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1.
Zhongguo Zhong Yao Za Zhi ; 45(16): 3908-3914, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32893588

RESUMO

G-quadruplex DNA has become an important target for tumor therapy and anti-tumor development. Modern pharmacology has proved that Macleaya cordata has anti-inflammatory, antibacterial, anti-tumor and other pharmacological effects. Affinity ultrafiltration method can screen active ingredients from compounds rapidly, but G-quadruplex DNA ligands are difficult to dissociate, which is a key step in conventional ultrafiltration method. In this paper, the filtrates after ultrafiltration were determined by HPLC-MS in substitution. The peaks with 20% reduction of MS response from the incubation vs control were considered to be ligand components to G-quadruplex. Two of the peaks with the relative abundance above 30% were identified as sanguinarine(SAN) and chelerine(CHE). Their circular dichroism conformations further proved that SAN and CHE are active ligands of HT4. In addition, another two gradients with high relative abundance were identified as protopine(PRO) and allpcryprotopine(ALL). The binding rate of SAN, CHE, PRO and ALL was calculated according to the HPLC-MS results, and the results showed a consistency with that of the molecular docking method. The proposed method can be used to screen active components from mixture.


Assuntos
Quadruplex G , Ultrafiltração , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Ligantes , Espectrometria de Massas , Simulação de Acoplamento Molecular
2.
Biosens Bioelectron ; 167: 112494, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32791468

RESUMO

G-quadruplex is a non-canonical nucleic acid structure formed by the folding of guanine rich DNA or RNA. The conformation and function of G-quadruplex are determined by a number of factors, including the number and polarity of nucleotide strands, the type of cations and the binding targets. Recent studies led to the discovery of additional advantageous attributes of G-quadruplex with the potential to be used in novel biosensors, such as improved ligand binding and unique folding properties. G-quadruplex based biosensor can detect various substances, such as metal ions, organic macromolecules, proteins and nucleic acids with improved affinity and specificity compared to standard biosensors. The recently developed G-quadruplex based biosensors include electrochemical and optical biosensors. A novel G-quadruplex based biosensors also show better performance and broader applications in the detection of a wide spectrum of pathogens, including SARS-CoV-2, the causative agent of COVID-19 disease. This review highlights the latest developments in the field of G-quadruplex based biosensors, with particular focus on the G-quadruplex sequences and recent applications and the potential of G-quadruplex based biosensors in SARS-CoV-2 detection.


Assuntos
Betacoronavirus , Técnicas Biossensoriais/métodos , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Quadruplex G , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/tendências , Técnicas de Laboratório Clínico/tendências , Colorimetria , Técnicas Eletroquímicas , Corantes Fluorescentes , Humanos , Pandemias
3.
Life Sci ; 258: 118252, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32791149

RESUMO

AIMS: This study aimed to analyze the impact of four synthesized benzoxazinone derivatives as screening drugs on c-Myc-overexpressed cancer cells (H7402, HeLa, SK-RC-42, SGC7901, and A549) and to explore their interaction mechanisms in detail. MATERIALS AND METHODS: Using morphological analysis, real-time cytotoxicity analysis, wound healing assay, reverse transcription PCR, electrophoretic mobility shift assay, and circular dichroism spectroscopy techniques. KEY FINDINGS: Results revealed that these four compounds could inhibit proliferation of SK-RC-42, SGC7901, and A549 cells in five cancer cell lines to varying degrees and significantly hinder migration. More importantly, the RT-PCR assay showed that the compounds could surprisingly downregulate the expression of c-Myc mRNA in a dose-dependent manner in the five cancer cells, which may be one of the causes of cancer cell proliferation in vitro inhibition. Further EMSA assays demonstrated that at the molecular level of DNA, four compounds can induce the formation of G-quadruplexes (G4-DNAs) in the c-Myc gene promoter. In addition, the CD result of compound 1 clearly indicates that it specifically induces a c-Myc GC-rich 36mer double-stranded DNA in the c-Myc promoter to form a G-quadruplex hybrid configuration. In conclusion, the compounds studied could dose-dependently inhibit the growth and migration of the cancer cells being investigated. This is positively associated with the reduction of overexpression of the c-Myc gene, which may be significantly regulated by the association of compounds with the G-quadruplexes produced in the c-Myc gene promoter region. SIGNIFICANCE: We conclude that three compounds merit further study, particularly against non-small-cell lung cancer, as leading compounds of anticancer drugs.


Assuntos
Antineoplásicos/administração & dosagem , Benzoxazinas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Quadruplex G/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células A549 , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7
4.
Nat Commun ; 11(1): 3907, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764578

RESUMO

Nucleic acids can fold into G-quadruplex (G4) structures that can fine-tune biological processes. Proteins are required to recognize G4 structures and coordinate their function. Here we identify Zuo1 as a novel G4-binding protein in vitro and in vivo. In vivo in the absence of Zuo1 fewer G4 structures form, cell growth slows and cells become UV sensitive. Subsequent experiments reveal that these cellular changes are due to reduced levels of G4 structures. Zuo1 function at G4 structures results in the recruitment of nucleotide excision repair (NER) factors, which has a positive effect on genome stability. Cells lacking functional NER, as well as Zuo1, accumulate G4 structures, which become accessible to translesion synthesis. Our results suggest a model in which Zuo1 supports NER function and regulates the choice of the DNA repair pathway nearby G4 structures.


Assuntos
Reparo do DNA/fisiologia , Quadruplex G , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
5.
Nat Commun ; 11(1): 4287, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855419

RESUMO

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.


Assuntos
Anormalidades Múltiplas/etiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Troca de Cromátide Irmã , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proliferação de Células , RNA Helicases DEAD-box/química , DNA Helicases/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estabilidade Proteica , Pseudogenes , RNA Helicases/genética , RNA Helicases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Síndrome , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
PLoS Genet ; 16(7): e1008917, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628663

RESUMO

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.


Assuntos
Quadruplex G , Malária/genética , Motivos de Nucleotídeos/genética , Plasmodium falciparum/genética , Aminoquinolinas/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma/efeitos dos fármacos , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Ácidos Picolínicos/farmacologia , Plasmodium falciparum/patogenicidade , Regiões Promotoras Genéticas/genética , Ribossomos/efeitos dos fármacos , Ribossomos/genética
7.
Nat Commun ; 11(1): 3392, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636376

RESUMO

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Quadruplex G , Estruturas R-Loop , Transcrição Genética , Proteínas Virais/genética , DNA/análise , DNA/química , RNA Polimerases Dirigidas por DNA/química , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , RNA/química , RNA Mensageiro/química , Sítio de Iniciação de Transcrição , Proteínas Virais/química
8.
Food Chem ; 333: 127343, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663746

RESUMO

Leuconostoc spp. are generally utilized as kimchi starters because of their beneficial effects on kimchi fermentation and sensory characteristics. We developed a DNAzyme-based colorimetric method for measuring the abundance of the kimchi starter Leuconostoc mesenteroides WiKim32. A primer set for loop-mediated isothermal amplification and target-specific DNAzyme was designed based on the WiKim32 nucleotide sequence. In the presence of the target amplicon, DNAzyme bound to it, resulting in negligible amounts of green product. In contrast, with the addition of hemin and in the absence of the target amplicon, DNAzyme fragments not bound to the target amplicon formed G-quadruplex-hemin conjugates, generating a visible green product by oxidizing 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt. There was no cross-reaction with other strains. The method had high detection sensitivity and quantitative capacity in kimchi samples without a requirement for DNA isolation. This strategy provides a rapid, sensitive, and simple detection method with possible industrial applications.


Assuntos
DNA Catalítico/metabolismo , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Leuconostoc mesenteroides/isolamento & purificação , Leuconostoc mesenteroides/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria , DNA Catalítico/química , Quadruplex G , Hemina/química , Leuconostoc mesenteroides/genética , Especificidade da Espécie
9.
PLoS Genet ; 16(6): e1008740, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542039

RESUMO

FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Quadruplex G , Mutação , RNA Helicases/genética , Animais , Sítios de Ligação , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Ligação Proteica , RNA Helicases/química , RNA Helicases/metabolismo , Células Sf9 , Spodoptera
10.
Cancer Sci ; 111(9): 3089-3099, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32579791

RESUMO

The telomere is the specialized nucleoprotein complex at the end of the chromosome. Its highly conserved 5'-TTAGGG-3' repeats and shelterin protein complexes form a protective loop structure to maintain the integrity and stability of linear chromosomes. Although human somatic cells gradually shorten telomeres to undergo senescence or crisis, cancer cells activate telomerase, or the recombination-based mechanism to maintain telomeres and exhibit immortality. As the most frequent non-coding mutations in cancer, gain-of-function mutations in the promoter region of the telomerase catalytic subunit, TERT, trigger telomerase activation. Promoter methylation and copy number gain are also associated with the enhanced TERT expression. Although telomerase inhibitors were pioneered from telomere-directed therapeutics, their efficacies are limited to cancer with short telomeres and some hematological malignancies. Other therapeutic approaches include a nucleoside analog incorporated to telomeres and TERT promoter-driven oncolytic adenoviruses. Tankyrase poly(ADP-ribose) polymerase, a positive regulator of telomerase, has been rediscovered as a target for Wnt-driven cancer. Meanwhile, telomeric nucleic acids form a higher-order structure called a G-quadruplex (G4). G4s are formed genome-wide and their dynamics affect various events, including replication, transcription, and translation. G4-stabilizing compounds (G4 ligands) exert anticancer effects and are in clinical investigations. Collectively, telomere biology has provided clues for deeper understanding of cancer, which expands opportunities to discover innovative anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Telômero/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/genética , Quadruplex G/efeitos dos fármacos , Terapia Genética , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Terapia Viral Oncolítica , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Encurtamento do Telômero/efeitos dos fármacos
11.
Nucleic Acids Res ; 48(12): 6458-6470, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32496520

RESUMO

The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Telômero/química , Quadruplex G , Humanos , Microscopia de Força Atômica , Desnaturação de Ácido Nucleico , Imagem Individual de Molécula
12.
J Phys Chem Lett ; 11(14): 5661-5667, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32536162

RESUMO

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Quadruplex G/efeitos dos fármacos , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , Betacoronavirus/efeitos dos fármacos , Dimerização , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Pandemias , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
13.
Food Chem ; 331: 127208, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554309

RESUMO

In this work, we have developed a simple, fast and visual Hg2+ detection strategy based on distance as readout on paper chip by the Hg2+-mediated formation of G-quadruplex-hemin DNAzymes. In the presence of Hg2+, the two oligonucleotides hybridize to form G-quadruplex DNA by T-Hg2+-T base pair, which was able to bind hemin to form the catalytically active G-quadruplex-hemin DNAzymes. Once DNAzymes were added to react with the precipitated 3,3,5,5-tetramethyl benzidine (TMB) immobilized on the sample area, a visible color band was produced, and the formed length was positively correlated with the concentration of Hg2+. This biosensor is capable of selectively detecting mercuric ions with good reproducibility and satisfactory dynamic range. The limit of detection was low to 0.23 nM. Therefore, this strategy not only provides a visual and quick screen of Hg2+, but also shows a promising future in monitoring analysis of other metal ions in POC diagnostic field.


Assuntos
Técnicas Biossensoriais/métodos , Quadruplex G , Mercúrio/análise , Colorimetria , Íons/química , Limite de Detecção , Microfluídica , Papel , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes
14.
Nucleic Acids Res ; 48(13): 7421-7438, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496517

RESUMO

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Quadruplex G , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Camundongos , Ligação Proteica , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química
15.
Sci Rep ; 10(1): 7635, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: covidwho-196615

RESUMO

Proteolytic cleavage of influenza A virus (IAV) hemagglutinin by host proteases is crucial for virus infectivity and spread. The transmembrane serine protease TMPRSS2 was previously identified as the essential protease that can cleave hemagglutinin of many subtypes of influenza virus and spike protein of coronavirus. Herein, we found that a guanine rich tract, capable of forming intramolecular G-quadruplex in the presence of potassium ions, in the promoter region of human TMPRSS2 gene was quite important for gene transcriptional activity, hence affecting its function. Furthermore, 7 new synthesized benzoselenoxanthene analogues were found to enable stabilizing such G-quadruplex. More importantly, compounds can down-regulate TMPRSS2 gene expression, especially endogenous TMPRSS2 protein levels, and consequently suppress influenza A virus propagation in vitro. Our results provide a new strategy for anti-influenza A virus infection by small molecules targeting the TMPRSS2 gene G-quadruplex and thus inhibiting TMPRSS2 expression, which is valuable for developing small molecule drugs against influenza A virus and also may be a potential candidate as anti- SARS-CoV-2 (Severe Acute Respiratory Syndrome CoV 2) lead molecules.


Assuntos
Vírus da Influenza A/crescimento & desenvolvimento , Compostos Organosselênicos , Serina Endopeptidases/genética , Linhagem Celular , Pegada de DNA , Descoberta de Drogas , Quadruplex G , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus da Influenza A/fisiologia , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Regiões Promotoras Genéticas , Transcrição Genética
16.
Sci Rep ; 10(1): 7635, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376987

RESUMO

Proteolytic cleavage of influenza A virus (IAV) hemagglutinin by host proteases is crucial for virus infectivity and spread. The transmembrane serine protease TMPRSS2 was previously identified as the essential protease that can cleave hemagglutinin of many subtypes of influenza virus and spike protein of coronavirus. Herein, we found that a guanine rich tract, capable of forming intramolecular G-quadruplex in the presence of potassium ions, in the promoter region of human TMPRSS2 gene was quite important for gene transcriptional activity, hence affecting its function. Furthermore, 7 new synthesized benzoselenoxanthene analogues were found to enable stabilizing such G-quadruplex. More importantly, compounds can down-regulate TMPRSS2 gene expression, especially endogenous TMPRSS2 protein levels, and consequently suppress influenza A virus propagation in vitro. Our results provide a new strategy for anti-influenza A virus infection by small molecules targeting the TMPRSS2 gene G-quadruplex and thus inhibiting TMPRSS2 expression, which is valuable for developing small molecule drugs against influenza A virus and also may be a potential candidate as anti- SARS-CoV-2 (Severe Acute Respiratory Syndrome CoV 2) lead molecules.


Assuntos
Vírus da Influenza A/crescimento & desenvolvimento , Compostos Organosselênicos , Serina Endopeptidases/genética , Linhagem Celular , Pegada de DNA , Descoberta de Drogas , Quadruplex G , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus da Influenza A/fisiologia , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Regiões Promotoras Genéticas , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 117(21): 11409-11420, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404420

RESUMO

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.


Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Quadruplex G , Células A549 , Acetilação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Expressão Gênica , Genes myc , Genoma Humano , Guanina/química , Guanina/metabolismo , Células HCT116 , Humanos , Oxirredução , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 11(1): 2661, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461552

RESUMO

RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma.


Assuntos
Quadruplex G , Glioblastoma/fisiopatologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica/fisiologia , Instabilidade Genômica/fisiologia , Humanos , RNA Mensageiro/metabolismo
19.
Chemistry ; 26(39): 8631-8638, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32428287

RESUMO

Numerous studies have shown compelling evidence that incorporation of an inversion of polarity site (IPS) in G-rich sequences can affect the topological and structural characteristics of G-quadruplexes (G4s). Herein, the influence of IPS on the formation of a previously studied intramolecular parallel G4 of d(G3 TG3 TG3 TG3 ) (TTT) and its stacked higher-order structures is explored. Insertion of 3'-3' or 5'-5' IPS did not change the parallel folding pattern of TTT. However, both the species and position of the IPS in TTT have a significant impact on the G4 stability and end-stacking through the alteration of G4-G4 interfaces properties. The data demonstrate that one base flip in each terminal G-tetrad can stabilize parallel G4s and facilitate intermolecular packing of monomeric G4s. Such modifications can also enhance the fluorescence and enzymatic performances by promoting interactions between parallel G4s with N-methyl mesoporphyrin IX (NMM) and hemin, respectively.


Assuntos
DNA Catalítico/química , Guanosina/química , Hemina/química , Mesoporfirinas/química , Quadruplex G , Estrutura Molecular
20.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
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