RESUMO
i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.
Assuntos
Citosina , DNA de Cadeia Simples , Motivos de Nucleotídeos , Citosina/química , Citosina/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Anticorpos/química , Anticorpos/metabolismo , DNA/química , DNA/metabolismo , Sequência de BasesRESUMO
Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.
Assuntos
Quadruplex G , Disostose Mandibulofacial , Animais , Humanos , DNA/metabolismo , Células HEK293 , Células HeLa , Disostose Mandibulofacial/genética , Disostose Mandibulofacial/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Naftiridinas/farmacologia , Ácidos Picolínicos/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , Quadruplex G , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA-SeqRESUMO
Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG4T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN (NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.
Assuntos
DNA/química , Quadruplex G , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/genética , DNA/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Porphyrins represent a valuable class of ligands for G-quadruplex nucleic acids. Herein, we evaluate the binding of cationic porphyrins metallated with gold(iii) to G-quadruplex DNA and we compare it with other porphyrin derivatives. The G-quadruplex stabilization capacity and the selectivity of the various porphyrins were evaluated by biophysical and biochemical assays. The porphyrins were also tested as inhibitors of telomerase. It clearly appeared that the insertion of gold(iii) ion in the center of the porphyrin increases the binding affinity of the porphyrin for the G-quadruplex target. Together with modelling studies, it is possible to propose that the insertion of the square planar gold(iii) ion adds an extra positive charge on the complex and decreases the electron density in the porphyrin aromatic macrocycle, both properties being in favour of stronger electrostatic and π-staking interactions.
RESUMO
Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme-drug-DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown in vitro that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2ß impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments. Mol Cancer Ther; 16(10); 2166-77. ©2017 AACR.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase II/administração & dosagem , Apoptose/efeitos dos fármacos , Proteína BRCA1/genética , Cromatina/genética , Vetores Genéticos/efeitos dos fármacos , Humanos , Neoplasias/genética , Neoplasias/patologia , Podofilotoxina/administração & dosagem , Podofilotoxina/análogos & derivados , Poliaminas/administração & dosagem , Rad51 Recombinase/genéticaRESUMO
G-quadruplexes (G4) are RNA and DNA secondary structures formed by the stacking of guanine quartets in guanine rich sequences. Quadruplex-prone motifs may be found in key genomic regions such as telomeres, ribosomal DNA, transcriptional activators and regulators or oncogene promoters. A number of proteins involved in various biological processes are able to interact with G4s. Among them, proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures. Mutations of these helicases are linked to genome instability and to increases in cancer risks. Here, we present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction. RHAU is an RNA helicase known as the major source of G4 resolution in HeLa cells. Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes in parallel and to screen for the optimal conditions for its activity. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Quadruplex G , RNA/metabolismo , RNA Helicases DEAD-box/genética , DNA/química , Ensaios de Triagem em Larga Escala , Humanos , Desnaturação de Ácido Nucleico , Potássio/química , Potássio/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , RNA/química , Estabilidade de RNA , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Telômero/química , Telômero/metabolismo , TemperaturaRESUMO
Four nickel(II)-salophen complexes containing alkyl-imidazolium chains connected at the ortho or meta positions were prepared: N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (1), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)phenylenediamine (2), N,N'-bis(2-hydroxy-3-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (3), and N,N'-bis(2-hydroxy-4-methyl-3H-imidazol-1-iumbenzylideneamino)methyl-3H-imidazol-1-iumphenylenediamine (4). They protect G-quadruplex DNA (G4 -DNA) against thermal denaturation and show KA values in the range of 7.4×10(5) to 4×10(7) m(-1) for G4 -DNA models. Complex 4 exhibits an IC50 value of 70â nm for telomerase inhibition.
Assuntos
Complexos de Coordenação/química , Níquel/química , Salicilatos/química , Telomerase/metabolismo , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quadruplex G/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Biossíntese de Proteínas/efeitos dos fármacos , Telomerase/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells. Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.
Assuntos
Quadruplex G/efeitos dos fármacos , Guanidina/química , Metaloporfirinas/farmacologia , Níquel/química , Porfirinas/química , Proteínas de Ligação a Telômeros/metabolismo , Telômero/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Cobalto/química , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Metaloporfirinas/síntese química , Metaloporfirinas/química , Estrutura Molecular , Transporte Proteico/efeitos dos fármacos , Complexo Shelterina , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 µM for G-quadruplexes and greater than 2 µM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.
Assuntos
Quadruplex G , Compostos Organometálicos/química , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese químicaRESUMO
To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Neoplasias/genética , Antígenos Nucleares/metabolismo , Instabilidade Cromossômica , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , Modelos GenéticosRESUMO
The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR-mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest. Surprisingly, increasing concentrations of 12459 leading to cell apoptosis induced a mechanism that bypasses the DNA damage signaling and leads to the dephosphorylation of Chk1 and γ-H2AX. We identified the phosphatase Protein Phosphatase Magnesium dependent 1D/Wild-type P53-Induced Phosphatase (PPM1D/WIP1) as a factor responsible for this dephosphorylation. SiRNA-mediated depletion of PPM1D/WIP1 reactivates the DNA damage signaling by 12459. In addition, PPM1D/WIP1 is activated by reactive oxygen species (ROS) induced by 12459. ROS generated by 12459 are sufficient to trigger an early DNA damage in A549 cells when PPM1D/WIP1 is depleted. However, ROS inactivation by N-acetyl cysteine (NAC) treatment does not change the apoptotic response induced by 12459. Because PPM1D expression was recently reported to modulate the recruitment of DNA repair molecules, our data would suggest a cycle of futile protection against 12459, thus leading to a delayed mechanism of cell death.
Assuntos
Dano ao DNA , Fosfoproteínas Fosfatases/metabolismo , Compostos de Quinolínio/farmacologia , Transdução de Sinais , Triazinas/farmacologia , Apoptose , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Senescência Celular , Quinase 1 do Ponto de Checagem , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Proteínas Quinases/metabolismo , Proteína Fosfatase 2C , Espécies Reativas de Oxigênio/metabolismo , Telômero/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.
Assuntos
DNA/química , Quadruplex G , Piridinas/química , Quinolinas/química , Telômero/química , Composição de Bases/efeitos dos fármacos , Composição de Bases/genética , Sequência de Bases , Bromo/química , Bromo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Halogenação , Humanos , Ligantes , Piridinas/metabolismo , Piridinas/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Interferência de RNA , Complexo Shelterina , Espectrometria de Massas por Ionização por Electrospray , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.
Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Antígenos Nucleares/genética , Fracionamento Celular , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Autoantígeno Ku , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
G-quadruplex nucleic acids are emerging as therapeutic targets for small molecules referred to as small-molecule G-quadruplex ligands. The porphyrin H(2)-TMPyP4 was early reported to be a suitable motif for G-quadruplex DNA recognition. It probably binds to G-quadruplex nucleic acid through π-π stacking with the external G-quartets. We explored chemical modifications of this porphyrin such as insertion of various metal ions in the centre of the aromatic core and addition of bulky substituents that may improve the specificity of the compound toward G-quadruplex DNA. Porphyrin metallation, affording a G4-ligand with two symmetric faces, allowed the conclusion that the presence of an axial water molecule perpendicular to the aromatic plane lowered but did not hamper π-π stacking interactions between the aromatic parts of the ligand on the one hand and the external G-quartet on the other. The charge introduced in the centre of the porphyrin had little influence on binding. Thus, the ionic channel in the centre of G-quadruplex nucleic acids was not found to provide clear additional molecular clues for G-quadruplex nucleic acids targeting by porphyrins tested in the present study. Furthermore, we confirmed the unique G-quadruplex selectivity of a porphyrin modified with four bulky substituents at the meso positions and showed that although the compound is not "drug-like" it was capable of entering cells in culture and mediated some of the typical cellular effects of small-molecule G-quadruplex ligands.
Assuntos
Inibidores Enzimáticos/química , Quadruplex G , Porfirinas/química , Porfirinas/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quadruplex G/efeitos dos fármacos , Humanos , Ligantes , Manganês/química , Manganês/metabolismo , Relação Estrutura-Atividade , Telomerase/antagonistas & inibidores , Telomerase/metabolismoRESUMO
Aquabirnaviruses (ABVs) cause serious diseases in a variety of fish species used worldwide in aquaculture and have been isolated from a variety of healthy fish and shellfish species. The type species of ABV is Infectious pancreatic necrosis virus (IPNV), which is the causative agent of a highly contagious disease in juvenile salmonid fish. Marine birnaviruses (MABVs) have been isolated from various marine fish and shellfish. In Korea, ABV infection has been identified in several fish and shellfish. The current study presents sequence data from nested polymerase chain reaction products of 3 ABV strains obtained from different species of asymptomatic aquarium fish collected from a private commercial aquarium in Korea. Phylogenetic analysis of these strains, based on the partial nucleotide sequence of the VP2/NS junction, placed them within the genogroup VII (95-99% bootstrap confidence), which also contains MABV. The subclinically infected fish may be a source of MABV infection for other susceptible fish species inside the aquarium and potentially represent a serious challenge for the management of MABV infections. Additionally, the presence of MABV in these subclinically infected aquarium fish imported from other countries indicates that there is a need for the establishment of appropriate quarantine practices.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Birnaviridae/virologia , Peixes , Vírus da Necrose Pancreática Infecciosa/classificação , Dados de Sequência Molecular , Filogenia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Telomeres protect chromosome ends from being recognized as double-stranded breaks. Telomeric function is ensured by the shelterin complex in which TRF2 protein is an essential player. The G-rich strand of telomere DNA can fold into G-quadruplex (G4) structure. Small molecules stabilizing G4 structures, named G4 ligands, have been shown to alter telomeric functions in human cells. In this study, we show that a guanine-rich RNA sequence located in the 5'-UTR region of the TRF2 mRNA (hereafter 91TRF2G) is capable of forming a stable quadruplex that causes a 2.8-fold decrease in the translation of a reporter gene in human cells, as compared to a mutant 5'-UTR unable to fold into G4. We also demonstrate that several highly selective G4 ligands, the pyridine dicarboxamide derivative 360A and bisquinolinium compounds Phen-DC(3) and Phen-DC(6), are able to bind the 91TRF2G:RNA sequence and to modulate TRF2 protein translation in vitro. Since the naturally occurring 5'-UTR TRF2:RNA G4 element was used here, which is conserved in several vertebrate orthologs, the present data substantiate a potential translational mechanism mediated by a G4 RNA motif for the downregulation of TRF2 expression.
Assuntos
Regiões 5' não Traduzidas , Quadruplex G , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Proteína 2 de Ligação a Repetições Teloméricas/genética , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Ligantes , Dados de Sequência Molecular , Estabilidade de RNARESUMO
DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA-PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA-PK end binding and activation step and (2) DNA-PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.
Assuntos
Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Instabilidade Genômica , Células HeLa , Humanos , Complexo ShelterinaRESUMO
In order to evaluate the genetic variability of the causative agent of cold water disease (CWD), plasmid profiling was used to characterize Flavobacterium (F.) psychrophilum isolates (n = 169). Size analysis of plasmids in F. psychrophilum isolates (n = 128) from several fish species demonstrated that six kinds of plasmids were harbored, and ayu isolates had different profiles compared to other isolates. Moreover, multiple isolates (n = 41) from CWD outbreaks in 2002 to 2003 at a single ayu farm were examined to determine differences between isolates from successive outbreaks and showed different profiles by the sources of seedlings.
Assuntos
Surtos de Doenças/veterinária , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/isolamento & purificação , Variação Genética/genética , Osmeriformes , Animais , DNA Bacteriano/genética , Eletroforese em Gel de Ágar/veterinária , Doenças dos Peixes/genética , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/genética , Japão , Plasmídeos/genéticaRESUMO
Diclofenac is a nonsteroidal anti-inflammatory drug widely used in Mexico where it is sold over the counter. It enters water bodies through municipal and industrial discharges, posing a risk to water systems and aquatic organisms. Diclofenac-enriched artificial sediment was used to evaluate the toxicity of this pharmaceutical on the sentinel species Hyalella azteca, using oxidative stress biomarkers in order to determine if the set of tests used in this study is a suitable early damage biomarker. The median lethal concentration (72-h LC(50)) was determined and oxidative stress was evaluated using lipid peroxidation, protein carbonyl content to evaluate oxidized protein content, and the activity of superoxide dismutase, catalase, and glutathione peroxidase. All biomarkers were significantly altered. Diclofenac induces oxidative stress in H. azteca and the set of tests used (lipid peroxidation, protein carbonyl content, antioxidant enzyme activities) constitutes an adequate early damage biomarker for evaluating the toxicity of this pharmaceutical group in aquatic species.