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1.
Nat Commun ; 15(1): 2096, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453913

RESUMO

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.


Assuntos
Sistemas CRISPR-Cas , Contenção de Riscos Biológicos , Humanos , Sistemas CRISPR-Cas/genética , Engenharia Genética , Bactérias/genética , Timidina
2.
Phys Chem Chem Phys ; 26(11): 8761-8766, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38419552

RESUMO

5-Fluorouracil is now routinely used in chemo- and radiotherapy. Incorporated within DNA, the molecule is bound to the sugar backbone, forming the 5-fluorouridine sub-unit investigated in the present work. For the clinical usage of the latter, no information exists on the mechanisms that control the radiosensitizing effect at the molecular level. As low energy (< 12 eV) electrons are abundantly produced along the radiation tracks during cancer treatment using beams of high energy particles, we study how these ballistic secondary electrons damage the sensitizing molecule. The salient result from our study shows that the N-glycosidic bonds are principally affected with a cross-section of approximately two orders of magnitude higher than the canonical thymidine, reflecting to some degree the surviving factor of radiation-treated carcinoma cells with and without 5-fluorouracil incorporation. This result may help in the comprehension of the radiosensitizing effect of the fluoro-substituted thymidine in DNA.


Assuntos
Elétrons , Radiossensibilizantes , Uridina/análogos & derivados , DNA/química , Radiossensibilizantes/química , Dano ao DNA , Timidina , Fluoruracila
3.
Cell Rep Med ; 5(3): 101434, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38387463

RESUMO

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.


Assuntos
Neoplasias Colorretais , Neoplasias Pancreáticas , Humanos , Proteína Supressora de Tumor p53/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo do DNA , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , DNA/uso terapêutico , Timidina/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética
4.
J Org Chem ; 89(6): 3747-3768, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38394362

RESUMO

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.


Assuntos
Ácidos Nucleicos , RNA Interferente Pequeno/química , DNA , Timidina , Uridina/química
6.
Tissue Eng Regen Med ; 21(2): 353-366, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37935935

RESUMO

BACKGROUND: Regeneration is a highly complex process that requires the coordination of numerous molecular events, and identifying the key ruler that governs is important to investigate. While it has been shown that TCTP is a multi-functional protein that regulates cell proliferation, differentiation, apoptosis, anti-apoptosis, stem cell maintenance, and immune responses, but only a few studies associated to regeneration have been reported. To investigate the multi-functional role of TCTP in regeneration, the earthworm Perionyx excavatus was chosen. METHODS: Through pharmacological suppression of TCTP, amputation, histology, molecular docking, and western blotting, the multi-function role of TCTP involved in regeneration is revealed. RESULTS: Amputational studies show that P. excavatus is a clitellum-independent regenerating earthworm resulting in two functional worms upon amputation. Arresting cell cycle at the G1/S boundary using 2 mM Thymidine confirms that P. excavatus execute both epimorphosis and morphallaxis regeneration mode. The pharmacological suppression of TCTP using buclizine results in regeneration suppression. Following the combinatorial injection of 2 mM Thymidine and buclizine, the earthworm regeneration is completely blocked, which suggests a critical functional role of TCTP in morphallaxis. The pharmacological inhibition of TCTP also suppresses the key proteins involved in regeneration: Wnt3a (stem cell marker), PCNA (cell proliferation) and YAP1 (Hippo signalling) but augments the expression of cellular stress protein p53. CONCLUSION: The collective results indicate that TCTP synchronously is involved in the process of stem cell activation, cell proliferation, morphallaxis, and organ development in the regeneration event.


Assuntos
Oligoquetos , Animais , Oligoquetos/metabolismo , Simulação de Acoplamento Molecular , Proliferação de Células , Regeneração , Timidina/metabolismo
7.
Carbohydr Res ; 535: 109012, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38157586

RESUMO

To understand the regioselectivity observed in the allylation of pyrimidine nucleosides and to identify the factors directing the reaction, a theoretical study of the regioselective allylation was carried out. Several key points were considered such as: the structure of the deprotonated nucleobase in the presence of Na+; the effect of the solvent on the dissociation and aggregation reactions of thymidine/Na+ ion pair; and the likely allylation reaction mechanisms involved. The results showed that the regioselectivity observed experimentally can be attributed to a greater stability of a dimeric form coupled to an increase of the reaction barrier in THF due to larger Na+ binding to the nucleobase.


Assuntos
Nucleosídeos de Pirimidina , Nucleosídeos de Pirimidina/química , Timidina
8.
ACS Chem Biol ; 18(12): 2535-2543, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38050717

RESUMO

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.


Assuntos
Malária , Parasitos , Animais , Desoxiuridina/química , Desoxiuridina/metabolismo , Timidina Quinase , Parasitos/metabolismo , Química Click , Azidas/química , DNA/química , Timidina , Proliferação de Células , Mamíferos/metabolismo
9.
J Int Med Res ; 51(12): 3000605231222244, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38140948

RESUMO

Herein, we describe a case of acute rhabdomyolysis in a man in his early 50s undergoing haemodialysis and receiving the antiviral drug, telbivudine, for chronic hepatitis B virus (HBV) infection. Following diagnosis by electromyography (EMG), magnetic resonance image (MRI) scans and laboratory data (i.e., elevated serum creatinine kinase (CK) and myoglobin) telbivudine was discontinued and the patient was treated with methylprednisolone. While his CK and myoglobin levels decreased rapidly, his muscle weakness and pain improved slowly. Learning points include: patients undergoing haemodialysis and concomitantly receiving antiviral treatment for HBV, should have their serum levels of CK and myoglobin monitored regularly; treatment with corticosteroids maybe required; relief from rhabdomyolysis-induced muscle weakness and pain may be slow due to nerve fibre damage.


Assuntos
Hepatite B Crônica , Rabdomiólise , Masculino , Humanos , Telbivudina/efeitos adversos , Hepatite B Crônica/complicações , Hepatite B Crônica/tratamento farmacológico , Antivirais/efeitos adversos , Mioglobina/efeitos adversos , Timidina/efeitos adversos , Rabdomiólise/induzido quimicamente , Rabdomiólise/tratamento farmacológico , Diálise Renal , Dor/tratamento farmacológico , Debilidade Muscular
10.
Aquat Toxicol ; 265: 106766, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37980847

RESUMO

Tritium is a betta emitter radionuclide. Being an isotope of hydrogen, it is easily transferred to different environmental compartments, and to human and non-human biota. Considering that tritium levels are expected to rise in the upcoming decades with the development of nuclear facilities producing tritium using fission processes, investigating the potential toxicity of tritium to human and non-human biota is necessary. Tritiated thymidine, an organic form of tritium, has been used in this study to assess its toxicity on fish embryo development. Zebrafish embryos (3.5 hpf; hours post fertilization) have been exposed to tritiated thymidine at three different activity concentrations (7.5; 40; 110 kBq/mL) for four days. These experiments highlighted that zebrafish development was affected by the exposure to organic tritium, with smaller larvae at 3 dpf after exposure to the two lowest dose rates (22 and 170 µGy/h), a delayed hatching after exposure to the two highest dose rates (170 and 470 µGy/h), an increase in the spontaneous tail movement (1 dpf) and a decrease in the heartbeat (3 dpf) after exposure to the highest dose rate. The results also highlighted an increase in ROS production in larvae exposed to the intermediate dose rate. A dysregulation of many genes, involved in apoptosis, DNA repair or oxidative stress, was also found after 1 day of exposure to the lowest tritium dose rate. Our results thus suggest that exposure to tritiated thymidine from a dose rate as low as 22 µGy/h can lead to sublethal effects, with an effect on the development, dysregulation of many genes and increase of the ROS production. This paper provides valuable information on toxic effects arising from the exposure of fish to an organic form of tritium, which was the main objective of this study.


Assuntos
Poluentes Químicos da Água , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Trítio/toxicidade , Espécies Reativas de Oxigênio , Poluentes Químicos da Água/toxicidade , Estresse Oxidativo , Larva , Timidina/farmacologia , Embrião não Mamífero
11.
Microbiol Spectr ; 11(6): e0054123, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-37888993

RESUMO

IMPORTANCE: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Estados Unidos , Cefoxitina/farmacologia , Cromatografia Líquida , National Cancer Institute (U.S.) , Espectrometria de Massas em Tandem , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Timidina
12.
Molecules ; 28(18)2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37764419

RESUMO

One of the most essential health problems is cancer, the first or second cause of death worldwide. Head and neck cancers are hard to detect due to non-specific symptoms. The treatment often relies on a combination of radio and chemotherapy. For this reason, the research of new anticancer compounds is fundamental. The natural and synthetic compounds with 1,4-naphthoquinone scaffold is characterized by high anticancer activity. The study aimed to evaluate the synthesis and anticancer activity of hybrids 1,4-naphthoquinone with thymidine derivatives. The series of compounds allows us to check the influence of the substituent in the C3' position of the thymidine moiety on the cytotoxicity against squamous cancer cell lines (SCC-9 and SCC-25) and submandibular gland cancer (A-253). An annexin V/propidium iodide (PI) co-staining assay shows that derivatives cause the apoptotic in SCC-25 and A-253 cell lines. The molecular docking study examined the interaction between the active site of the BCL-2 protein and the hybrids.


Assuntos
Antineoplásicos , Neoplasias de Cabeça e Pescoço , Humanos , Simulação de Acoplamento Molecular , Antineoplásicos/química , Timidina/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Proliferação de Células , Estrutura Molecular , Apoptose , Relação Estrutura-Atividade
13.
Int J Antimicrob Agents ; 62(3): 106909, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37419291

RESUMO

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Humanos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos , Endopeptidase K/farmacologia , Pepsina A/farmacologia , Tripsina/farmacologia , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Inflamatórios/farmacologia , Bactérias , Biofilmes , Timidina/farmacologia , Testes de Sensibilidade Microbiana
14.
Life Sci Alliance ; 6(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37328191

RESUMO

Base-J (ß-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.


Assuntos
Proteínas de Transporte , Timina , Uracila/química , Uracila/metabolismo , DNA , Timidina/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-37235455

RESUMO

Nucleoside analogs are an important, well-established class of clinically useful medicinal agents that exhibit potent antimicrobial activity. Thus, we designed to explore the synthesis and spectral characterization of 5'-O-(myristoyl)thymidine esters (2-6) for in vitro antimicrobial, molecular docking, molecular dynamics, SAR, and POM analyses. An unimolar myristoylation of thymidine under controlled conditions furnished the 5'-O-(myristoyl)thymidine and it was further converted into four 3'-O-(acyl)-5'-O-(myristoyl)thymidine analogs. The chemical structures of the synthesized analogs were ascertained by analyzing their physicochemical, elemental, and spectroscopic data. In vitro antimicrobial tests along with PASS, prediction indicated expectant antibacterial functionality of these thymidine esters compared to the antifungal activities. In support of this observation, their molecular docking studies have been performed against lanosterol 14α-demethylase (CYP51A1) and Aspergillus flavus (1R51) and significant binding affinities and non-bonding interactions were observed. The stability of the protein-ligand complexes was monitored by a 100 ns MD simulation and found the stable conformation and binding mode in a stimulating environment of thymidine esters. Pharmacokinetic predictions were studied to assess their ADMET properties and showed promising results in silico. SAR investigation indicated that acyl chains, lauroyl (C-12) and myristoyl (C-14), combined with deoxyribose, were most effective against the tested bacterial and fungal pathogens. The POM analyses provide the structural features responsible for their combined antibacterial/antifungal activity and provide guidelines for further modifications, with the aim of improving each activity and selectivity of designed drugs targeting potentially drug-resistant microorganisms. It also opens avenues for the development of newer antimicrobial agents targeting bacterial and fungal pathogens.


A novel series of 5´-O-(myristoyl)thymidine derivatives were synthesized and characterized by FTIR, 1H-NMR, 2D-NMR, 13C-NMR, mass and physicochemical studies.In vitro antimicrobial susceptibility revealed that alkyl chain and aromatic substituents can improve the antimicrobial efficacy of the thymidine structure which was also supported by PASS enumeration.Molecular docking study against lanosterol 14α-demethylase (CYP51A1) and Aspergillus flavus (1R51) exhibited a promising binding score and interaction in the catalytic active site.A 100ns MD simulation revealed the stable conformation and binding pattern in a stimulating environment of thymidine derivatives.ADMET analysis revealed that most of the compounds are non-toxic and most of them have an inhibitory property to the CYP1A2 and CYP3A4In silico and POM analyses provide substantial ideas about the structural features responsible for their combined antibacterial/antifungal agents and provide guidelines for further modifications.


Assuntos
Anti-Infecciosos , Antifúngicos , Antifúngicos/química , Simulação de Acoplamento Molecular , Antibacterianos/química , Bactérias , Ésteres/química , Timidina/farmacologia , Estrutura Molecular , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 296: 122691, 2023 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-37023654

RESUMO

DNA alkylation is caused by long-term exposure of cells to the environmental and endogenous alkylating agents, which can also lead to DNA mutations and therefore trigger some cancers. Since O4-methylthymidine (O4-meT), mismatched with guanine (G), is the most common but not easily repaired alkylated nucleoside, monitoring O4-meT can help to effectively reduce the occurrence of carcinogenesis. In this work, the modified G-analogues are selected as the fluorescence probe to monitor the existence of O4-meT according to its pairing characteristics. The photo-physical properties of considered G-analogues formed by ring expansion or addition of fluorophores were studied in detail. It is found that, compared with natural G, the absorption peaks of these fluorescence analogues are red-shifted (>55 nm) and the luminescence is enhanced by π-conjugation. Especially, the xG has a large Stokes shift (65 nm) with fluorescence insensitive to natural cytosine (C) and retains efficient emission after pairing, while it is sensitive to O4-meT and the quenching phenomenon occurs due to the excited state intermolecular charge transfer. Accordingly, the xG can be used as a fluorescent probe to identify the O4-meT in solution. In addition, the direct use of deoxyguanine fluorescent analogue for monitoring O4-meT was evaluated by the effects of ligating deoxyribose on absorption and fluorescence emission.


Assuntos
DNA , Nucleosídeos , Fluorescência , Timidina
17.
Radiat Res ; 199(4): 373-384, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36921298

RESUMO

Tritium is found in the environment under three forms: free in the water, gaseous, and bound to organic matter. Once internalized in living organisms, it can be found in two forms: tissue free water tritium (TFWT) and organically bound tritium (OBT). This study aims to better understand OBT internalization in living organisms and to show the complementarity between experimental procedures and microdosimetry simulations that have often been used to obtain more information on imparted energy to cell nuclei. To do so, tritiated thymidine, an organic form of tritium, was chosen and zebrafish embryos [3.5 h post fertilization (hpf)] were exposed to a range of activity concentrations (2.21 × 103 to 5.95 × 105 Bq/mL). First, individual zebrafish embryos were sampled after different exposure times (1 to 96 h) to qualify the internalization kinetics. Then, the barrier role of the chorion was assessed after 2 days of exposure. Lastly, individual zebrafish embryos were sampled after 1 and 4 days of exposure to measure the internalization in the whole fish and its DNA, but also to highlight a possible link between the internal dose rate and the external activity concentration. Microdosimetry simulations were also made to quantify the imparted energy that could occur in the zebrafish cells after exposure to tritium. Results showed that when bound to thymidine, tritium rapidly incorporates in zebrafish early life stages, with the internalization being almost complete after 24 h. Results also showed that while the chorion acted as a barrier to prevent thymidine from entering the embryos, significant levels could still be measured in the whole organisms as well as in DNA. This study also highlighted that when the external activity concentration increased, the internal dose rate increased as well, following a sigmoidal trend. Microdosimetry simulations highlighted that the size and shape of the cell matters, and that the smallest cells seem to be at the greater risk, with only low-energy electrons inducing energy depositions. A linear fit was also found between the mean energy deposited and the logarithm of the radius of the cell, thus showing that the quantity of deposited energy is proportional to the radius of the cell. While this study highlighted important internalization pattern, it will also be used as the starting point of a study focusing on the toxic effects of tritiated thymidine on zebrafish in its early life stages.


Assuntos
Água , Peixe-Zebra , Animais , Trítio , Timidina/metabolismo , Projetos de Pesquisa
18.
Nucleic Acids Res ; 51(10): 4881-4898, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-36971122

RESUMO

UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by ∼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 µM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA , DNA/química , Timidina , Raios Ultravioleta
19.
Nat Genet ; 55(4): 568-580, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36959362

RESUMO

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.


Assuntos
Telomerase , Humanos , Telomerase/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Nucleotídeos/genética , Homeostase do Telômero/genética , Timidina , Telômero/genética
20.
J Cell Biol ; 222(4)2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36795402

RESUMO

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.


Assuntos
Replicação do DNA , Espectrometria de Massas em Tandem , Humanos , DNA/genética , Escherichia coli/genética , Timidina , Núcleo Celular/genética , Mitocôndrias/genética
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