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1.
Genome Biol ; 25(1): 115, 2024 05 06.
Article in English | MEDLINE | ID: mdl-38711126

ABSTRACT

BACKGROUND: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. RESULTS: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays. CONCLUSIONS: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.


Subject(s)
Chromatin , Dinoflagellida , Pentoxyl , Pentoxyl/analogs & derivatives , Dinoflagellida/genetics , Dinoflagellida/metabolism , Chromatin/metabolism , Pentoxyl/metabolism , Genome, Protozoan
2.
Nano Lett ; 23(22): 10625-10632, 2023 Nov 22.
Article in English | MEDLINE | ID: mdl-37930759

ABSTRACT

5-Hydroxymethyluracil (5hmU) is an oxidation derivative of thymine in the genomes of various organisms and may serve as both an epigenetic mark and a cancer biomarker. However, the current 5hmU assays usually have drawbacks of laborious procedures, low specificity, and unsatisfactory sensitivity. Herein, we demonstrate the click chemistry-mediated hyperbranched amplification-driven dendritic nanoassembly for genome-wide analysis of 5hmU in breast cell lines and human breast tissues. The proposed strategy possesses good selectivity, ultralow background, and high sensitivity with a detection limit of 83.28 aM. This method can accurately detect even a 0.001% 5hmU level in the mixture. Moreover, it can determine 5hmU at single-cell level and distinguish the expressions of 5hmU in tissues of normal persons and breast cancer patients, holding great promise in 5hmU-related biological research and clinical diagnosis.


Subject(s)
DNA , Pentoxyl , Humans , DNA/metabolism , Pentoxyl/metabolism , Cell Line
3.
Molecules ; 28(5)2023 Feb 24.
Article in English | MEDLINE | ID: mdl-36903362

ABSTRACT

For many decades, uracil has been an antineoplastic agent used in combination with tegafur to treat various human cancers, including breast, prostate, and liver cancer. Therefore, it is necessary to explore the molecular features of uracil and its derivatives. Herein, the molecule's 5-hydroxymethyluracil has been thoroughly characterized by NMR, UV-Vis, and FT-IR spectroscopy by means of experimental and theoretical analysis. Density functional theory (DFT) using the B3LYP method at 6-311++G(d,p) was computed to achieve the optimized geometric parameters of the molecule in the ground state. For further investigation and computation of the NLO, NBO, NHO analysis, and FMO, the improved geometrical parameters were utilized. The potential energy distribution was used to allocate the vibrational frequencies using the VEDA 4 program. The NBO study determined the relationship between the donor and acceptor. The molecule's charge distribution and reactive regions were highlighted using the MEP and Fukui functions. Maps of the hole and electron density distribution in the excited state were generated using the TD-DFT method and PCM solvent model in order to reveal electronic characteristics. The energies and diagrams for the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) were also provided. The HOMO-LUMO band gap estimated the charge transport within the molecule. When examining the intermolecular interactions in 5-HMU, Hirshfeld surface analysis was used, and fingerprint plots were also produced. The molecular docking investigation involved docking 5-HMU with six different protein receptors. Molecular dynamic simulation has given a better idea of the binding of the ligand with protein.


Subject(s)
Molecular Dynamics Simulation , Spectrum Analysis, Raman , Humans , Molecular Docking Simulation , Molecular Conformation , Spectroscopy, Fourier Transform Infrared , Static Electricity , Thermodynamics , Spectrophotometry, Ultraviolet , Pentoxyl , Quantum Theory
4.
Methods Mol Biol ; 2198: 169-181, 2021.
Article in English | MEDLINE | ID: mdl-32822031

ABSTRACT

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.


Subject(s)
DNA/immunology , Immunohistochemistry/methods , 5-Methylcytosine/metabolism , Animals , Antibodies/metabolism , Cell Nucleus/metabolism , Cytosine/analogs & derivatives , Cytosine/analysis , DNA/genetics , DNA Methylation/immunology , Epigenesis, Genetic/genetics , Humans , Pentoxyl/analogs & derivatives , Pentoxyl/analysis , Peroxidase/chemistry
5.
Methods Mol Biol ; 2198: 269-284, 2021.
Article in English | MEDLINE | ID: mdl-32822038

ABSTRACT

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.


Subject(s)
Flow Cytometry/methods , Pentoxyl/analogs & derivatives , Single-Cell Analysis/methods , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/analysis , 5-Methylcytosine/blood , Chromatography, Liquid , Cytosine/metabolism , DNA/genetics , DNA Methylation/physiology , Epigenesis, Genetic/physiology , Humans , Oxidation-Reduction , Pentoxyl/analysis , Pentoxyl/blood , Pentoxyl/metabolism , Tandem Mass Spectrometry , Thymine/metabolism
6.
J Am Chem Soc ; 142(6): 2889-2896, 2020 02 12.
Article in English | MEDLINE | ID: mdl-31986025

ABSTRACT

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.


Subject(s)
5-Methylcytosine/analogs & derivatives , Hydrogels/chemistry , Microfluidics , Pentoxyl/analogs & derivatives , Single-Cell Analysis/methods , 5-Methylcytosine/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , DNA/genetics , Epigenesis, Genetic , Female , Humans , Neoplasm Invasiveness , Pentoxyl/metabolism
8.
Int J Mol Sci ; 20(20)2019 Oct 10.
Article in English | MEDLINE | ID: mdl-31658654

ABSTRACT

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.


Subject(s)
DNA Replication/drug effects , DNA/biosynthesis , DNA/drug effects , Oxidative Stress , Pyrimidines/pharmacology , DNA Damage , DNA Repair , DNA-Directed DNA Polymerase/metabolism , HIV-1 , Humans , Mutagens/chemistry , Mutagens/metabolism , Mutagens/pharmacology , Oxidation-Reduction , Pentoxyl/analogs & derivatives , Pentoxyl/chemistry , Pentoxyl/metabolism , Pentoxyl/pharmacology , Pyrimidines/chemistry , Pyrimidines/metabolism , Thermodynamics , Uracil/analogs & derivatives , Uracil/chemistry , Uracil/metabolism , Uracil/pharmacology
9.
Sci Rep ; 8(1): 6860, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29717169

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans , DNA Glycosylases/metabolism , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Uracil-DNA Glycosidase/metabolism , Animals , Apoptosis , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , DNA Damage/genetics , DNA Glycosylases/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Germ Cells/metabolism , Longevity/genetics , Loss of Function Mutation , Pentoxyl/analogs & derivatives , Pentoxyl/metabolism , Uracil-DNA Glycosidase/genetics
10.
Org Biomol Chem ; 16(9): 1527-1535, 2018 02 28.
Article in English | MEDLINE | ID: mdl-29431832

ABSTRACT

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).


Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/biosynthesis , DNA/chemistry , Light , Nucleotides/chemistry , Pentoxyl/analogs & derivatives , Photochemical Processes , Models, Molecular , Nucleic Acid Conformation , Pentoxyl/chemistry
11.
Nucleic Acids Res ; 46(4): 1624-1634, 2018 02 28.
Article in English | MEDLINE | ID: mdl-29378012

ABSTRACT

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.


Subject(s)
DNA/chemistry , Telomere/chemistry , Adenine/analogs & derivatives , Adenine/chemistry , Cytosine/chemistry , DNA Damage , Humans , Pentoxyl/analogs & derivatives , Pentoxyl/chemistry , Uracil/chemistry
12.
Chem Commun (Camb) ; 53(99): 13253-13255, 2017 Dec 12.
Article in English | MEDLINE | ID: mdl-29184924

ABSTRACT

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.


Subject(s)
Cytosine/metabolism , DNA-Directed RNA Polymerases/genetics , Epigenesis, Genetic/genetics , Escherichia coli/enzymology , Pentoxyl/analogs & derivatives , Transcription, Genetic/genetics , Cytosine/chemistry , DNA-Directed RNA Polymerases/metabolism , Pentoxyl/chemistry , Pentoxyl/metabolism
13.
Genome Biol ; 18(1): 23, 2017 01 30.
Article in English | MEDLINE | ID: mdl-28137275

ABSTRACT

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.


Subject(s)
Chromosome Mapping/methods , DNA, Protozoan/genetics , Leishmania/genetics , Pentoxyl/analogs & derivatives , DNA, Protozoan/chemistry , Glucosides/chemistry , Leishmania/chemistry , Pentoxyl/chemistry , Uracil/analogs & derivatives , Uracil/chemistry
14.
Orig Life Evol Biosph ; 47(1): 3-11, 2017 Mar.
Article in English | MEDLINE | ID: mdl-27029792

ABSTRACT

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.


Subject(s)
5-Methylcytosine/analogs & derivatives , Evolution, Chemical , Pentoxyl/analogs & derivatives , Polymerization , Water/chemistry , 5-Methylcytosine/chemistry , Hot Temperature , Origin of Life , Pentoxyl/chemistry
15.
Free Radic Biol Med ; 101: 378-383, 2016 12.
Article in English | MEDLINE | ID: mdl-27833031

ABSTRACT

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.


Subject(s)
5-Methylcytosine/analogs & derivatives , Ascorbic Acid/pharmacology , DNA/metabolism , Pentoxyl/analogs & derivatives , 5-Methylcytosine/agonists , 5-Methylcytosine/metabolism , Ascorbic Acid/metabolism , Cytosine/agonists , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA Methylation/drug effects , HCT116 Cells , Humans , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Pentoxyl/agonists , Pentoxyl/metabolism , Proto-Oncogene Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Thymine/agonists , Thymine/metabolism
16.
Acta Crystallogr C Struct Chem ; 72(Pt 5): 379-88, 2016 05 01.
Article in English | MEDLINE | ID: mdl-27146565

ABSTRACT

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.


Subject(s)
Pentoxyl/analogs & derivatives , Thiouracil/analogs & derivatives , Uracil/analogs & derivatives , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Conformation , Pentoxyl/chemistry
17.
Mutat Res Rev Mutat Res ; 767: 59-66, 2016.
Article in English | MEDLINE | ID: mdl-27036066

ABSTRACT

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.


Subject(s)
DNA Repair/genetics , DNA/genetics , Pentoxyl/analogs & derivatives , 5-Methylcytosine/chemistry , Animals , Bacteriophages/genetics , Humans , Hydroxylation/physiology , Mice , Oxidation-Reduction , Pentoxyl/chemistry , Pentoxyl/metabolism , Rats , Reactive Oxygen Species/metabolism , Thymine/chemistry , Thymine/metabolism
18.
Nucleic Acids Res ; 44(8): 3713-27, 2016 05 05.
Article in English | MEDLINE | ID: mdl-26843430

ABSTRACT

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.


Subject(s)
5-Methylcytosine/metabolism , DNA Mismatch Repair , DNA Repair , Animals , Cell Line , Cell Line, Tumor , DNA/chemistry , DNA/metabolism , Gene Expression , Humans , Mice , Mice, Knockout , MutS Homolog 2 Protein/genetics , Pentoxyl/analogs & derivatives , Pentoxyl/metabolism , Promoter Regions, Genetic , Uracil/metabolism
19.
Nucleic Acids Res ; 44(5): 2085-92, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26578595

ABSTRACT

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.


Subject(s)
DNA/chemistry , Molecular Dynamics Simulation , Pentoxyl/analogs & derivatives , Thymine/chemistry , DNA-Binding Proteins/chemistry , Epigenesis, Genetic , Hydrophobic and Hydrophilic Interactions , Nanopores , Nucleic Acid Denaturation , Oxidation-Reduction , Pentoxyl/chemistry , Protein Binding , Surface Properties
20.
PLoS One ; 10(12): e0144859, 2015.
Article in English | MEDLINE | ID: mdl-26660343

ABSTRACT

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.


Subject(s)
Cytosine/analogs & derivatives , DNA/metabolism , Epigenesis, Genetic , Pentoxyl/analogs & derivatives , 5-Methylcytosine/analogs & derivatives , Animals , Brain Chemistry , Chromatography, High Pressure Liquid , Cytosine/metabolism , DNA/genetics , DNA Methylation , Dioxygenases/genetics , Dioxygenases/metabolism , Gene Expression , Kidney/chemistry , Kidney/metabolism , Liver/chemistry , Liver/metabolism , Lung/chemistry , Lung/metabolism , Male , Myocardium/chemistry , Myocardium/metabolism , Organ Specificity , Pentoxyl/metabolism , Rats , Rats, Wistar , Swine , Tandem Mass Spectrometry , Thymus Gland/chemistry , Thymus Gland/metabolism
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