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1.
Nat Neurosci ; 23(6): 718-729, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367065

RESUMO

DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state-effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.


Assuntos
Adenosina Desaminase/metabolismo , Adenosina Desaminase/fisiologia , DNA Forma Z/fisiologia , Extinção Psicológica/fisiologia , Edição de RNA/fisiologia , Animais , DNA Forma Z/metabolismo , Medo , Aprendizagem/fisiologia , Camundongos , Córtex Pré-Frontal/metabolismo , RNA Interferente Pequeno/farmacologia
2.
J Photochem Photobiol B ; 198: 111580, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31394353

RESUMO

Heavy metal acclimation of bacteria is of particular interest in many aspects. It could add to our understanding of adaptation strategies applied by bacteria, as well as help us in devising ways to use such adaptive bacteria for bioremediation. In this study, we have explored the changes in the DNA of an aquatic Gordonia sp. acclimated to silver, cadmium, and lead. We have measured the changes in the DNA extracted from the acclimated bacteria by using ATR-FTIR coupled with unsupervised and supervised pattern recognition algorithms. Although whole-cell FTIR studies do reveal nucleic acid changes, the special care should be taken when considering marker nucleic acid bands in such spectra, as various other cell or tissue constituents also yield IR bands in the same region. An FTIR study on isolated DNA can be used to avoid this problem. The IR spectral profiles of the DNA molecules revealed significant changes in the backbone and sugar conformations of upon acclimation. We then further analyzed the DNA's global cytosine-methylation patterns of the heavy metal-acclimated bacteria. We aimed to find out whether epigenetic mechanisms operate in bacteria for survival and growth in inhibitory heavy metal concentrations or not. We found hypermethylation in Cd acclimation but hypomethylation for both Pb and Ag in Gordonia sp. Our results imply that changes in the conformational and methylation states of DNA seem to let bacteria to thrive in otherwise inhibitory conditions and mark the involvement of epigenetic modulation in acclimation processes.


Assuntos
Metilação de DNA , DNA Forma Z/química , Gordonia (Bactéria)/química , Metais Pesados/metabolismo , Açúcares/química , Cádmio/química , Cádmio/metabolismo , Cádmio/toxicidade , Análise por Conglomerados , Análise Discriminante , Gordonia (Bactéria)/efeitos dos fármacos , Gordonia (Bactéria)/metabolismo , Chumbo/química , Chumbo/metabolismo , Chumbo/toxicidade , Metais Pesados/química , Metais Pesados/toxicidade , Testes de Sensibilidade Microbiana , Análise de Componente Principal , Prata/química , Prata/metabolismo , Prata/toxicidade , Espectroscopia de Infravermelho com Transformada de Fourier
3.
Nucleic Acids Res ; 47(16): 8899-8912, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31361900

RESUMO

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.


Assuntos
Antibióticos Antineoplásicos/química , Cromomicina A3/química , DNA Forma Z/química , Dactinomicina/química , Substâncias Intercalantes/química , Oligodesoxirribonucleotídeos/química , Antibióticos Antineoplásicos/metabolismo , Pareamento Incorreto de Bases , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cromomicina A3/metabolismo , Cristalização , Cristalografia por Raios X , DNA Forma Z/metabolismo , Dactinomicina/metabolismo , Humanos , Substâncias Intercalantes/metabolismo , Modelos Moleculares , Oligodesoxirribonucleotídeos/síntese química , Soluções
4.
Int J Biol Macromol ; 137: 337-345, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247230

RESUMO

The most remarkable conformational transition in nature is the B-to-Z transition of DNA which not only contributes for epigenetic regulation but also is exploited to create several advanced nanomaterials for sensing and nanomechanics. The present communication focuses on the intrinsic factors that control the La3+/Ce3+-induced B-to-Z transition in self-assembled branched DNA (bDNA) nanostructures. The transition is sensitive even to two nucleotide change in the loop length and overhang sequences. Predominantly, bDNA structures having 3 T loop length are more sensitive towards helical switching than the 5 T bearing structures. Particularly, bDNA US-17, US-19 and US-23 having 3 T in the loop are showing B-Z transition in presence of LaCl3. Interestingly, with 'GATC' overhangs both La3+/Ce3+-induced B-to-Z transition was noticed in bDNA structures US-21 and US-22 (having 3 T and 5 T in the loop, respectively). The lanthanide-induced B-Z transition in bDNA is reversed with treatment of EDTA. Isothermal titration calorimetry (ITC) experiments show that the binding mode of lanthanide salts to bDNA followed an entropically and enthalpically favorable process. Further, for the first time ITC data suggests the B-to-Z transition in bDNA is a cooperative shift from exothermic to endothermic.


Assuntos
Sequência de Bases , DNA de Forma B/química , DNA Forma Z/química , Conformação de Ácido Nucleico , Fenômenos Biofísicos , Calorimetria , Dicroísmo Circular , Termodinâmica
5.
Chem Res Toxicol ; 32(5): 899-909, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-30821442

RESUMO

One response to oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in a gene promoter is regulation of mRNA expression suggesting an epigenetic-like role for OG. A proposed mechanism involves G oxidation within a potential G-quadruplex-forming sequence (PQS) in the promoter, enabling a structural shift from B-DNA to a G-quadruplex fold (G4). When OG was located in the coding vs template strand, base excision repair led to an on/off transcriptional switch. Herein, a G-rich, potential Z-DNA-forming sequence (PZS) comprised of a d(GC) n repeat was explored to determine whether oxidation in this motif was also a transcriptional switch. Bioinformatic analysis found 1650 PZSs of length >10 nts in the human genome that were overrepresented in promoters and 5'-UTRs. Studies in human cells transfected with a luciferase reporter plasmid in which OG was synthesized in a PZS context in the promoter found that a coding strand OG increased expression and a template strand OG decreased expression. The initial base excision repair product of OG, an abasic site (AP), was also found to yield similar expression changes as OG. Biophysical studies on model Z-DNA strands found OG favored a shift in the equilibrium to Z-DNA from B-DNA, while an AP disrupted Z-DNA to favor a hairpin, placing AP in the loop where it is a poor substrate for the endonuclease APE1. Overall, the impact of OG and AP in a PZS on gene expression was similar to that in a PQS but reduced in magnitude.


Assuntos
Adutos de DNA/metabolismo , DNA Forma Z/metabolismo , Expressão Gênica/fisiologia , Guanina/química , Regiões Promotoras Genéticas/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Adutos de DNA/química , Adutos de DNA/genética , DNA Forma Z/química , DNA Forma Z/genética , Quadruplex G , Genoma/fisiologia , Guanina/análogos & derivados , Humanos , Oxirredução , Estresse Oxidativo/genética
6.
Commun Biol ; 2: 7, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729177

RESUMO

Left-handed Z-DNA/Z-RNA is bound with high affinity by the Zα domain protein family that includes ADAR (a double-stranded RNA editing enzyme), ZBP1 and viral orthologs regulating innate immunity. Loss-of-function mutations in ADAR p150 allow persistent activation of the interferon system by Alu dsRNAs and are causal for Aicardi-Goutières Syndrome. Heterodimers of ADAR and DICER1 regulate the switch from RNA- to protein-centric immunity. Loss of DICER1 function produces age-related macular degeneration, a different type of Alu-mediated disease. The overlap of Z-forming sites with those for the signal recognition particle likely limits invasion of primate genomes by Alu retrotransposons.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , DNA Forma Z/genética , DNA Forma Z/metabolismo , Malformações do Sistema Nervoso/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Elementos Alu/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , RNA Helicases DEAD-box/genética , DNA Forma Z/química , Humanos , Mutação com Perda de Função , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética
7.
ACS Chem Biol ; 14(2): 245-255, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30592616

RESUMO

Human RNA editing enzyme ADAR1 deaminates adenosine in pre-mRNA to yield inosine. The Zα domain of human ADAR1 (hZαADAR1) binds specifically to left-handed Z-RNA as well as Z-DNA and stabilizes the Z-conformation. To answer the question of how hZαADAR1 can induce both the B-Z transition of DNA and the A-Z transition of RNA, we investigated the structure and dynamics of hZαADAR1 in complex with 6-base-pair Z-DNA or Z-RNA. We performed chemical shift perturbation and relaxation dispersion experiments on hZαADAR1 upon binding to Z-DNA as well as Z-RNA. Our study demonstrates the unique dynamics of hZαADAR1 during the A-Z transition of RNA, in which the hZαADAR1 protein forms a thermodynamically stable complex with Z-RNA, similar to Z-DNA, but kinetically converts RNA to the Z-form more slowly than DNA. We also discovered some distinct structural features of hZαADAR1 in the Z-RNA binding conformation. Our results suggest that the A-Z transition of RNA facilitated by hZαADAR1 displays unique structural and dynamic features that may be involved in targeting ADAR1 for a role in recognition of RNA substrates.


Assuntos
Adenosina Desaminase/química , DNA Forma Z/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Ligação a RNA/química , RNA/genética , Humanos
8.
Biochem Biophys Res Commun ; 508(4): 1215-1220, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30558789

RESUMO

The crystal structure of BZ-junction reveals that left-handed Z-DNA stabilized by Z-DNA binding domain (Zα) is continuously stacked to right-handed B-DNA with AT bases' extrusion in the junction site. However, this structure might not fully represent the BZ-junction in solution due to the possibility of the junction formation either by crystal packing or Zα interaction. Therefore, we investigated BZ-junction in solution with chemical Z-DNA inducers using CD and 2-aminopurine base-extrusion assay. We confirmed the formation of Z-DNA and BZ-junction with base-extrusion by chemical Z-DNA inducers. However, neither typical Z-DNA nor base-extrusion could be detected with some inducers such as spermine, suggesting that the energy barrier for the formation of the BZ junction might vary depending on the Z-DNA induction conditions.


Assuntos
DNA de Forma B/química , DNA Forma Z/química , Conformação de Ácido Nucleico , 2-Aminopurina/química , Temperatura Baixa , Temperatura Alta , Oligonucleotídeos/química
9.
Bioorg Med Chem ; 27(2): 364-369, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30545733

RESUMO

We synthesized several DNA oligonucleotides containing one or several 2'-O-methyl-8-methyl guanosine (m8Gm) and demonstrated that these oligonucleotides not only stabilize the Z-DNA with a wide range of sequences under low salt conditions but also possess high thermal stability. Using artificial nucleobase-containing oligonucleotides, we studied the interaction of the Zα domain with Z-DNA. Furthermore, we showed that the m8Gm-contained oligonucleotides allow to study the photochemical reaction of Z-DNA.


Assuntos
DNA Forma Z/química , Guanosina/análogos & derivados , Guanosina/química , Oligodesoxirribonucleotídeos/química , DNA Forma Z/síntese química , DNA Forma Z/metabolismo , DNA Forma Z/efeitos da radiação , Proteínas de Fluorescência Verde/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/efeitos da radiação , Oxirredução , Ligação Proteica , Temperatura de Transição , Raios Ultravioleta
11.
Molecules ; 23(11)2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30355979

RESUMO

Z-DNA is stabilized by various Z-DNA binding proteins (ZBPs) that play important roles in RNA editing, innate immune response, and viral infection. In this review, the structural and dynamics of various ZBPs complexed with Z-DNA are summarized to better understand the mechanisms by which ZBPs selectively recognize d(CG)-repeat DNA sequences in genomic DNA and efficiently convert them to left-handed Z-DNA to achieve their biological function. The intermolecular interaction of ZBPs with Z-DNA strands is mediated through a single continuous recognition surface which consists of an α3 helix and a ß-hairpin. In the ZBP-Z-DNA complexes, three identical, conserved residues (N173, Y177, and W195 in the Zα domain of human ADAR1) play central roles in the interaction with Z-DNA. ZBPs convert a 6-base DNA pair to a Z-form helix via the B-Z transition mechanism in which the ZBP first binds to B-DNA and then shifts the equilibrium from B-DNA to Z-DNA, a conformation that is then selectively stabilized by the additional binding of a second ZBP molecule. During B-Z transition, ZBPs selectively recognize the alternating d(CG)n sequence and convert it to a Z-form helix in long genomic DNA through multiple sequence discrimination steps. In addition, the intermediate complex formed by ZBPs and B-DNA, which is modulated by varying conditions, determines the degree of B-Z transition.


Assuntos
DNA Forma Z/química , Proteínas de Ligação a DNA/química , DNA/química , Modelos Moleculares , Termodinâmica , Algoritmos , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Conformação Proteica , Relação Estrutura-Atividade
12.
Sci Rep ; 8(1): 14828, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287873

RESUMO

Dimethyl sulfoxide (DMSO) is a small molecule with polar, aprotic and amphiphilic properties. It serves as a solvent for many polar and nonpolar molecules and continues to be one of the most used solvents (vehicle) in medical applications and scientific research. To better understand the cellular effects of DMSO within the concentration range commonly used as a vehicle (0.1-1.5%, v/v) for cellular treatments, we applied Attenuated Total Reflectance (ATR) Fourier Transform Infrared (FT-IR) spectroscopy to DMSO treated and untreated epithelial colon cancer cells. Both unsupervised (Principal Component Analysis-PCA) and supervised (Linear Discriminant Analysis-LDA) pattern recognition/modelling algorithms applied to the IR data revealed total segregation and prominent differences between DMSO treated and untreated cells at whole, lipid and nucleic acid regions. Several of these data were supported by other independent techniques. Further IR data analyses of macromolecular profile indicated comprehensive alterations especially in proteins and nucleic acids. Protein secondary structure analysis showed predominance of ß-sheet over α-helix in DMSO treated cells. We also observed for the first time, a reduction in nucleic acid level upon DMSO treatment accompanied by the formation of Z-DNA. Molecular docking and binding free energy studies indicated a stabilization of Z-DNA in the presence of DMSO. This alternate DNA form may be related with the specific actions of DMSO on gene expression, differentiation, and epigenetic alterations. Using analytical tools combined with molecular and cellular biology techniques, our data indicate that even at very low concentrations, DMSO induces a number of changes in all macromolecules, which may affect experimental outcomes where DMSO is used as a solvent.


Assuntos
Neoplasias do Colo/patologia , Dimetil Sulfóxido/metabolismo , Células Epiteliais/fisiologia , Algoritmos , Neoplasias do Colo/metabolismo , Simulação por Computador , DNA Forma Z/metabolismo , Células HCT116 , Humanos , Simulação de Acoplamento Molecular , Complexos Multiproteicos/metabolismo , Análise de Componente Principal , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Receptores de Reconhecimento de Padrão/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 10): 603-609, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279310

RESUMO

Crystals of left-handed Z-DNA [d(CGCGCG)]2 diffract X-rays to beyond 1 Šresolution, feature a small unit cell (∼18 × 31 × 44 Å) and are well hydrated, with around 90 water molecules surrounding the duplex in the asymmetric unit. The duplex shows regular hydration patterns in the narrow minor groove, on the convex surface and around sugar-phosphate backbones. Therefore, Z-DNA offers an ideal case to test the benefits of low-temperature neutron diffraction data collection to potentially determine the donor-acceptor patterns of first- and second-shell water molecules. Nucleic acid fragments pose challenges for neutron crystallography because water molecules are located on the surface rather than inside sequestered spaces such as protein active sites or channels. Water molecules can be expected to display dynamic behavior, particularly in cases where water is not part of an inner shell and directly coordinated to DNA atoms. Thus, nuclear density maps based on room-temperature diffraction data with a resolution of 1.6 Šdid not allow an unequivocal determination of the orientations of water molecules. Here, cryo-neutron diffraction data collection for a Z-DNA crystal on the Macromolecular Neutron Diffractometer at the Spallation Neutron Source at Oak Ridge National Laboratory and the outcome of an initial refinement of the structure are reported. A total of 12 diffraction images were recorded with an exposure time of 3.5 h per image, whereby the crystal was static for each diffraction image with a 10° ϕ rotation between images. Initial refinements using these neutron data indicated the positions and orientations of 30 water molecules within the first hydration shell of the DNA molecule. This experiment constitutes a state-of-the-art approach and is the first attempt to our knowledge to determine the low-temperature neutron structure of a DNA crystal.


Assuntos
DNA Forma Z/química , Água/química , Temperatura Baixa , Cristalização , Cristalografia , Ligação de Hidrogênio , Difração de Nêutrons
14.
Nucleic Acids Res ; 46(22): 11806-11821, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30304469

RESUMO

The Z-DNA binding domain (Zα), derived from the human RNA editing enzyme ADAR1, can induce and stabilize the Z-DNA conformation. However, the biological function of Zα/Z-DNA remains elusive. Herein, we sought to identify proteins associated with Zα to gain insight into the functional network of Zα/Z-DNA. By pull-down, biophysical and biochemical analyses, we identified a novel Zα-interacting protein, MBD3, and revealed that Zα interacted with its C-terminal acidic region, an aspartate (D)/glutamate (E)-rich domain, with high affinity. The D/E-rich domain of MBD3 may act as a DNA mimic to compete with Z-DNA for binding to Zα. Dimerization of MBD3 via intermolecular interaction of the D/E-rich domain and its N-terminal DNA binding domain, a methyl-CpG-binding domain (MBD), attenuated the high affinity interaction of Zα and the D/E-rich domain. By monitoring the conformation transition of DNA, we found that Zα could compete with the MBD domain for binding to the Z-DNA forming sequence, but not vice versa. Furthermore, co-immunoprecipitation experiments confirmed the interaction of MBD3 and ADAR1 in vivo. Our findings suggest that the interplay of Zα and MBD3 may regulate the transition of the DNA conformation between B- and Z-DNA and thereby modulate chromatin accessibility, resulting in alterations in gene expression.


Assuntos
Adenosina Desaminase/química , DNA Forma Z/química , Proteínas de Ligação a DNA/química , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/química , Sítios de Ligação , Bioquímica , Ilhas de CpG , Reagentes para Ligações Cruzadas/química , DNA/química , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
15.
Artigo em Inglês | MEDLINE | ID: mdl-30188765

RESUMO

Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (mC) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5'-thiol oligonucleotide embedded with 5'-mCGmCGmCGmCGm CG-3' was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.


Assuntos
5-Metilcitosina/química , DNA Forma Z/química , Proteínas de Ligação a DNA/análise , Técnicas Biossensoriais/métodos , Ligação Proteica
16.
Nucleic Acids Res ; 46(19): 10504-10513, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30184200

RESUMO

BZ junctions, which connect B-DNA to Z-DNA, are necessary for local transformation of B-DNA to Z-DNA in the genome. However, the limited information on the junction-forming sequences and junction structures has led to a lack of understanding of the structural diversity and sequence preferences of BZ junctions. We determined three crystal structures of BZ junctions with diverse sequences followed by spectroscopic validation of DNA conformation. The structural features of the BZ junctions were well conserved regardless of sequences via the continuous base stacking through B-to-Z DNA with A-T base extrusion. However, the sequence-dependent structural heterogeneity of the junctions was also observed in base step parameters that are correlated with steric constraints imposed during Z-DNA formation. Further, circular dichroism and fluorescence-based analysis of BZ junctions revealed that a base extrusion was only found at the A-T base pair present next to a stable dinucleotide Z-DNA unit. Our findings suggest that Z-DNA formation in the genome is influenced by the sequence preference for BZ junctions.


Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , DNA/química , Conformação de Ácido Nucleico , Domínios Proteicos , Proteínas de Ligação a RNA/química , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Pareamento de Bases , Sequência de Bases , Dicroísmo Circular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA de Forma B/genética , DNA de Forma B/metabolismo , DNA Forma Z/genética , DNA Forma Z/metabolismo , Humanos , Modelos Moleculares , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
17.
J Vet Sci ; 19(6): 759-770, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30173491

RESUMO

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.


Assuntos
Infecções por Adenoviridae/veterinária , Proteínas do Capsídeo/genética , Adenovirus A das Aves , Infecções por Adenoviridae/virologia , Animais , Embrião de Galinha/virologia , Galinhas/virologia , Clonagem Molecular , DNA Forma Z/genética , Adenovirus A das Aves/genética , Adenovirus A das Aves/isolamento & purificação , Adenovirus A das Aves/patogenicidade , Fígado/patologia , Fígado/virologia , Malásia , Filogenia , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/virologia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA , Organismos Livres de Patógenos Específicos
18.
Int J Mol Sci ; 19(4)2018 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-29617273

RESUMO

Recognition of unusual left-handed Z-DNA by specific binding of small molecules is crucial for understanding biological functions in which this particular structure participates. Recent investigations indicate that zinc cationic porphyrin (ZnTMPyP4) is promising as a probe for recognizing Z-DNA due to its characteristic chiroptical properties upon binding with Z-DNA. However, binding mechanisms of the ZnTMPyP4/Z-DNA complex remain unclear. By employing time-resolved UV-visible absorption spectroscopy in conjunction with induced circular dichroism (ICD), UV-vis, and fluorescence measurements, we examined the binding interactions of ZnTMPyP4 towards B-DNA and Z-DNA. For the ZnTMPyP4/Z-DNA complex, two coexisting binding modes were identified as the electrostatic interaction between pyridyl groups and phosphate backbones, and the major groove binding by zinc(II) coordinating with the exposed guanine N7. The respective contribution of each mode is assessed, allowing a complete scenario of binding modes revealed for the ZnTMPyP4/Z-DNA. These interaction modes are quite different from those (intercalation and partial intercalation modes) for the ZnTMPyP4/B-DNA complex, thereby resulting in explicit differentiation between B-DNA and Z-DNA. Additionally, the binding interactions of planar TMPyP4 to DNA were also investigated as a comparison. It is shown that without available virtual orbitals to coordinate, TMPyP4 binds with Z-DNA solely in the intercalation mode, as with B-DNA, and the intercalation results in a structural transition from Z-DNA to B-ZNA. These results provide mechanistic insights for understanding ZnTMPyP4 as a probe of recognizing Z-DNA and afford a possible strategy for designing new porphyrin derivatives with available virtual orbitals for the discrimination of B-DNA and Z-DNA.


Assuntos
DNA/química , Metaloporfirinas/química , Conformação de Ácido Nucleico , DNA/metabolismo , DNA de Forma B/química , DNA de Forma B/metabolismo , DNA Forma Z/química , DNA Forma Z/metabolismo , Metaloporfirinas/metabolismo , Porfirinas/química , Porfirinas/metabolismo , Ligação Proteica , Análise Espectral
19.
Org Biomol Chem ; 16(13): 2198-2209, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29532848

RESUMO

Base modifications are known to affect the structure and function of DNA. C8-guanine adducts from various carcinogenic compounds have been shown to be potent Z-DNA inducers. Hence, it has been hypothesized that Z-DNA plays a role in cancer and other genetic diseases. In this comprehensive review, Z-DNA and the effect of prevalent C8-guanine adducts on the B-Z transition are addressed. The discoveries of Z-DNA binding proteins including ADAR1, E3L, DLM1, and PKZ have suggested the relevance of Z-DNA in living systems. In addition, increasing evidence on the Z-DNA connection to gene transcription and inhibition reveals potential biological functions of the left-handed DNA. Finally, C8-guanine adducts that promote Z-DNA formation can be used as a tool to explore the Z-DNA function and its role in carcinogenesis.


Assuntos
Adutos de DNA/metabolismo , DNA Forma Z/metabolismo , Guanina/química , Neoplasias/genética , Animais , Carcinógenos/química , Adutos de DNA/química , DNA Forma Z/química , DNA Forma Z/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Humanos
20.
Nucleic Acids Res ; 46(8): 4129-4137, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29584891

RESUMO

Left-handed Z-DNA is an extraordinary conformation of DNA, which can form by special sequences under specific biological, chemical or physical conditions. Human ADAR1, prototypic Z-DNA binding protein (ZBP), binds to Z-DNA with high affinity. Utilizing single-molecule FRET assays for Z-DNA forming sequences embedded in a long inactive DNA, we measure thermodynamic populations of ADAR1-bound DNA conformations in both GC and TG repeat sequences. Based on a statistical physics model, we determined quantitatively the affinities of ADAR1 to both Z-form and B-form of these sequences. We also reported what pathways it takes to induce the B-Z transition in those sequences. Due to the high junction energy, an intermediate B* state has to accumulate prior to the B-Z transition. Our study showing the stable B* state supports the active picture for the protein-induced B-Z transition that occurs under a physiological setting.


Assuntos
Adenosina Desaminase/metabolismo , DNA de Forma B/química , DNA Forma Z/química , Proteínas de Ligação a RNA/metabolismo , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Estatísticos
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