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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502300

RESUMO

Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = -0.81, p < 0.0001), MTHFR protein expression (r = -0.8; p = 0.01), and MTHFR enzyme activity (r = -0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion-induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion-induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight into the significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies.


Assuntos
Carcinoma Hepatocelular/patologia , Segregação de Cromossomos , DNA de Neoplasias/genética , Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Uracila/metabolismo , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Instabilidade Cromossômica , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Polimorfismo Genético , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
2.
ACS Chem Biol ; 16(9): 1701-1708, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34427431

RESUMO

In this study, we provide experimental (Protein Data Bank (PDB) inspection) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the involvement of charge assisted chalcogen bonding (ChB) interactions in the recognition and folding mechanisms of S-adenosylmethionine (SAM) riboswitches. Concretely, an initial PDB search revealed several examples where ChBs between S-adenosyl methionine (SAM)/adenosyl selenomethionine (EEM) molecules and uracil (U) bases belonging to RNA take place. While these interactions are usually described as a merely Coulombic attraction between the positively charged S/Se group and RNA, theoretical calculations indicated that the σ holes of S and Se are involved. Moreover, computational models shed light on the strength and directionality properties of the interaction, which was also further characterized from a charge-density perspective using Bader's "Atoms in Molecules" (AIM) theory, Non-Covalent Interaction plot (NCIplot) visual index, and Natural Bonding Orbital (NBO) analyses. As far as our knowledge extends, this is the first time that ChBs in SAM-RNA complexes have been systematically analyzed, and we believe the results might be useful for scientists working in the field of RNA engineering and chemical biology as well as to increase the visibility of the interaction among the biological community.


Assuntos
Calcogênios/química , S-Adenosilmetionina/química , Selênio/química , Enxofre/química , Bases de Dados de Proteínas , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Teoria Quântica , RNA/metabolismo , Riboswitch , Selenometionina/química , Eletricidade Estática , Termodinâmica , Uracila/metabolismo
3.
Chemphyschem ; 22(15): 1622-1630, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34101319

RESUMO

DFT calculations are employed to quantify the influence of the presence, number, nature, and position of posttranscriptional methylation on stacking strength of RNA bases. We carry out detailed potential energy scans of the variation in stacking energies with characteristic geometrical parameters in three categories of forty stacked dimers - canonical base homodimers (N||N), methylated base homodimers (mN||mN) and heterodimers of canonical bases and methylated counterparts (N||mN). Our analysis reveals that neutral methylation invariably enhances the stacking of bases. Further, N||mN stacking is stronger than mN||mN stacking and charged N||mN exhibit strongest stacking among all dimers. This indicates that methylations greatly enhance stacking when dispersed in RNA sequences containing identical bases. Comparison of stacks involving singly- and doubly-methylated purines reveal that incremental methylation enhances the stacking in neutral dimers. Although methylation at the carbon position of neutral pyrimidine dimers greatly enhances the stacking, methylation on the 5-membered ring imparts better stacking compared to methylation on the 6-membered ring in adenine dimers. However, methylation at the ring nitrogen (N1 ) provides better stacking than the amino group (N2 ) in guanine dimers. Our results thus highlight subtle structural effects of methylation on RNA base stacking and will enhance our understanding of the physicochemical principles of RNA structure and dynamics.


Assuntos
Processamento Pós-Transcricional do RNA , RNA/química , RNA/metabolismo , Adenina/química , Adenina/metabolismo , Citosina/química , Citosina/metabolismo , Teoria da Densidade Funcional , Dimerização , Guanina/química , Guanina/metabolismo , Metilação , Uracila/química , Uracila/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063755

RESUMO

Energetically unfavorable Watson-Crick (WC)-like tautomeric forms of nucleobases are known to introduce spontaneous mutations, and contribute to replication, transcription, and translation errors. Recent NMR relaxation dispersion techniques were able to show that wobble (w) G•U mispair exists in equilibrium with the short-lived, low-population WC-like enolic tautomers. Presently, we have investigated the wG•U → WC-like enolic reaction pathway using various theoretical methods: quantum mechanics (QM), molecular dynamics (MD), and combined quantum mechanics/molecular mechanics (QM/MM). The previous studies on QM gas phase calculations were inconsistent with experimental data. We have also explored the environmental effects on the reaction energies by adding explicit water. While the QM-profile clearly becomes endoergic in the presence of water, the QM/MM-profile remains consistently endoergic in the presence and absence of water. Hence, by including microsolvation and QM/MM calculations, the experimental data can be explained. For the G•Uenol→ Genol•U pathway, the latter appears to be energetically more favorable throughout all computational models. This study can be considered as a benchmark of various computational models of wG•U to WC-like tautomerization pathways with and without the environmental effects, and may contribute on further studies of other mispairs as well.


Assuntos
Guanina/metabolismo , RNA/genética , Uracila/metabolismo , Pareamento Incorreto de Bases/genética , Pareamento de Bases/genética , Simulação por Computador , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação Puntual/genética , Teoria Quântica
5.
Microbiol Res ; 249: 126773, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33940365

RESUMO

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.


Assuntos
Agrobacterium tumefaciens/genética , Hypocreales/genética , Transformação Genética , Antifúngicos/farmacologia , Quitinases/genética , Quitinases/metabolismo , DNA Bacteriano/genética , Genes Fúngicos , Genes Reporter , Hypocreales/efeitos dos fármacos , Hypocreales/metabolismo , Mutagênese Insercional , Mutação , Uracila/metabolismo , Uridina/metabolismo
6.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805725

RESUMO

Monocarboxylate transporters (MCTs) are of great research interest for their role in cancer cell metabolism and their potential ability to transport pharmacologically relevant compounds across the membrane. Each member of the MCT family could potentially provide novel therapeutic approaches to various diseases. The major differences among MCTs are related to each of their specific metabolic roles, their relative substrate and inhibitor affinities, the regulation of their expression, their intracellular localization, and their tissue distribution. MCT4 is the main mediator for the efflux of L-lactate produced in the cell. Thus, MCT4 maintains the glycolytic phenotype of the cancer cell by supplying the molecular resources for tumor cell proliferation and promotes the acidification of the extracellular microenvironment from the co-transport of protons. A promising therapeutic strategy in anti-cancer drug design is the selective inhibition of MCT4 for the glycolytic suppression of solid tumors. A small number of studies indicate molecules for dual inhibition of MCT1 and MCT4; however, no selective inhibitor with high-affinity for MCT4 has been identified. In this study, we attempt to approach the structural characteristics of MCT4 through an in silico pipeline for molecular modelling and pharmacophore elucidation towards the identification of specific inhibitors as a novel anti-cancer strategy.


Assuntos
Antineoplásicos/química , Transportadores de Ácidos Monocarboxílicos/química , Proteínas Musculares/química , Floretina/química , Pirimidinonas/química , Quercetina/química , Reserpina/análogos & derivados , Tiofenos/química , Uracila/análogos & derivados , Animais , Antineoplásicos/metabolismo , Sítios de Ligação , Transporte Biológico , Desenho de Fármacos , Glicólise/fisiologia , Humanos , Ácido Láctico/química , Ácido Láctico/metabolismo , Simulação de Acoplamento Molecular , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Floretina/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pirimidinonas/metabolismo , Quercetina/metabolismo , Reserpina/química , Reserpina/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Tiofenos/metabolismo , Uracila/química , Uracila/metabolismo
7.
Biochemistry ; 60(19): 1485-1497, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33929180

RESUMO

Editing of the pre-mRNA of the DNA repair glycosylase NEIL1 results in substitution of a Lys with Arg in the lesion recognition loop of the enzyme. Unedited (UE, Lys242) NEIL1 removes thymine glycol lesions in DNA ∼30 times faster than edited (Ed, Arg242) NEIL1. Herein, we evaluated recognition and excision mediated by UE and Ed NEIL1 of 5-hydroxyuracil (5-OHU), a highly mutagenic lesion formed via oxidation of cytosine. Both NEIL1 isoforms catalyzed low levels of 5-OHU excision in single-stranded DNA, bubble and bulge DNA contexts and in duplex DNA base paired with A. Removal of 5-OHU in base pairs with G, T, and C was found to be faster and proceed to a higher overall extent with UE than with Ed NEIL1. In addition, the presence of mismatches adjacent to 5-OHU magnified the hampered activity of the Ed isoform. However, Ed NEIL1 was found to exhibit higher affinity for 5-OHU:G and 5-OHU:C duplexes than UE NEIL1. These results suggest that NEIL1 plays an important role in detecting and capturing 5-OHU lesions in inappropriate contexts, in a manner that does not lead to excision, to prevent mutations and strand breaks. Indeed, inefficient removal of 5-OHU by NEIL1 from 5-OHU:A base pairs formed during replication would thwart mutagenesis. Notably, nonproductive engagement of 5-OHU by Ed NEIL1 suggests the extent of 5-OHU repair will be reduced under cellular conditions, such as inflammation, that increase the extent of NEIL1 RNA editing. Tipping the balance between the two NEIL1 isoforms may be a significant factor leading to genome instability.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Edição de RNA/genética , DNA/genética , Reparo do DNA , DNA de Cadeia Simples , Humanos , Oxirredução , Timina/análogos & derivados , Uracila/análogos & derivados , Uracila/metabolismo
8.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921666

RESUMO

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.


Assuntos
Uracila/metabolismo , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Metilação de DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Epigênese Genética/genética , Epigenômica/métodos , Humanos
9.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918885

RESUMO

The appearance of uracil in the deoxyuridine moiety of DNA is among the most frequently occurring genomic modifications. Three different routes can result in genomic uracil, two of which do not require specific enzymes: spontaneous cytosine deamination due to the inherent chemical reactivity of living cells, and thymine-replacing incorporation upon nucleotide pool imbalances. There is also an enzymatic pathway of cytosine deamination with multiple DNA (cytosine) deaminases involved in this process. In order to describe potential roles of genomic uracil, it is of key importance to utilize efficient uracil-DNA detection methods. In this review, we provide a comprehensive and critical assessment of currently available uracil detection methods with special focus on genome-wide mapping solutions. Recent developments in PCR-based and in situ detection as well as the quantitation of genomic uracil are also discussed.


Assuntos
DNA , Genoma , Uracila , Animais , DNA/química , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Testes Genéticos , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização In Situ , Nucleotídeos , Reação em Cadeia da Polimerase , Transdução de Sinais , Uracila/química , Uracila/metabolismo , Uracila-DNA Glicosidase/metabolismo
10.
J Biol Chem ; 296: 100651, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839153

RESUMO

The Rid protein family (PF14588, IPR006175) is divided into nine subfamilies, of which only the RidA subfamily has been characterized biochemically. RutC, the founding member of one subfamily, is encoded in the pyrimidine utilization (rut) operon that encodes a pathway that allows Escherichia coli to use uracil as a sole nitrogen source. Results reported herein demonstrate that RutC has 3-aminoacrylate deaminase activity and facilitates one of the reactions previously presumed to occur spontaneously in vivo. RutC was active with several enamine-imine substrates, showing similarities and differences in substrate specificity with the canonical member of the Rid superfamily, Salmonella enterica RidA. Under standard laboratory conditions, a Rut pathway lacking RutC generates sufficient nitrogen from uracil for growth of E. coli. These results support a revised model of the Rut pathway and provide evidence that Rid proteins may modulate metabolic fitness, rather than catalyzing essential functions.


Assuntos
Acrilatos/metabolismo , Aminoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Aminoidrolases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Família , Nitrogênio/metabolismo , Oxirredutases/genética , Fosfato de Piridoxal/metabolismo , Salmonella enterica/enzimologia , Especificidade por Substrato , Uracila/metabolismo
11.
Biochemistry ; 60(14): 1120-1132, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33755421

RESUMO

Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of ∼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 Šacross the protein.


Assuntos
Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Uracila/análogos & derivados , Animais , Domínio Catalítico , Di-Hidrouracila Desidrogenase (NADP)/química , Sinergismo Farmacológico , Ligação Proteica , Suínos , Uracila/metabolismo , Uracila/farmacologia
12.
Nucleic Acids Res ; 49(7): 3948-3966, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33784377

RESUMO

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.


Assuntos
DNA Glicosilases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Uracila/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica , Proteínas Recombinantes/metabolismo
13.
J Mol Biol ; 433(10): 166923, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33713677

RESUMO

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.


Assuntos
Produtos do Gene gag/química , Guanina/química , Vírus dos Macacos de Mason-Pfizer/química , RNA Viral/química , Uracila/química , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Viral da Expressão Gênica , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Guanina/metabolismo , Interações Hospedeiro-Patógeno , Vírus dos Macacos de Mason-Pfizer/genética , Vírus dos Macacos de Mason-Pfizer/metabolismo , Conformação de Ácido Nucleico , Papio , Ligação Proteica , Conformação Proteica , Pegadas de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , Uracila/metabolismo
14.
Genes (Basel) ; 12(2)2021 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573186

RESUMO

Uracil is an unavoidable aberrant base in DNA sequences, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires multiple biochemical steps with conformational changes of DNA that inhibit DNA replication and interfere with transcription. However, the relevance of uracil in DNA for cellular physiology and transcriptional regulation is not fully understood. We investigated the functional roles of SMUG1 using knock-down (KD) and knock-out (KO) models. The proliferation ratio of SMUG1 KD and KO cells was decreased compared to WT control cells, and the cell cycle was arrested in the G2/M phases before the transition to mitosis. The apoptotic cell death was increased in KD and KO cell lines through the increase of BAX and active caspase 3 expression. Phospho-gamma-H2AX expression, which reflected accumulated DNA damage, was also increased in KO cells. Moreover, the apoptotic cells by DNA damage accumulation were markedly increased in SMUG1 KD and KO cells after ultraviolet C irradiation. Transcriptomic analysis using RNA-seq revealed that SMUG1 was involved in gene sets expression including cell cycle transition and chromatin silencing. Together, the results implicate SMUG1 as a critical factor in cell cycle and transcriptional regulation.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Uracila-DNA Glicosidase/genética , Uracila/metabolismo , Apoptose/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Dano ao DNA , Reparo do DNA/genética , Replicação do DNA/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Uracila-DNA Glicosidase/antagonistas & inibidores
15.
Curr Genet ; 67(2): 283-294, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33386486

RESUMO

Yeast Apn2 is an AP endonuclease and DNA 3'-diesterase that belongs to the Exo III family with homology to the E. coli exonuclease III, Schizosaccharomyces pombe eth1, and human AP endonucleases APEX1 and APEX2. In the absence of Apn1, the major AP endonuclease in yeast, Apn2 can cleave the DNA backbone at an AP lesion initiating the base excision repair pathway. To study the role and relative contribution of Apn2, we took advantage of a reporter system that was previously used to delineate how uracil-derived AP sites are repaired. At this reporter, disruption of the Apn1-initiated base excision repair pathway led to a significant elevation of A:T to C:G transversions. Here we show that such highly elevated A:T to C:G transversion mutations associated with uracil residues in DNA are abolished when apn1∆ yeast cells are grown in glucose as the primary carbon source. We also show that the disruption of Apn2, either by the complete gene deletion or by the mutation of a catalytic residue, results in a similarly reduced rate of the uracil-associated mutations. Overall, our results indicate that Apn2 activity is regulated by the glucose repression pathway in yeast.


Assuntos
Carbono/metabolismo , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Humanos , Mutagênese/genética , Mutação , Saccharomyces cerevisiae/genética , Uracila/metabolismo
16.
Methods Mol Biol ; 2198: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822020

RESUMO

Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.


Assuntos
DNA/metabolismo , Epigênese Genética , Epigenômica , Espectrometria de Massas , Animais , DNA/genética , DNA/urina , Metilação de DNA , Epigenômica/métodos , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem , Uracila/metabolismo , Urinálise/métodos
17.
DNA Repair (Amst) ; 97: 103028, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33254084

RESUMO

Uracil-DNA glycosylase (UDG) is a highly conserved DNA repair enzyme that acts as a key component in the base excision repair pathway to correct hydrolytic deamination of cytosine making it critical to genome integrity in living organisms. We report here a non-labeled, non-radio-isotopic and very specific method to measure UDG activity. Oligodeoxyribonucleotide duplex containing a site-specific G:U mismatch that is hydrolyzed by UDG then subjected to Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectrometry analysis. A protocol was developed to maintain the AP product in DNA without strand break then the cleavage of uracil was identified by the mass change from uracil substrate to AP product. From UDG kinetic analysis, for G:U substrate the Km is 50 nM, Vmax is 0.98 nM/s and Kcat = 9.31 s-1. The method was applied to uracil glycosylase inhibitor measurement with an IC50 value of 7.6 pM. Single-stranded and double-stranded DNAs with uracil at various positions of the substrates were also tested for UDG activity albeit with different efficiencies. The simple, rapid, quantifiable, scalable and versatile method has potential to be the reference method for monofunctional glycosylase measurement, and can also be used as a tool for glycosylase inhibitors screening.


Assuntos
Reparo do DNA , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Uracila-DNA Glicosidase/metabolismo , Uracila/análise , Dano ao DNA , Escherichia coli/genética , Cinética , Uracila/metabolismo
18.
J Biol Chem ; 296: 100114, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33234590

RESUMO

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten's ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.


Assuntos
Benzilaminas/química , Benzilaminas/metabolismo , Miosinas/metabolismo , Uracila/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/química , Fosforilação/fisiologia , Uracila/química , Uracila/metabolismo
19.
Angew Chem Int Ed Engl ; 60(8): 4175-4182, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33142013

RESUMO

Synthetic nucleic acids, with four non-canonical nucleobases, can function as genetic materials. A comprehensive analysis of PCR amplification, transcription, reverse transcription, and cloning was done to screen for alternative genetic monomers. A small library of six modified nucleobases was selected: the modified 2'-deoxyribonucleoside (dZTPs) and ribonucleoside (rZTPs) triphosphates of 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine or inosine together with 5-fluorocytosine or 5-bromocytosine. The fragments composed of one to four modified nucleotides (denoted as DZA) have been successfully recognized and transcribed to natural or modified RNA (denoted as RZA) by T7 RNA polymerase. The fully modified RZA fragment could be reverse transcribed and then amplified in the presence of various dZTPs. Noticeably, modified fragments could function as genetic templates in vivo by encoding the 678 base pair gene of a fluorescent protein in bacteria. These results demonstrate the existence of a fully simulated genetic circuit that uses synthetic materials.


Assuntos
Ácidos Nucleicos/metabolismo , Biologia Sintética/métodos , Citosina/análogos & derivados , Citosina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Uracila/análogos & derivados , Uracila/metabolismo , Proteínas Virais/metabolismo
20.
Chembiochem ; 22(4): 672-678, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33034934

RESUMO

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2'- and 3'-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4'-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariantes Associadas à Mucosa/metabolismo , Ribitol/análogos & derivados , Uracila/análogos & derivados , Humanos , Cinética , Ligantes , Ativação Linfocitária , Ribitol/química , Ribitol/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Uracila/química , Uracila/metabolismo
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