidos/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , ADN/metabolismo , ADN/genéticaRESUMEN
TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.
Asunto(s)
Biopelículas , Desoxirribonucleasas , Planarias , Animales , Planarias/genética , Planarias/fisiología , Planarias/enzimología , Biopelículas/efectos de los fármacos , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/farmacología , Secuencia de AminoácidosRESUMEN
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Asunto(s)
Bacterias , Proteínas Bacterianas , Bacteriófagos , Evasión Inmune , Multimerización de Proteína , Bacterias/genética , Bacterias/inmunología , Bacterias/metabolismo , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Bacteriófagos/genética , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Desoxirribonucleasas/ultraestructura , ADN Viral/química , ADN Viral/metabolismo , ADN Viral/ultraestructuraRESUMEN
BACKGROUND: The Epstein-Barr virus (EBV) is a prevalent oncovirus associated with a variety of human illnesses. BGLF5, an EBV DNase with alkaline nuclease (AN) activity, plays important roles in the viral life cycle and progression of human malignancies and has been suggested as a possible diagnostic marker and target for cancer therapy. Methods used conventionally for the detection of AN activity, radioactivity-based nuclease activity assay and DNA digestion detection by gel electrophoresis, are not suitable for screening AN inhibitors; the former approach is unsafe, and the latter is complicated. In the present study, a fluorescence-based nuclease activity assay was used to screen several natural compounds and identify an EBV DNase inhibitor. RESULTS: Fluorescence-based nuclease activity assays, in which the DNA substrate is labelled with PicoGreen dye, are cheaper, safer, and easier to perform. Herein, the results of the fluorescence-based nuclease activity assay were consistent with the results of the two conventional methods. In addition, the PicoGreen-labelling method was applied for the biochemical characterisation of viral nucleases. Using this approach, we explored EBV DNase inhibitors. After several rounds of screening, emodin, an anthraquinone derivative, was found to possess significant anti-EBV DNase activity. We verified the efficacy of emodin using the conventional DNA-cleavage assay. Furthermore, using comet assay and micronucleus formation detection, we confirmed that emodin can inhibit DNase-induced DNA damage and genomic instability. Additionally, emodin treatment inhibited EBV production. CONCLUSIONS: Using a PicoGreen-mediated nuclease activity assay, we successfully demonstrated that emodin has the potential to inhibit EBV DNase nuclease activity. Emodin also inhibits EBV DNase-related biological functions, suggesting that it is a potential inhibitor of EBV DNase.
Asunto(s)
Emodina , Infecciones por Virus de Epstein-Barr , Humanos , Emodina/farmacología , Herpesvirus Humano 4/genética , ADN , Desoxirribonucleasas/química , Desoxirribonucleasas/genéticaRESUMEN
Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.
Asunto(s)
Desoxirribonucleasas , Ectothiorhodospiraceae , Desoxirribonucleasas/química , Tolerancia a la Sal , ADN/química , ADN/genética , Dominios Proteicos , Bacterias/genética , Ectothiorhodospiraceae/genéticaRESUMEN
Nucleases are ubiquitous hydrolytic enzymes that cleave phosphodiester bond of DNA (DNases), RNA (RNases), or protein-RNA/DNA (phosphodiesterases), within the strand (endonucleases) or from the end (exonucleases) [...].
Asunto(s)
Desoxirribonucleasas , Endonucleasas , Desoxirribonucleasas/química , Hidrolasas Diéster Fosfóricas , ADN/química , ARN/químicaRESUMEN
(1) Double-stranded DNA (dsDNA) and deoxyribonuclease (DNase) as surrogate parameters for accumulating inflammatory hazards are insufficiently studied in resuscitation research. (2) Blood samples of 76 individuals after CA were analyzed 24 and 96 h after ICU admission. Plasma levels of dsDNA, interleukin-8, and monocyte chemoattractant protein-1 and activity of DNase were assessed along with baseline characteristics, intensive care measures, and outcome data. DsDNA/DNase ratio was used as main prognostication parameter. After calculating an optimal empirical cut-off for outcome prediction (death or Cerebral Performance Category ≥3 at 6 months), multivariable logistic regression was applied. (3) Using receiver operating characteristic (ROC) analysis, an area under the curve (AUC) of 0.65 (95% CI 0.50-0.79) was found for dsDNA/DNase after 24 h versus 0.83 (95% CI 0.73-0.92) after 96 h (p = 0.03). The empirical cut-off for dsDNA/DNase ratio after 96 h was 149.97 (Youden). DsDNA/DNase ratio was associated with unfavorable outcome at six months (aOR 1.006, 95% CI 1.0017-1.0094, p = 0.005). In multivariable analysis, the association of dsDNA/DNase ratio independently predicted outcome as a continuous variable (aOR 1.004, 95% CI 1.0004-1.0079, p = 0.029) after adjusting for potential confounders. (4) DsDNA/DNase ratio at 96 h demonstrates good predictive performance for estimating outcome after CA.
Asunto(s)
ADN , Desoxirribonucleasas , Paro Cardíaco , Humanos , Desoxirribonucleasas/sangre , Desoxirribonucleasas/química , ADN/sangre , ADN/química , Paro Cardíaco/diagnóstico , Valor Predictivo de las Pruebas , Resucitación , PronósticoRESUMEN
This study presents the preparation of a novel tetra-substituted phthalonitrile (1), namely, 3,6-bis(hexyloxy)-4,5-bis(4-(trifluoromethoxy)phenoxy)phthalonitrile (1) and its metal-free (2)/metal {M = Zn (3), Cu (4), Co (5), Lu(CH3COO) (6), Lu (7)} phthalocyanines. A series of various spectroscopic methods (UV-vis, FT-IR, mass, and 1H NMR spectroscopy) were performed for the characterization of the newly synthesized compounds. The potential of compounds 2, 3, and 6 as photosensitizing materials for photodynamic and sonophotodynamic therapies was evaluated by photophysical, photochemical, and sonochemical methods. The highest singlet quantum yields were obtained for the zinc phthalocyanine derivative 3 by performing photochemical and sonochemical methods. In addition, several biological activities of the new compounds 1-7 were investigated. The newly synthesized phthalocyanines exhibited excellent DPPH scavenging activity and also DNA nuclease activity. The antimicrobial activity of the new compounds was evaluated by the disc diffusion assay. Effective microbial cell viability inhibition was observed with phthalocyanine macromolecules. The photodynamic antimicrobial therapy of the phthalocyanines showed 100% bacterial inhibition when compared to the control. They also exhibited significant biofilm inhibition activity against S. aureus and P. aeruginosa. These results indicate that new phthalocyanines are promising photodynamic antimicrobial therapies for the treatment of infectious diseases.
Asunto(s)
Antiinfecciosos/farmacología , Antioxidantes/farmacología , Isoindoles/farmacología , Metales/farmacología , Fármacos Fotosensibilizantes/farmacología , Antiinfecciosos/química , Antioxidantes/química , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Compuestos de Bifenilo/química , Candida parapsilosis/efectos de los fármacos , Candida parapsilosis/crecimiento & desarrollo , Candida tropicalis/efectos de los fármacos , Candida tropicalis/crecimiento & desarrollo , Desoxirribonucleasas/química , Halogenación , Isoindoles/química , Metales/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Picratos/química , Oxígeno Singlete/químicaRESUMEN
The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we demonstrate that a trinuclear coordination complex based on a dinuclear Fe(III)Zn(II) core designed for biomimicry of the hydrolytic enzyme kidney bean purple acid phosphatase, containing an additional pendant arm coordinating a Pd(II) ion, has the ability to interact with DNA and to promote its hydrolytic cleavage. These results were found through analysis of plasmid DNA interaction and cleavage by the trinuclear complex 1 and its derivatives 2 and 3, in addition to the analysis of alteration in the DNA structure in the presence of the complexes through circular dichroism and DNA footprinting techniques. The suggested covalent interaction of the palladium-containing complex with DNA was analysed using an electrophoretic mobility assay, circular dichroism, high resolution gel separation techniques and kinetic analysis. This is a new and promising metal complex targeted to nucleic acids and acting in two separate ways: strong DNA interaction and hydrolytic cleavage.
Asunto(s)
Complejos de Coordinación/química , División del ADN , Desoxirribonucleasas/química , Metales/química , Plásmidos/químicaRESUMEN
Polymer nanoparticle hydrogels made of deoxyribonucleic acid and silica have been prepared and shown to display shear thinning and self-healing properties, sustained release of cargo and enzymatic degradation.
Asunto(s)
ADN/química , Hidrogeles/química , Nanopartículas/química , Polímeros/química , ADN/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Hidrogeles/metabolismo , Nanopartículas/metabolismo , Polímeros/metabolismo , Dióxido de Silicio/química , Dióxido de Silicio/metabolismoRESUMEN
DNase is a powerful tool for a series of molecular biology applications. Developing a strategy for large-scale production of DNase with high purity and activity is critical for scientific research. In this study, a previously uncharacterized gene with nuclease activity was found in Trichogramma pretiosum genome. Pichia pastoris GS115 was preferred as the host to overcome the issues related to prokaryotic expression. Under the optimal conditions, the activity of T. pretiosum DNase (Tp-DNase) reached 1940 U/mL of culture supernatant in fed-batch fermentation. Using ion-exchange chromatography and adsorption chromatography, Tp-DNase was produced with a purity of >99% and molecular weight of 45 kDa. In vitro DNA degradation experiments showed that Tp-DNase could effectively degrade dsDNA, and its activity was slightly higher than that of bovine pancreas DNase I under the same conditions. Moreover, Tp-DNase can be used to eliminate nucleic acid contamination and improve the accuracy of nucleic acid detection.
Asunto(s)
ADN/química , Desoxirribonucleasas/química , Himenópteros/química , Proteínas de Insectos/química , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Animales , Cromatografía por Intercambio Iónico , Clonación Molecular , ADN/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Expresión Génica , Himenópteros/enzimología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Cinética , Peso Molecular , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.
Asunto(s)
Proteínas Bacterianas/química , Desoxirribonucleasas/química , Staphylococcus aureus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalización , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Bacteriano/genética , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Conformación Proteica , Dominios Proteicos , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMEN
In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Sistemas CRISPR-Cas/inmunología , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas/metabolismo , Endorribonucleasas/metabolismo , Oligorribonucleótidos/metabolismo , ARN/metabolismo , Staphylococcus/enzimología , Staphylococcus/inmunología , Nucleótidos de Adenina/inmunología , Adenosina Trifosfato/metabolismo , Bacteriófagos/inmunología , Bacteriófagos/fisiología , Biocatálisis , Dominio Catalítico , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Endorribonucleasas/química , Endorribonucleasas/genética , Activación Enzimática , Ligandos , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Oligorribonucleótidos/inmunología , Plásmidos/genética , Plásmidos/metabolismo , Multimerización de Proteína , Rotación , Staphylococcus/crecimiento & desarrollo , Staphylococcus/virología , Especificidad por SustratoRESUMEN
The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.
Asunto(s)
Proteínas Bacterianas/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Desoxirribonucleasas/química , Proteínas de Escherichia coli/química , Exonucleasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo Celular , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Humanos , Hidrólisis , Proteína Homóloga de MRE11/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Zinc/químicaRESUMEN
Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds-ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3'-overhang while Bax1 interacts with the 5'-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.
Asunto(s)
ADN Helicasas/química , Reparación del ADN , Proteínas de Unión al ADN/química , ADN/química , Desoxirribonucleasas/química , Disparidad de Par Base , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Sulfolobaceae/enzimología , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismoRESUMEN
Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3',5'-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein-ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein-ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction "route" on the free energy surface, which was consistent with a flux analysis.
Asunto(s)
Proteínas Bacterianas/química , Desoxirribonucleasas/química , Staphylococcus/enzimología , Proteínas Bacterianas/metabolismo , Desoxirribonucleasas/metabolismo , Cinética , Ligandos , Simulación de Dinámica Molecular , Staphylococcus/químicaRESUMEN
In this study, the new N, N, O tridentate donor water soluble isoniazid based biopolymer Schiff base ligand and their Co (II), Cu (II), Zn (II) metal complexes were prepared. The compounds were designed for potential biological application such as antibacterial, antifungal, anti-inflammatory, total antioxidant, antidiabetic and DNA binding studies. The synthesized compounds were illuminated in different light sources of various spectra were used to explore the functional groups of Biopolymer derivatives. Thermal degradation, thermal stability and percentage of mass loss for the prepared compounds were investigated through thermo gravimetric and differential thermal (TGA-DTA) analyses. Crystalline structure of synthesized biopolymer derivatives were explored by X-ray diffraction (XRD) studies, the crystallinity of chitosan is gradually decreased after the Schiff base and complex formation. Surface morphology and structures of the prepared compounds were examined using SEM analysis. The magnetic moment and magnetism of the metal complexes were studied using Vibrating-sample magnetometer (VSM). Antidiabetic studies of Biopolymer Schiff base and metal complexes were carried out by α-amylose inhibitory method. DNA nuclease activities of synthesized metal complexes were investigated by Ultra-Violet (UV) and viscometry methods. The Cu (II) complexes showed better DNA binding results than Co (II) and Zn (II) complexes.
Asunto(s)
Quitosano/análogos & derivados , Cobalto/química , Complejos de Coordinación/química , Cobre/química , Isoniazida/química , Bases de Schiff/química , Zinc/química , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Fenómenos Químicos , Técnicas de Química Sintética , Quitosano/química , Desoxirribonucleasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ligandos , Porosidad , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Análisis Espectral , Termogravimetría , Difracción de Rayos X , alfa-Amilasas/químicaRESUMEN
Molecular computing offers a powerful framework for in situ biosensing and signal processing at the nanoscale. However, for in vivo applications, the use of conventional DNA components can lead to false positive signals being generated due to degradation of circuit components by nuclease enzymes. Here, we use hybrid chiral molecules, consisting of both l- and d-nucleic acid domains, to implement leakless signal translators that enable d-nucleic acid signals to be detected by hybridization and then translated into a robust l-DNA signal for further analysis. We show that our system is robust to false positive signals even if the d-DNA components are degraded by nucleases, thanks to circuit-level robustness. This work thus broadens the scope and applicability of DNA-based molecular computers for practical, in vivo applications.
Asunto(s)
Computadores Moleculares , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Animales , Secuencia de Bases , Bovinos , Medios de Cultivo/química , Fragmentación del ADN , Desoxirribonucleasas/química , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Biosíntesis de Proteínas , Recombinación Genética , Albúmina Sérica BovinaRESUMEN
The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3'-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3'-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.