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1.
Methods Mol Biol ; 2198: 169-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822031

RESUMO

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.


Assuntos
DNA/imunologia , Imuno-Histoquímica/métodos , 5-Metilcitosina/metabolismo , Animais , Anticorpos/metabolismo , Núcleo Celular/metabolismo , Citosina/análogos & derivados , Citosina/análise , DNA/genética , Metilação de DNA/imunologia , Epigênese Genética/genética , Humanos , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/análise , Peroxidase/química
2.
Methods Mol Biol ; 2198: 269-284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822038

RESUMO

5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.


Assuntos
Citometria de Fluxo/métodos , Pentoxil (Uracila)/análogos & derivados , Análise de Célula Única/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , 5-Metilcitosina/sangue , Cromatografia Líquida , Citosina/metabolismo , DNA/genética , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Humanos , Oxirredução , Pentoxil (Uracila)/análise , Pentoxil (Uracila)/sangue , Pentoxil (Uracila)/metabolismo , Espectrometria de Massas em Tandem , Timina/metabolismo
3.
J Am Chem Soc ; 142(6): 2889-2896, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31986025

RESUMO

5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.


Assuntos
5-Metilcitosina/análogos & derivados , Hidrogéis/química , Microfluídica , Pentoxil (Uracila)/análogos & derivados , Análise de Célula Única/métodos , 5-Metilcitosina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA/genética , Epigênese Genética , Feminino , Humanos , Invasividade Neoplásica , Pentoxil (Uracila)/metabolismo
5.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658654

RESUMO

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2'-deoxyribo-5-hydroxyuridin (HdU) and 2'-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF-), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA/biossíntese , DNA/efeitos dos fármacos , Estresse Oxidativo , Pirimidinas/farmacologia , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , HIV-1 , Humanos , Mutagênicos/química , Mutagênicos/metabolismo , Mutagênicos/farmacologia , Oxirredução , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo , Pentoxil (Uracila)/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Termodinâmica , Uracila/análogos & derivados , Uracila/química , Uracila/metabolismo , Uracila/farmacologia
6.
Sci Rep ; 8(1): 6860, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717169

RESUMO

In Caenorhabditis elegans, two DNA glycosylases, UNG-1 and NTH-1, and two AP endonucleases, APN-1 and EXO-3, have been characterized from the base-excision repair (BER) pathway that repairs oxidatively modified DNA bases. UNG-1 removes uracil, while NTH-1 can remove 5-hydroxymethyluracil (5-hmU), an oxidation product of thymine, as well as other lesions. Both APN-1 and EXO-3 can incise AP sites and remove 3'-blocking lesions at DNA single strand breaks, and only APN-1 possesses 3'- to 5'-exonulease and nucleotide incision repair activities. We used C. elegans mutants to study the role of the BER pathway in processing 5-hmU. We observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. These phenotypes were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We show that partially purified UNG-1 can act on 5-hmU lesion in vitro. We propose that UNG-1 removes 5-hmU incorporated into the genome and the resulting AP site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1 creating a genotoxic 3'-blocking lesion that requires the action of APN-1.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Dano ao DNA/genética , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Células Germinativas/metabolismo , Longevidade/genética , Mutação com Perda de Função , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/metabolismo , Uracila-DNA Glicosidase/genética
7.
Org Biomol Chem ; 16(9): 1527-1535, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29431832

RESUMO

Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Luz , Nucleotídeos/química , Pentoxil (Uracila)/análogos & derivados , Processos Fotoquímicos , Modelos Moleculares , Conformação de Ácido Nucleico , Pentoxil (Uracila)/química
8.
Nucleic Acids Res ; 46(4): 1624-1634, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29378012

RESUMO

i-Motif (iM) is a four stranded DNA structure formed by cytosine-rich sequences, which are often present in functionally important parts of the genome such as promoters of genes and telomeres. Using electronic circular dichroism and UV absorption spectroscopies and electrophoretic methods, we examined the effect of four naturally occurring DNA base lesions on the folding and stability of the iM formed by the human telomere DNA sequence (C3TAA)3C3T. The results demonstrate that the TAA loop lesions, the apurinic site and 8-oxoadenine substituting for adenine, and the 5-hydroxymethyluracil substituting for thymine only marginally disturb the formation of iM. The presence of uracil, which is formed by enzymatic or spontaneous deamination of cytosine, shifts iM formation towards substantially more acidic pH values and simultaneously distinctly reduces iM stability. This effect depends on the position of the damage sites in the sequence. The results have enabled us to formulate additional rules for iM formation.


Assuntos
DNA/química , Telômero/química , Adenina/análogos & derivados , Adenina/química , Citosina/química , Dano ao DNA , Humanos , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/química , Uracila/química
9.
Chem Commun (Camb) ; 53(99): 13253-13255, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29184924

RESUMO

DNA templates containing 5-hydroxymethyluracil or 5-hydroxymethylcytosine were used in an in vitro transcription assay with RNA polymerase from Escherichia coli. A strong enhancement of transcription was observed from DNA containing the Pveg promoter whereas a decrease was observed from DNA containing the rrnB P1 promoter, suggesting that they may act as epigenetic marks.


Assuntos
Citosina/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Epigênese Genética/genética , Escherichia coli/enzimologia , Pentoxil (Uracila)/análogos & derivados , Transcrição Genética/genética , Citosina/química , RNA Polimerases Dirigidas por DNA/metabolismo , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo
10.
Genome Biol ; 18(1): 23, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-28137275

RESUMO

BACKGROUND: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. RESULTS: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(ß-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases. CONCLUSIONS: By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.


Assuntos
Mapeamento Cromossômico/métodos , DNA de Protozoário/genética , Leishmania/genética , Pentoxil (Uracila)/análogos & derivados , DNA de Protozoário/química , Glucosídeos/química , Leishmania/química , Pentoxil (Uracila)/química , Uracila/análogos & derivados , Uracila/química
11.
Orig Life Evol Biosph ; 47(1): 3-11, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27029792

RESUMO

On early Earth, a primitive polymer that could spontaneously form from likely available precursors may have preceded both RNA and DNA as the first genetic material. Here, we report that heated aqueous solutions containing 5-hydroxymethyluracil (HMU) result in oligomers of uracil, heated solutions containing 5-hydroxymethylcytosine (HMC) result in oligomers of cytosine, and heated solutions containing both HMU and HMC result in mixed oligomers of uracil and cytosine. Oligomerization of hydroxymethylated pyrimidines, which may have been abundant on the primitive Earth, might have been important in the development of simple informational polymers.


Assuntos
5-Metilcitosina/análogos & derivados , Evolução Química , Pentoxil (Uracila)/análogos & derivados , Polimerização , Água/química , 5-Metilcitosina/química , Temperatura Alta , Origem da Vida , Pentoxil (Uracila)/química
12.
Free Radic Biol Med ; 101: 378-383, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27833031

RESUMO

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10-100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10-100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.


Assuntos
5-Metilcitosina/análogos & derivados , Ácido Ascórbico/farmacologia , DNA/metabolismo , Pentoxil (Uracila)/análogos & derivados , 5-Metilcitosina/agonistas , 5-Metilcitosina/metabolismo , Ácido Ascórbico/metabolismo , Citosina/agonistas , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA/efeitos dos fármacos , Células HCT116 , Humanos , Oxigenases de Função Mista/metabolismo , Oxirredução , Pentoxil (Uracila)/agonistas , Pentoxil (Uracila)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Timina/agonistas , Timina/metabolismo
13.
Acta Crystallogr C Struct Chem ; 72(Pt 5): 379-88, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27146565

RESUMO

In order to examine the preferred hydrogen-bonding pattern of various uracil derivatives, namely 5-(hydroxymethyl)uracil, 5-carboxyuracil and 5-carboxy-2-thiouracil, and for a conformational study, crystallization experiments yielded eight different structures: 5-(hydroxymethyl)uracil, C5H6N2O3, (I), 5-carboxyuracil-N,N-dimethylformamide (1/1), C5H4N2O4·C3H7NO, (II), 5-carboxyuracil-dimethyl sulfoxide (1/1), C5H4N2O4·C2H6OS, (III), 5-carboxyuracil-N,N-dimethylacetamide (1/1), C5H4N2O4·C4H9NO, (IV), 5-carboxy-2-thiouracil-N,N-dimethylformamide (1/1), C5H4N2O3S·C3H7NO, (V), 5-carboxy-2-thiouracil-dimethyl sulfoxide (1/1), C5H4N2O3S·C2H6OS, (VI), 5-carboxy-2-thiouracil-1,4-dioxane (2/3), 2C5H4N2O3S·3C6H12O3, (VII), and 5-carboxy-2-thiouracil, C10H8N4O6S2, (VIII). While the six solvated structures, i.e. (II)-(VII), contain intramolecular S(6) O-H...O hydrogen-bond motifs between the carboxy and carbonyl groups, the usually favoured R2(2)(8) pattern between two carboxy groups is formed in the solvent-free structure, i.e. (VIII). Further R2(2)(8) hydrogen-bond motifs involving either two N-H...O or two N-H...S hydrogen bonds were observed in three crystal structures, namely (I), (IV) and (VIII). In all eight structures, the residue at the ring 5-position shows a coplanar arrangement with respect to the pyrimidine ring which is in agreement with a search of the Cambridge Structural Database for six-membered cyclic compounds containing a carboxy group. The search confirmed that coplanarity between the carboxy group and the cyclic residue is strongly favoured.


Assuntos
Pentoxil (Uracila)/análogos & derivados , Tiouracila/análogos & derivados , Uracila/análogos & derivados , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Pentoxil (Uracila)/química
14.
Mutat Res Rev Mutat Res ; 767: 59-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27036066

RESUMO

The aim of this review is to describe the reactions which lead to generation of 5-hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-hydroxymethyluracil is epigenetic mark. The level of 5-hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-hydroxymethyluracil from DNA. 5-hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-hydroxymethyluracil may be either a regulatory or erroneous compound.


Assuntos
Reparo do DNA/genética , DNA/genética , Pentoxil (Uracila)/análogos & derivados , 5-Metilcitosina/química , Animais , Bacteriófagos/genética , Humanos , Hidroxilação/fisiologia , Camundongos , Oxirredução , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Timina/química , Timina/metabolismo
15.
Nucleic Acids Res ; 44(8): 3713-27, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-26843430

RESUMO

Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM.


Assuntos
5-Metilcitosina/metabolismo , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Animais , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS/genética , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/metabolismo , Regiões Promotoras Genéticas , Uracila/metabolismo
16.
Nucleic Acids Res ; 44(5): 2085-92, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26578595

RESUMO

Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation experiments and molecular dynamics simulations that provide insight into the impact of this modification on the structure and dynamics of DNA. When transported through ultrathin solid-state nanopores, short DNA fragments containing thymine modifications were found to exhibit distinct, reproducible features in their transport characteristics that differentiate them from unmodified molecules. Molecular dynamics simulations suggest that 5-hydroxymethyluracil alters the flexibility and hydrophilicity of the DNA molecules, which may account for the differences observed in our nanopore translocation experiments. The altered physico-chemical properties of DNA produced by the thymine modifications may have implications for recognition and processing of such modifications by regulatory DNA-binding proteins.


Assuntos
DNA/química , Simulação de Dinâmica Molecular , Pentoxil (Uracila)/análogos & derivados , Timina/química , Proteínas de Ligação a DNA/química , Epigênese Genética , Interações Hidrofóbicas e Hidrofílicas , Nanoporos , Desnaturação de Ácido Nucleico , Oxirredução , Pentoxil (Uracila)/química , Ligação Proteica , Propriedades de Superfície
17.
PLoS One ; 10(12): e0144859, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26660343

RESUMO

BACKGROUND: Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. RESULTS: Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine, as well as 5-hydroxymethyl-2'-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2'-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2'-deoxycytidine and its product 5-formyl-2'-deoxycytidine, as well as between 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). CONCLUSIONS: Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2'-deoxycytidine and/or 5-carboxyl-2'-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2'-deoxycytidine which defines the active demethylation process.


Assuntos
Citosina/análogos & derivados , DNA/metabolismo , Epigênese Genética , Pentoxil (Uracila)/análogos & derivados , 5-Metilcitosina/análogos & derivados , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Citosina/metabolismo , DNA/genética , Metilação de DNA , Dioxigenases/genética , Dioxigenases/metabolismo , Expressão Gênica , Rim/química , Rim/metabolismo , Fígado/química , Fígado/metabolismo , Pulmão/química , Pulmão/metabolismo , Masculino , Miocárdio/química , Miocárdio/metabolismo , Especificidade de Órgãos , Pentoxil (Uracila)/metabolismo , Ratos , Ratos Wistar , Suínos , Espectrometria de Massas em Tandem , Timo/química , Timo/metabolismo
18.
Nucleic Acids Res ; 43(20): 10026-38, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26429971

RESUMO

TET proteins play a vital role in active DNA demethylation in mammals and thus have important functions in many essential cellular processes. The chemistry for the conversion of 5mC to 5hmC, 5fC and 5caC catalysed by TET proteins is similar to that of T to 5hmU, 5fU and 5caU catalysed by thymine-7-hydroxylase (T7H) in the nucleotide anabolism in fungi. Here, we report the crystal structures and biochemical properties of Neurospora crassa T7H. T7H can bind the substrates only in the presence of cosubstrate, and binding of different substrates does not induce notable conformational changes. T7H exhibits comparable binding affinity for T and 5hmU, but 3-fold lower affinity for 5fU. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Our data reveal the molecular basis for substrate specificity and catalytic mechanism of T7H and provide new insights into the molecular mechanism of substrate recognition and catalysis of TET proteins.


Assuntos
Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Biocatálise , Domínio Catalítico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese , Neurospora crassa/enzimologia , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo , Ligação Proteica , Especificidade por Substrato , Timina/química , Timina/metabolismo , Uracila/análogos & derivados , Uracila/química , Uracila/metabolismo
19.
Biomarkers ; 20(5): 287-91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26329524

RESUMO

CONTEXT: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer. OBJECTIVES: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-hmUra). MATERIALS AND METHODS: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited. RESULTS: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio). CONCLUSION: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.


Assuntos
Biomarcadores Tumorais/urina , Neoplasias Colorretais/urina , Guanina/análogos & derivados , Pentoxil (Uracila)/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina/análogos & derivados , Idoso , Feminino , Guanina/urina , Humanos , Masculino , Pessoa de Meia-Idade , Pentoxil (Uracila)/urina
20.
Nucleic Acids Res ; 43(19): 9541-52, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26358812

RESUMO

Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G ·: T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme-product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Timina DNA Glicosilase/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo , Ligação Proteica , Timina/metabolismo , Timina DNA Glicosilase/metabolismo , Uracila/metabolismo
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