205 results on '"Pentoxyl"'
Search Results
2. Differentiated Visualization of Single-Cell 5-Hydroxymethylpyrimidines with Microfluidic Hydrogel Encoding
- Author
-
Jing Xue, Yongxi Zhao, Xu Yu, Chunhai Fan, Min Bai, Feng Chen, and Zhang Jin
- Subjects
Lysis ,Microfluidics ,Cell ,Breast Neoplasms ,Computational biology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Genome ,Catalysis ,Epigenesis, Genetic ,Pentoxyl ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cell Line, Tumor ,medicine ,Humans ,Neoplasm Invasiveness ,Gene ,Hydrogels ,DNA ,General Chemistry ,0104 chemical sciences ,genomic DNA ,medicine.anatomical_structure ,chemistry ,Cell culture ,5-Methylcytosine ,Agarose ,Female ,Single-Cell Analysis - 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.
- Published
- 2020
3. Quantum Computational, Spectroscopic (FT-IR, FT-Raman, NMR, and UV-Vis) Hirshfeld Surface and Molecular Docking-Dynamics Studies on 5-Hydroxymethyluracil (Monomer and Trimer).
- Author
-
Kumar M, Jaiswar G, Afzal M, Muddassir M, Alarifi A, Fatima A, Siddiqui N, Ayub R, Abduh NAY, Saeed WS, and Javed S
- Subjects
- Humans, Molecular Docking Simulation, Molecular Conformation, Spectroscopy, Fourier Transform Infrared, Static Electricity, Thermodynamics, Spectrophotometry, Ultraviolet, Pentoxyl, Quantum Theory, Molecular Dynamics Simulation, Spectrum Analysis, Raman
- 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.
- Published
- 2023
- Full Text
- View/download PDF
4. Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry
- Author
-
Lidia, Gackowska, Anna, Labejszo, and Daniel, Gackowski
- Subjects
Pentoxyl ,Cytosine ,Tandem Mass Spectrometry ,5-Methylcytosine ,Humans ,DNA ,DNA Methylation ,Single-Cell Analysis ,Flow Cytometry ,Oxidation-Reduction ,Thymine ,Chromatography, Liquid ,Epigenesis, Genetic - 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.
- Published
- 2020
5. Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry
- Author
-
Abdulkadir, Abakir and Alexey, Ruzov
- Subjects
Cell Nucleus ,Pentoxyl ,Cytosine ,5-Methylcytosine ,Animals ,Humans ,DNA ,DNA Methylation ,Immunohistochemistry ,Antibodies ,Epigenesis, Genetic ,Peroxidase - 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.
- Published
- 2020
6. UNG-1 and APN-1 are the major enzymes to efficiently repair 5-hydroxymethyluracil DNA lesions in C. elegans
- Author
-
J. Richard Wagner, Arturo Papaluca, Dindial Ramotar, and H. Uri Saragovi
- Subjects
0301 basic medicine ,DNA Repair ,Science ,Longevity ,Mutant ,Apoptosis ,Article ,DNA Glycosylases ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Loss of Function Mutation ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Animals ,AP site ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Uracil-DNA Glycosidase ,Endodeoxyribonucleases ,Multidisciplinary ,biology ,Wild type ,Uracil ,Endonucleases ,biology.organism_classification ,Thymine ,Cell biology ,Germ Cells ,030104 developmental biology ,chemistry ,DNA glycosylase ,Medicine ,DNA ,DNA Damage - 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.
- Published
- 2018
7. i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions
- Author
-
Zuzana Dvorakova, Petra Školáková, Daniel Renčiuk, Iva Kejnovská, Michaela Vorlíčková, Janos Sagi, and Klára Bednárová
- Subjects
0301 basic medicine ,DNA damage ,Adenine ,Deamination ,Uracil ,DNA ,Telomere ,Biology ,Thymine ,Pentoxyl ,Cytosine ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Chemical Biology and Nucleic Acid Chemistry ,chemistry ,Biochemistry ,Genetics ,Humans ,AP site ,Gene ,DNA Damage - 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.
- Published
- 2018
8. Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA
- Author
-
Zuzana Vaníková, Petr Klán, Sona Bohacova, Lenka Slavetinska Postova, Michal Hocek, and Lucie Ludvíková
- Subjects
Models, Molecular ,Light ,DNA polymerase ,Stereochemistry ,DNA-Directed DNA Polymerase ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Pentoxyl ,chemistry.chemical_compound ,Nucleotide ,Physical and Theoretical Chemistry ,Polymerase ,chemistry.chemical_classification ,biology ,Nucleotides ,010405 organic chemistry ,Oligonucleotide ,Organic Chemistry ,Uracil ,DNA ,Photochemical Processes ,0104 chemical sciences ,chemistry ,biology.protein ,Nucleic acid ,Nucleic Acid Conformation ,Uracil nucleotide - Abstract
Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxy-methyl) 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 mono-phosphates 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).
- Published
- 2018
9. Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA
- Author
-
Joanna Rzeszowska-Wolny, Marta Starczak, Daniel Gackowski, Ewelina Zarakowska, Maciej Gawronski, Marek Foksinski, Martyna Modrzejewska, Magdalena Skonieczna, and Ryszard Olinski
- Subjects
0301 basic medicine ,Spectrometry, Mass, Electrospray Ionization ,Ascorbic Acid ,Biochemistry ,Mixed Function Oxygenases ,Pentoxyl ,Cytosine ,03 medical and health sciences ,chemistry.chemical_compound ,Proto-Oncogene Proteins ,Physiology (medical) ,Humans ,Demethylation ,chemistry.chemical_classification ,5-Hydroxymethylcytosine ,Vitamin C ,DNA ,DNA Methylation ,HCT116 Cells ,Ascorbic acid ,Thymine ,030104 developmental biology ,Enzyme ,chemistry ,5-Methylcytosine ,Oxidation-Reduction ,Intracellular - 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.
- Published
- 2016
10. 5-formylcytosine and 5-hydroxymethyluracil as surrogate markers of TET2 and SF3B1 mutations in myelodysplastic syndrome, respectively
- Author
-
Tomas Radivoyevitch, Maciej Gawronski, Cassandra M Kerr, Marta Starczak, Ewelina Zarakowska, Daniel Gackowski, Alexey Ruzov, Ryszard Olinski, Abdulkadir Abakir, and Jaroslaw P. Maciejewski
- Subjects
Extramural ,Hematology ,Biology ,Phosphoproteins ,Dioxygenases ,DNA-Binding Proteins ,Pentoxyl ,Cytosine ,5-formylcytosine ,Myelodysplastic Syndromes ,Proto-Oncogene Proteins ,Mutation (genetic algorithm) ,Mutation ,Cancer research ,Humans ,RNA Splicing Factors ,Online Only Articles ,Biomarkers - Published
- 2019
11. Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis
- Author
-
Viktor Brabec, Ondrej Hrabina, and Olga Novakova
- Subjects
0301 basic medicine ,DNA Repair ,DNA polymerase ,2’-deoxyribo-5-hydroxyuridin ,translesion dna synthesis ,DNA-Directed DNA Polymerase ,01 natural sciences ,Pentoxyl ,lcsh:Chemistry ,chemistry.chemical_compound ,lcsh:QH301-705.5 ,Spectroscopy ,Klenow fragment ,biology ,food and beverages ,General Medicine ,Computer Science Applications ,Biochemistry ,Thermodynamics ,Oxidation-Reduction ,DNA Replication ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,Humans ,Physical and Theoretical Chemistry ,Uracil ,2’-deoxyribo-5-hydroxymethyl- uridin ,oxidized nucleotides ,Molecular Biology ,DNA synthesis ,010405 organic chemistry ,Microscale thermophoresis ,fungi ,Organic Chemistry ,dna polymerases ,DNA ,microscale thermophoresis ,Reverse transcriptase ,0104 chemical sciences ,Oxidative Stress ,Pyrimidines ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,HIV-1 ,biology.protein ,DNA polymerase I ,Thymidine ,DNA Damage ,Mutagens - Abstract
Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2&rsquo, deoxyribo-5-hydroxyuridin (HdU) and 2&rsquo, 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 &eta, (pol&eta, ), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF&ndash, ), 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.
- Published
- 2019
12. 5-(Hydroxymethyl)uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase
- Author
-
Hana Šanderová, Libor Krásný, Michal Hocek, Dragana Vítovská, Martina Janoušková, Fabrizia Nici, Zuzana Vaníková, and Soňa Boháčová
- Subjects
0301 basic medicine ,Transcription, Genetic ,Response element ,RNA polymerase II ,Catalysis ,Epigenesis, Genetic ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosine ,Transcription (biology) ,RNA polymerase ,Materials Chemistry ,Escherichia coli ,Polymerase ,biology ,General transcription factor ,Chemistry ,Metals and Alloys ,Promoter ,General Chemistry ,DNA-Directed RNA Polymerases ,Molecular biology ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,030104 developmental biology ,Biochemistry ,Ceramics and Composites ,biology.protein ,Transcription factor II D - 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.
- Published
- 2017
13. Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA
- Author
-
Edwin Pozharski, Christopher T. Coey, Alexander C. Drohat, Shuja Shafi Malik, and Kristen M. Varney
- Subjects
Models, Molecular ,Stereochemistry ,Base Pair Mismatch ,Deamination ,Biology ,Crystallography, X-Ray ,Pentoxyl ,chemistry.chemical_compound ,Catalytic Domain ,Genetics ,Humans ,Uracil ,Nuclear Magnetic Resonance, Biomolecular ,Nucleic Acid Enzymes ,Base excision repair ,DNA ,Thymine DNA Glycosylase ,Thymine ,chemistry ,Biochemistry ,DNA glycosylase ,Thymine-DNA glycosylase ,Protein Binding - Abstract
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 A) 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.
- Published
- 2015
14. Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling
- Author
-
Miao Yu, Chun-Xiao Song, and Chuan He
- Subjects
Epigenomics ,Mammals ,Deamination ,DNA ,Base excision repair ,DNA Methylation ,Biology ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,Pentoxyl ,chemistry.chemical_compound ,DNA demethylation ,Biochemistry ,chemistry ,DNA methylation ,Click chemistry ,Animals ,Molecular Biology ,Cytosine - Abstract
How DNA demethylation is achieved in mammals is still under extensive investigation. One proposed mechanism is deamination of 5-hydroxymethylcytosine to form 5-hydroxymethyluracil (5hmU), followed by base excision repair to replace the mismatched 5hmU with cytosine. In this process, 5hmU:G mispair serves as a key intermediate and its localization and distribution in mammalian genome could be important information to investigate the proposed pathway. Here we describe a selective labeling method to map mismatched 5hmU. After converting other cytosine modifications to 5-carboxylcytosines, a biotin tag is installed onto mismatched 5hmU through β-glucosyltransferase-catalyzed glucosylation and click chemistry. The enriched 5hmU-containing DNA fragments can be subject to subsequent sequencing to reveal the distribution of 5hmU:G mispair with base-resolution information acquired.
- Published
- 2015
15. Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania
- Author
-
Kawasaki, Fumiko, Beraldi, Dario, Hardisty, Robyn E, McInroy, Gordon R, van Delft, Pieter, Balasubramanian, Shankar, van Delft, Pieter [0000-0002-4726-9467], Balasubramanian, Shankar [0000-0002-0281-5815], and Apollo - University of Cambridge Repository
- Subjects
Leishmania ,Pentoxyl ,5-Hydroxymethyluracil (5hmU) ,Glucosides ,Research ,Base J ,5-Formyluracil (5fU) ,Chromosome Mapping ,DNA, Protozoan ,Uracil ,Genome-wide mapping ,Leishmania major ,Leishmania donovani - 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-(β-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. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1150-1) contains supplementary material, which is available to authorized users.
- Published
- 2017
16. Thermodynamics of organic molecule adsorption on sorbents modified with 5-hydroxy-6-methyluracil by inverse gas chromatography
- Author
-
Vladimir Yu. Gus’kov, Sergey P. Ivanov, Florida Kh. Kudasheva, and Yulia Yu. Gainullina
- Subjects
Chromatography, Gas ,Sorbent ,Surface Properties ,Entropy ,Molecular Conformation ,Supramolecular chemistry ,Thermodynamics ,Crystallography, X-Ray ,Biochemistry ,Analytical Chemistry ,Pentoxyl ,chemistry.chemical_compound ,Adsorption ,Alkanes ,Inverse gas chromatography ,Porosity ,Chromatography ,Internal energy ,Chemistry ,Hydrogen bond ,Silica gel ,Organic Chemistry ,Hydrogen Bonding ,General Medicine ,Alcohols - Abstract
The thermodynamic features of organic molecule adsorption from the gaseous phase of sorbents modified with 5-hydroxy-6-methyluracil (HMU) were studied. Molar internal energy and entropy of adsorption variation analyses showed that with every type surface, except for silica gel, layers of supramolecular structure have cavities equal in size with the ones revealed in HMU crystals by X-ray diffraction. Adsorption thermodynamics on HMU-modified sorbents depended on the amount of impregnated HMU and on the polarity, but not the porosity, of the initial sorbent. Polarity of the modified surface increased as a function of HMU quantity and initial sorbent mean pore size, but become appreciably lower if the initial surface is capable of hydrogen bonding.
- Published
- 2014
17. Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome
- Author
-
Shuo Liu, Jessica Lopes da Rosa-Spiegler, Robert Sabatini, Whitney Bullard, and Yinsheng Wang
- Subjects
Trypanosoma brucei brucei ,Base J ,Protozoan Proteins ,Context (language use) ,macromolecular substances ,DNA and Chromosomes ,Biology ,medicine.disease_cause ,Biochemistry ,Genome ,Epigenesis, Genetic ,Substrate Specificity ,Pentoxyl ,Gene Knockout Techniques ,chemistry.chemical_compound ,Glucosides ,parasitic diseases ,medicine ,RNA, Messenger ,Uracil ,Molecular Biology ,Mutation ,fungi ,Cell Biology ,DNA, Protozoan ,Thymine ,carbohydrates (lipids) ,chemistry ,Glucosyltransferases ,Mutagenesis, Site-Directed ,biology.protein ,RNA Interference ,Glucosyltransferase ,Genome, Protozoan ,RNA, Protozoan ,DNA - Abstract
O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.
- Published
- 2014
18. Polymerase Synthesis of Photocaged DNA Resistant against Cleavage by Restriction Endonucleases
- Author
-
Michal Hocek and Zuzana Vaníková
- Subjects
DNA clamp ,biology ,DNA polymerase ,Ultraviolet Rays ,DNA polymerase II ,General Chemistry ,DNA ,DNA Restriction Enzymes ,DNA-Directed DNA Polymerase ,General Medicine ,Molecular biology ,Catalysis ,Restriction fragment ,Pentoxyl ,chemistry.chemical_compound ,Restriction enzyme ,chemistry ,Biochemistry ,biology.protein ,Flap endonuclease ,Polymerase - Abstract
5-[(2-Nitrobenzyl)oxymethyl]-2′-deoxyuridine 5′-O-triphosphate was used for polymerase (primer extension or PCR) synthesis of photocaged DNA that is resistant to the cleavage by restriction endonucleases. Photodeprotection of the caged DNA released 5-hydroxymethyluracil-modified nucleic acids, which were fully recognized and cleaved by restriction enzymes.
- Published
- 2014
19. A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS–UHPLC analysis of four urinary biomarkers
- Author
-
Berta Rodrigues Mendes, Jorge A. M. Pereira, Pedro Silva, José S. Câmara, and Isabel Mendonça
- Subjects
Adult ,Male ,Analyte ,Accuracy and precision ,Ultra high pressure liquid chromatography ,Formic acid ,Hypercholesterolemia ,Microextraction by packed sorbent (MEPS) ,Urine ,Analytical Chemistry ,Pentoxyl ,Faculdade de Ciências Exatas e da Engenharia ,chemistry.chemical_compound ,Limit of Detection ,Risk Factors ,Malondialdehyde ,Humans ,Cardiovascular diseases (CVDs) ,Chromatography, High Pressure Liquid ,Solid Phase Microextraction ,Detection limit ,Reproducibility ,Chromatography ,Elution ,Extraction (chemistry) ,Oxidative stress biomarkers (OSBs) ,Deoxyguanosine ,Reproducibility of Results ,Hydrogen-Ion Concentration ,Middle Aged ,Uric Acid ,Oxidative Stress ,chemistry ,8-Hydroxy-2'-Deoxyguanosine ,Case-Control Studies ,Calibration ,Hypertension ,Disease Progression ,Female ,Urinary metabolomic profile ,Biomarkers - Abstract
In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS/UHPLC-PDA method were also very satisfactory. Finally, the application of the methodology to the determination of target biomarkers in normal subjects and CVDs patients' revealed that the DNA adducts 5-HMUra and 8-oxodG levels are much more abundant in CVDs patients while no statistic differences were obtain for MDA and UAc. This result points to the importance of 5-HMUra and 8-oxodG as biomarkers of CVDs risk progression and further epidemiological studies are needed to explore the importance of this correlation.
- Published
- 2013
20. Side-by-Side Comparison of DNA Damage Induced by Low-Energy Electrons and High-Energy Photons with Solid TpTpT Trinucleotide
- Author
-
Yeunsoo Park, J. Richard Wagner, Guru S. Madugundu, Anita R. Peoples, and Léon Sanche
- Subjects
DNA damage ,Radical ,Oligonucleotides ,Analytical chemistry ,Electrons ,Tandem mass spectrometry ,Photochemistry ,Article ,Ionizing radiation ,Pentoxyl ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Radiation, Ionizing ,Ionization ,Materials Chemistry ,Physical and Theoretical Chemistry ,Uracil ,Chromatography, High Pressure Liquid ,Photons ,Oligonucleotide ,Surfaces, Coatings and Films ,Thymine ,chemistry ,DNA ,DNA Damage - Abstract
The genotoxic effects of high-energy ionizing radiation have been largely attributed to the ionization of H2O leading to hydroxyl radicals and the ionization of DNA leading mostly to damage through base radical cations. However, the contribution of low-energy electrons (LEEs; ≤ 10 eV), which involves subionization events, has been considered to be less important than that of hydroxyl radicals and base radical cations. Here, we compare the ability of LEEs and high-energy X-ray photons to induce DNA damage using dried thin films of TpTpT trinucleotide as a simple and representative model for DNA damage. The main radiation-induced damage of TpTpT as measured by high-performance liquid chromatography (HPLC) with UV detection and HPLC coupled to tandem mass spectrometry analyses included thymine release (-Thy), strand breaks (pT, Tp, pTpT, TpTp, and TpT), and the formation of base modifications [5,6-dihydrothymine (5,6-dhT), 5-hydroxymethyluracil (5-hmU), and 5-formyluracil (5-fU)]. The global profile of products was very similar for both types of radiation indicating converging pathways of formation. The percent damage of thymine release, fragmentation, and base modification was 20, 19, and 61 for high-energy X-rays, respectively, compared to 35, 13, and 51 for LEEs (10 eV). Base release was significantly lower for X-rays. In both cases, phosphodiester bond cleavage gave mononucleotides (pT and Tp) and dinucleotides (pTpT and TpTp) containing a terminal phosphate as the major fragments. For base modifications, the ratio of reductive (5,6-dhT) to oxidative products (5-hmU plus 5-fU) was 0.9 for high-energy X-rays compared to 1.7 for LEEs. These results indicate that LEEs give a similar profile of products compared to ionizing radiation.
- Published
- 2013
21. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation
- Author
-
Inga R. Grin and Alexander A. Ishchenko
- Subjects
0301 basic medicine ,DNA Repair ,DNA repair ,Gene Expression ,Biology ,Genome Integrity, Repair and Replication ,DNA Mismatch Repair ,Cell Line ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,Animals ,Humans ,Promoter Regions, Genetic ,Uracil ,Mice, Knockout ,Base excision repair ,DNA ,Very short patch repair ,5-Methylcytosine ,030104 developmental biology ,DNA demethylation ,MutS Homolog 2 Protein ,Biochemistry ,chemistry ,DNA glycosylase ,DNA mismatch repair ,030217 neurology & neurosurgery ,Nucleotide excision repair - 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.
- Published
- 2016
22. Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation
- Author
-
Ashok S. Bhagwat, Samuel Hong, Xing Zhang, Xiaodong Cheng, and Hideharu Hashimoto
- Subjects
Models, Molecular ,Guanine ,Deamination ,Biology ,Gene Regulation, Chromatin and Epigenetics ,010402 general chemistry ,01 natural sciences ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosine ,Catalytic Domain ,Genetics ,Humans ,030304 developmental biology ,0303 health sciences ,Uracil ,DNA ,Thymine DNA Glycosylase ,0104 chemical sciences ,Thymine ,DNA demethylation ,chemistry ,Biochemistry ,DNA glycosylase ,Biocatalysis ,Thymine-DNA glycosylase - Abstract
The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.
- Published
- 2012
23. Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation
- Author
-
Xiaodong Cheng, Hideharu Hashimoto, and Xing Zhang
- Subjects
Models, Molecular ,Base Pair Mismatch ,Guanine ,Deamination ,Gene Regulation, Chromatin and Epigenetics ,Biology ,DNA Glycosylases ,MBD4 ,Pentoxyl ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Genetics ,Animals ,030304 developmental biology ,0303 health sciences ,Endodeoxyribonucleases ,030302 biochemistry & molecular biology ,DNA ,Protein Structure, Tertiary ,Very short patch repair ,Thymine ,Biochemistry ,chemistry ,DNA glycosylase ,Cytosine ,Nucleotide excision repair - Abstract
The mammalian DNA glycosylase--methyl-CpG binding domain protein 4 (MBD4)--is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson-Crick polar edge of thymine or 5hmU via the O2, N3 and O4 atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C1' of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.
- Published
- 2012
24. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation
- Author
-
Xiaodong Cheng, Hideharu Hashimoto, Anup K. Upadhyay, Paula M. Vertino, Xing Zhang, Shelley B. Howerton, Yiwei Liu, and Yanqi Chang
- Subjects
DNA (Cytosine-5-)-Methyltransferase 1 ,DNA Replication ,Deamination ,Gene Regulation, Chromatin and Epigenetics ,Biology ,Pentoxyl ,Cytosine ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,Humans ,DNA (Cytosine-5-)-Methyltransferases ,030304 developmental biology ,0303 health sciences ,DNA replication ,Molecular biology ,Thymine DNA Glycosylase ,3. Good health ,Thymine ,DNA-Binding Proteins ,chemistry ,CpG site ,DNA glycosylase ,030220 oncology & carcinogenesis ,5-Methylcytosine ,Thymine-DNA glycosylase ,DNA - Abstract
Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.
- Published
- 2012
25. Far-UV Photochemical Bond Cleavage of n-Amyl Nitrite: Bypassing a Repulsive Surface
- Author
-
Sanghamitra Deb, Rasmus Y. Brogaard, Martin Rosenberg, Peter M. Weber, Yao Zhang, Michael P. Minitti, and Theis I. Sølling
- Subjects
Photolysis ,Free Radicals ,Surface Properties ,Ultraviolet Rays ,Chemistry ,Photoelectron Spectroscopy ,Radical ,Photodissociation ,Photoionization ,Nitric Oxide ,Photochemistry ,Mass Spectrometry ,Dissociation (chemistry) ,Homolysis ,Pentoxyl ,Kinetics ,Ionization ,Potential energy surface ,Quantum Theory ,Amyl Nitrite ,Physical and Theoretical Chemistry ,Bond cleavage - Abstract
We have investigated the deep-UV photoinduced, homolytic bond cleavage of amyl nitrite to form NO and pentoxy radicals. One-color multiphoton ionization with ultrashort laser pulses through the S(2) state resonance gives rise to photoelectron spectra that reflect ionization from the S(1) state. Time-resolved pump-probe photoionization measurements show that upon excitation at 207 nm, the generation of NO in the v = 2 state is delayed, with a rise time of 283 (16) fs. The time-resolved mass spectrum shows the NO to be expelled with a kinetic energy of 1.0 eV, which is consistent with dissociation on the S(1) state potential energy surface. Combined, these observations show that the first step of the dissociation reaction involves an internal conversion from the S(2) to the S(1) state, which is followed by the ejection of the NO radical on the predissociative S(1) state potential energy surface.
- Published
- 2012
26. Enigmatic 5-hydroxymethyluracil: Oxidatively modified base, epigenetic mark or both?
- Author
-
Ryszard Olinski, Marta Starczak, and Daniel Gackowski
- Subjects
0301 basic medicine ,DNA Repair ,Guanine ,DNA repair ,Health, Toxicology and Mutagenesis ,Deamination ,Biology ,Hydroxylation ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Genetics ,Animals ,Humans ,Bacteriophages ,Epigenetics ,5-Hydroxymethylcytosine ,DNA ,Thymine ,Rats ,5-Methylcytosine ,030104 developmental biology ,Biochemistry ,chemistry ,Reactive Oxygen Species ,Oxidation-Reduction - 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.
- Published
- 2015
27. Base J Glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA
- Author
-
Whitney Bullard, Pengcheng Wang, Robert Sabatini, Yinsheng Wang, and Laura Cliffe
- Subjects
Leishmania ,Mutant ,Base J ,Protozoan Proteins ,Biology ,DNA, Protozoan ,Telomere ,Molecular biology ,DNA sequencing ,Article ,Thymine ,Pentoxyl ,chemistry.chemical_compound ,Kinetics ,chemistry ,Biochemistry ,Glucosyltransferases ,biology.protein ,Parasitology ,Glucosyltransferase ,Thymidine ,Molecular Biology ,DNA - Abstract
Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.
- Published
- 2015
28. Hydroxymethyluracil modifications enhance the flexibility and hydrophilicity of double-stranded DNA
- Author
-
James Wilson, Aleksei Aksimentiev, Meni Wanunu, Spencer Carson, and Peter Weigele
- Subjects
0301 basic medicine ,HMG-box ,Surface Properties ,Biology ,Molecular Dynamics Simulation ,Nucleic Acid Denaturation ,Epigenesis, Genetic ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,Nanopores ,Genetics ,A-DNA ,chemistry.chemical_classification ,DNA ligase ,Gene regulation, Chromatin and Epigenetics ,DNA ,Thymine ,DNA-Binding Proteins ,Nanopore ,030104 developmental biology ,Biochemistry ,chemistry ,Biophysics ,Hydrophobic and Hydrophilic Interactions ,Oxidation-Reduction ,Protein Binding - 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.
- Published
- 2015
29. Urinary 5-hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer
- Author
-
Ryszard Olinski, Martyna Modrzejewska, Zbigniew Banaszkiewicz, Rafal Rozalski, Agnieszka Siomek-Gorecka, Daniel Gackowski, and Marta Starczak
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Guanine ,Colorectal cancer ,DNA damage ,DNA repair ,Health, Toxicology and Mutagenesis ,Urinary system ,Clinical Biochemistry ,Inflammation ,medicine.disease_cause ,Biochemistry ,Gastroenterology ,Pentoxyl ,Internal medicine ,medicine ,Biomarkers, Tumor ,Humans ,Aged ,Receiver operating characteristic ,business.industry ,Area under the curve ,Middle Aged ,medicine.disease ,8-Hydroxy-2'-Deoxyguanosine ,Female ,medicine.symptom ,business ,Colorectal Neoplasms ,Oxidative stress - Abstract
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
- Published
- 2015
30. Selective Chemical Labeling of Natural T Modifications in DNA
- Author
-
Hardisty, Robyn E, Kawasaki, Fumiko, Sahakyan, Aleksandr B, Balasubramanian, Shankar, Sahakyan, Aleksandr [0000-0002-8343-3594], Balasubramanian, Shankar [0000-0002-0281-5815], and Apollo - University of Cambridge Repository
- Subjects
Models, Molecular ,Pentoxyl ,Base Sequence ,Oligodeoxyribonucleotides ,Nucleic Acid Conformation ,DNA ,Uracil ,Thymine - Abstract
We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.
- Published
- 2015
31. Selective Chemical Labeling of Natural T Modifications in DNA
- Author
-
Robyn E, Hardisty, Fumiko, Kawasaki, Aleksandr B, Sahakyan, and Shankar, Balasubramanian
- Subjects
Models, Molecular ,Pentoxyl ,Base Sequence ,Oligodeoxyribonucleotides ,Communication ,Nucleic Acid Conformation ,DNA ,Uracil ,Thymine - Abstract
We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.
- Published
- 2015
32. Spontaneous Oligomerization of Nucleotide Alternatives in Aqueous Solutions
- Author
-
Christopher H. House, Jason P. Dworkin, Michael P. Callahan, and Karen E. Smith
- Subjects
0301 basic medicine ,Hot Temperature ,Origin of Life ,01 natural sciences ,Polymerization ,Pentoxyl ,03 medical and health sciences ,chemistry.chemical_compound ,0103 physical sciences ,Organic chemistry ,Nucleotide ,010303 astronomy & astrophysics ,Ecology, Evolution, Behavior and Systematics ,chemistry.chemical_classification ,Aqueous solution ,Evolution, Chemical ,RNA ,Water ,Uracil ,General Medicine ,Combinatorial chemistry ,5-Methylcytosine ,030104 developmental biology ,chemistry ,Space and Planetary Science ,Cytosine ,DNA - 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.
- Published
- 2015
33. DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the hNEIL1 and hNTH1 enzymes in human cells
- Author
-
Akira Yasui, Masashi Takao, Qiu-Mei Zhang, Shuji Yonei, Shin Ichiro Yonekura, and Hiroshi Sugiyama
- Subjects
DNA, Complementary ,DNA Repair ,Mutant ,Oligonucleotides ,Biology ,medicine.disease_cause ,Biochemistry ,DNA Glycosylases ,Pentoxyl ,Deoxyribonuclease (Pyrimidine Dimer) ,chemistry.chemical_compound ,Escherichia coli ,medicine ,Humans ,Cloning, Molecular ,Uracil ,Molecular Biology ,Oligonucleotide ,Hydrogen Peroxide ,Cell Biology ,Base excision repair ,Molecular biology ,Thymine ,chemistry ,DNA glycosylase ,Mutation ,DNA ,Nucleotide excision repair - Abstract
Bacteria and eukaryotes possess redundant activities that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases excise damaged bases to initiate the base excision repair pathway. hOgg1 and hNTH1, homologues of E. coli MutM and Nth, respectively, had been identified and characterized in human cells. Recent works revealed that human cells have three orthologues of E. coli Nei, hNEIL1, hNEIL2 and hNEIL3. In the present experiments, hNEIL1 protected the E. coli nth nei mutant from lethal effect of hydrogen peroxide and high frequency of spontaneous mutations under aerobic conditions. Furthermore, hNEIL1 efficiently cleaved double stranded oligonucleotides containing 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU) in vitro via beta- and delta-elimination reactions. Similar activities were detected with hNTH1. These results indicate that hNEIL1 and hNTH1 are DNA glycosylases that excise 5-foU and 5-hmU as efficiently as Tg in human cells.
- Published
- 2005
34. Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase
- Author
-
Shunsuke Izumi, Eiji Ohmae, Hiroshi Ide, Hiroaki Terato, Katsuo Katayanagi, Tamon Tanaka, and Mayumi Matsubara
- Subjects
DNA Repair ,Pyrimidine ,Stereochemistry ,DNA Mutational Analysis ,Molecular Sequence Data ,Xenopus Proteins ,Biology ,Catalysis ,DNA Glycosylases ,Pentoxyl ,chemistry.chemical_compound ,Genetics ,Humans ,Amino Acid Sequence ,Uracil ,Uracil-DNA Glycosidase ,Mutagenesis ,Articles ,Thymine ,Kinetics ,chemistry ,Biochemistry ,Structural Homology, Protein ,DNA glycosylase ,Uracil-DNA glycosylase ,Mutagenesis, Site-Directed ,Cytosine ,DNA ,DNA Damage - Abstract
Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.
- Published
- 2004
35. Qualitative and Quantitative Analyses of the Decomposition Products that Arise from the Exposure of Thymine to Monochromatic Ultrasoft X Rays and60Co Gamma Rays in the Solid State
- Author
-
Akinari Yokoya, Kentaro Fujii, and Ken Akamatsu
- Subjects
Time Factors ,DNA Repair ,Nitrogen ,Biophysics ,Radiation ,Spectral line ,law.invention ,Pentoxyl ,chemistry.chemical_compound ,law ,Radiology, Nuclear Medicine and imaging ,Irradiation ,Cobalt Radioisotopes ,Uracil ,Chromatography, High Pressure Liquid ,Photons ,Chemistry ,X-Rays ,Radiochemistry ,Gamma ray ,Dose-Response Relationship, Radiation ,DNA ,Synchrotron ,Thymine ,Oxygen ,Models, Chemical ,Gamma Rays ,Spectrophotometry ,Calibration ,Monochromatic color ,Synchrotrons ,DNA Damage - Abstract
HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 gamma rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the gamma-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by gamma radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and gamma rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.
- Published
- 2004
36. Surface Salt Bridges Modulate DNA Wrapping by the Type II DNA-Binding Protein TF1
- Author
-
Anne Grove
- Subjects
Anions ,Models, Molecular ,HMG-box ,Glutamic Acid ,Bacterial genome size ,Biology ,Biochemistry ,DNA-binding protein ,Pentoxyl ,Viral Proteins ,chemistry.chemical_compound ,Cations ,Bacteriophages ,Thermotoga maritima ,Electrophoresis, Agar Gel ,Ions ,Aspartic Acid ,Alanine ,Lysine ,DNA ,DNA-Binding Proteins ,Kinetics ,Cross-Linking Reagents ,chemistry ,Glutaral ,Mutation ,Electrophoresis, Polyacrylamide Gel ,Salts ,Dimerization ,Thymine ,Bacillus subtilis ,Plasmids ,Protein Binding - Abstract
The histone-like protein HU is involved in compaction of the bacterial genome. Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA. Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported. TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine. On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations. The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1. These data are consistent with complex formation linked to disruption of surface salt bridges. Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA. We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA. Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.
- Published
- 2003
37. Structure and Specificity of the Vertebrate Anti-Mutator Uracil-DNA Glycosylase SMUG1
- Author
-
Karl A. Haushalter, Gregory L. Verdine, Laurence H. Pearl, Timothy R. Waters, and Jane E.A. Wibley
- Subjects
Models, Molecular ,DNA Repair ,Base Pair Mismatch ,Protein Conformation ,Molecular Sequence Data ,Deamination ,Biology ,Xenopus Proteins ,DNA Glycosylases ,Substrate Specificity ,Pentoxyl ,chemistry.chemical_compound ,Cytosine ,Mice ,Xenopus laevis ,Animals ,Humans ,Amino Acid Sequence ,Uracil-DNA Glycosidase ,Base Pairing ,N-Glycosyl Hydrolases ,Molecular Biology ,Mice, Knockout ,Transition (genetics) ,Base Sequence ,Molecular Structure ,Sequence Homology, Amino Acid ,Uracil ,Cell Biology ,Molecular biology ,Thymine ,chemistry ,Biochemistry ,DNA glycosylase ,Uracil-DNA glycosylase ,Mutation ,DNA ,DNA Damage - Abstract
Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.
- Published
- 2003
- Full Text
- View/download PDF
38. The Role of Surface-Exposed Lysines in Wrapping DNA about the Bacterial Histone-Like Protein HU
- Author
-
Tatiana C. Saavedra and Anne Grove
- Subjects
Molecular Sequence Data ,Sequence (biology) ,Bacillus Phages ,Biochemistry ,Histones ,Pentoxyl ,Viral Proteins ,chemistry.chemical_compound ,Bacterial Proteins ,Mutant protein ,Amino Acid Sequence ,Binding site ,Conserved Sequence ,Aspartic Acid ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Lysine ,Molecular biology ,Thymine ,Bacillus subtilis bacteriophage SPO1 ,DNA-Binding Proteins ,DNA binding site ,Kinetics ,Histone ,chemistry ,DNA, Viral ,biology.protein ,Nucleic Acid Conformation ,DNA ,Bacillus subtilis - Abstract
Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.
- Published
- 2002
39. In silico studies to explore the mutagenic ability of 5-halo/oxy/li-oxy-uracil bases with guanine of DNA base pairs
- Author
-
Kalyanashis Jana and Bishwajit Ganguly
- Subjects
Guanine ,Base pair ,Stereochemistry ,Static Electricity ,Nucleobase ,Pentoxyl ,chemistry.chemical_compound ,Cytosine ,Halogens ,A-DNA ,Computer Simulation ,Physical and Theoretical Chemistry ,Uracil ,Base Pairing ,Polymerase ,biology ,Models, Genetic ,Chemistry ,Mutagenesis ,Hydrogen Bonding ,DNA ,biology.protein - Abstract
DNA nucleobases are reactive in nature and undergo modifications by deamination, oxidation, alkylation, or hydrolysis processes. Many such modified bases are susceptible to mutagenesis when formed in cellular DNA. The mutagenesis can occur by mispairing with DNA nucleobases by a DNA polymerase during replication. We have performed a study of mispairing of DNA bases with unnatural bases computationally. 5-Halo uracils have been studied as mispairs in mutagenesis; however, the reports on their different forms are scarce in the literature. The stability of mispairs with keto form, enol form, and ionized form of 5-halo-uracil has been computed with the M06-2X/6-31+G** level of theory. The enol form of 5-halo-uracil showed remarkable stability toward DNA mispair compared to the corresponding keto and ionized forms. (F)U-G mispair showed the highest stability in the series and (Halo)(U(enol/ionized)-G mispair interactions energies are more stable than the natural G-C basepair of DNA. To enhance the stability of DNA mispairs, we have introduced the hydroxyl group in the place of halogen atoms, which provides additional hydrogen-bonding interactions in the system while forming the 5-membered ring. The study has been further extended with lithiated 5-hydroxymethyl-uracil to stabilize the DNA mispair. (CH2OLi)U(ionized)-G mispair has shown the highest stability (ΔG = -32.4 kcal/mol) with multi O-Li interactions. AIM (atoms in molecules) and EDA (energy decomposition analysis) analysis has been performed to examine the nature of noncovalent interactions in such mispairs. EDA analysis has shown that electrostatic energy mainly contributes toward the interaction energy of mispairs. The higher stability achieved in these studied mispairs can play a pivotal role in the mutagenesis and can help to attain the mutation for many desired biological processes.
- Published
- 2014
40. Comparison of the absolute level of epigenetic marks 5-methylcytosine, 5-hydroxymethylcytosine, and 5-hydroxymethyluracil between human leukocytes and sperm
- Author
-
Jolanta, Guz, Daniel, Gackowski, Marek, Foksinski, Rafal, Rozalski, and Ryszard, Olinski
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,DNA ,DNA Methylation ,Deoxycytidine ,Spermatozoa ,Epigenesis, Genetic ,Up-Regulation ,Pentoxyl ,Cytosine ,Tandem Mass Spectrometry ,5-Methylcytosine ,Leukocytes ,Humans ,Poland ,Biomarkers ,Chromatography, High Pressure Liquid ,Thymidine - Abstract
5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 10⁴ deoxynucleosides, and 5.209 versus 0.492 per 10⁶ deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome.
- Published
- 2014
41. The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence
- Author
-
Agus Darwanto, Jacob A. Theruvathu, Chia Wei Hsu, and Lawrence C. Sowers
- Subjects
DNA Repair ,Telomere-Binding Proteins ,Biology ,Genome Integrity, Repair and Replication ,Shelterin Complex ,Pentoxyl ,03 medical and health sciences ,Genetics ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,AP site ,Uracil ,Uracil-DNA Glycosidase ,030304 developmental biology ,Telomere-binding protein ,0303 health sciences ,Binding Sites ,Base Sequence ,030302 biochemistry & molecular biology ,Base excision repair ,DNA ,Telomere ,Molecular biology ,Very short patch repair ,DNA glycosylase ,Uracil-DNA glycosylase ,Fluorouracil ,Schizosaccharomyces pombe Proteins ,Abasic Site Formation ,Nucleotide excision repair ,Protein Binding - Abstract
Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.
- Published
- 2014
42. [Effect of pyramidon, pentoxyl, butazolidine, and dimedrol on therapeutic action of penicillin in experimental staphylococcal inflammatory foci in white mice]
- Author
-
V N, SOLOV'EV
- Subjects
Pentoxyl ,Mice ,Antipyretics ,Diphenhydramine ,Staphylococcus ,Animals ,Penicillins ,Aminopyrine ,Micrococcus - Published
- 2014
43. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA
- Author
-
Nada Raddaoui, Mirko Wagner, Udo Müller, Heinrich Leonhardt, Caterina Brandmayr, Markus Müller, Olga Kotljarova, Cornelia G. Spruijt, Gengo Kashiwazaki, Barbara Steigenberger, Jessica Steinbacher, Stylianos Michalakis, Olesea Kosmatchev, Silvia K. Laube, Michiel Vermeulen, Fabio Spada, David Eisen, Stefan Schiesser, David Schuermann, Benjamin Hackner, Toni Pfaffeneder, Matthias Truss, Primo Schär, and Thomas Carell
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Pyrimidine ,DNA repair ,Molecular Sequence Data ,Deamination ,Gene Expression ,Biology ,Chromatin remodeling ,Dioxygenases ,Pentoxyl ,chemistry.chemical_compound ,Cytosine ,Mice ,Proto-Oncogene Proteins ,Animals ,Molecular Biology ,Transcription factor ,Embryonic Stem Cells ,Carbon Isotopes ,Base Sequence ,Proteomics and Chromatin Biology ,Cell Biology ,DNA ,Chromatin Assembly and Disassembly ,Thymine ,DNA-Binding Proteins ,Biochemistry ,chemistry ,5-Methylcytosine ,Stem cell ,Oxidation-Reduction ,Chromatography, Liquid ,Protein Binding ,Transcription Factors - Abstract
Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
- Published
- 2014
44. Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1
- Author
-
George W. Teebor, Stuart M. Brown, Yuliang Ma, Dina R. Marenstein, Archie Cummings, Thomas A. Neubert, Robert J. Boorstein, and Michael K. Chan
- Subjects
DNA, Complementary ,Molecular Sequence Data ,Biology ,Biochemistry ,DNA Glycosylases ,law.invention ,Pentoxyl ,chemistry.chemical_compound ,law ,Complementary DNA ,Animals ,Amino Acid Sequence ,Uracil-DNA Glycosidase ,N-Glycosyl Hydrolases ,Molecular Biology ,5-hydroxymethyluracil DNA N-glycosylase activity ,Gel electrophoresis ,Molecular mass ,Cell Biology ,Molecular biology ,chemistry ,DNA glycosylase ,Uracil-DNA glycosylase ,Recombinant DNA ,Cattle ,Electrophoresis, Polyacrylamide Gel ,DNA - Abstract
Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20x the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.
- Published
- 2001
45. Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53
- Author
-
George W. Teebor, Li-Jun Mi, Robert Horowitz, Robert J. Boorstein, and Wenren Chaung
- Subjects
G2 Phase ,Cancer Research ,Programmed cell death ,DNA Repair ,DNA repair ,DNA damage ,Apoptosis ,HL-60 Cells ,Phosphatidylserines ,Biology ,Pentoxyl ,Mice ,chemistry.chemical_compound ,Cricetulus ,DNA Nucleotidylexotransferase ,Cricetinae ,Animals ,Humans ,Lung ,Cell Cycle ,Cell Membrane ,DNA ,General Medicine ,Base excision repair ,Fibroblasts ,Cell cycle ,Genes, p53 ,Molecular biology ,Bromodeoxyuridine ,Terminal deoxynucleotidyl transferase ,chemistry ,Mutation ,Tumor Suppressor Protein p53 ,Fluorescein-5-isothiocyanate ,DNA Damage ,Thymidine - Abstract
We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.
- Published
- 2001
46. An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts
- Author
-
Valaiporn Rusmintratip and Lawrence C. Sowers
- Subjects
Cell Extracts ,Guanine ,DNA Repair ,DNA damage ,Base pair ,DNA repair ,Deamination ,Biology ,Cell Line ,DNA Glycosylases ,Pentoxyl ,chemistry.chemical_compound ,Humans ,Base Pairing ,N-Glycosyl Hydrolases ,Multidisciplinary ,Adenine ,Biological Sciences ,Fibroblasts ,Thymine ,Biochemistry ,chemistry ,DNA glycosylase ,DNA ,DNA Damage ,HeLa Cells - Abstract
The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10 7 . The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.
- Published
- 2000
47. NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA
- Author
-
Antonietta Pepe, David R. Kearns, Luciano Mayol, Hai M. Vu, HAI M., Vu, Pepe, A., Mayol, Luciano, and Kearns, D. R.
- Subjects
Models, Molecular ,Base pair ,Biology ,Pentoxyl ,chemistry.chemical_compound ,Genetics ,Molecule ,Base Pairing ,Nuclear Magnetic Resonance, Biomolecular ,Base Sequence ,Hydrogen bond ,Chemical shift ,Hydrogen Bonding ,DNA ,Nuclear magnetic resonance spectroscopy ,Thymine ,Solutions ,Crystallography ,chemistry ,Biochemistry ,Helix ,Nucleic Acid Conformation ,Protons ,Software ,Research Article - Abstract
Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1. We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy. The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned. Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond. Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d. was 0.66 A (all atoms) for the internal 15 bp. The helical twist was 38.5 degrees per step. The base pairs were situated directly on the helix axis (X-displacement = -0.2 A). All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees. The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ). Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.
- Published
- 1999
48. Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine and 5-(hydroxymethyl) uracil in Smokers
- Author
-
Faranak Firoozi, Jean Hee, Alain Favier, Jean Cadet, Michelle Tripier, Véronique Ducros, Sandrine Pourcelot, and Henri Faure
- Subjects
Adult ,Male ,medicine.medical_specialty ,Free Radicals ,Stereochemistry ,Urinary system ,Biochemistry ,Pentoxyl ,Excretion ,Nicotine ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Deoxyguanosine ,Hydroxymethyl ,Creatinine ,Smoking ,8-Hydroxy-2'-deoxyguanosine ,Uracil ,General Medicine ,Middle Aged ,Endocrinology ,chemistry ,8-Hydroxy-2'-Deoxyguanosine ,Case-Control Studies ,Oxidation-Reduction ,DNA Damage ,medicine.drug - Abstract
Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54+/-2.07 vs. 6.70+/-1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16+/-0.35 vs. 1.35+/-0.50 nmol/mmol creatinine, p = 0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r(p) = 0.44), BMI (r(p) = -0.27) and nicotine derivatives (r(p) = 0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r(p) = 0.36), nicotine derivatives (r(p) = 0.29), BMI (r(p) = -0.24) on the other.
- Published
- 1999
49. Clinical variability in three Danish patients with dihydropyrimidine dehydrogenase deficiency all homozygous for the same mutation
- Author
-
Peter Vreken, A. B. P. Van Kuilenburg, S. Kjaergaard, Ernst Christensen, A. H. van Gennip, I. Cezanne, H. Horlyk, V. Faurholt Pedersen, and Other departments
- Subjects
Male ,medicine.medical_specialty ,Biology ,Pentoxyl ,Danish ,Dihydropyrimidine dehydrogenase deficiency ,Internal medicine ,Genetics ,medicine ,Humans ,Child ,Uracil ,Cells, Cultured ,Dihydrouracil Dehydrogenase (NADP) ,Genetics (clinical) ,Cell Line, Transformed ,Netherlands ,Homozygote ,Infant ,medicine.disease ,Human genetics ,language.human_language ,Endocrinology ,Child, Preschool ,Mutation ,Mutation (genetic algorithm) ,language ,Female ,Oxidoreductases ,Thymine - Published
- 1998
50. Oxidative DNA Damage Levels in Blood from Women at High Risk for Breast Cancer are Associated with Dietary intakes of Meats, Vegetables, and Fruits
- Author
-
Lance K. Heilbrun, Virginia E. Uhley, Zora Djuric, Samir Lababidi, Janice B. Depper, Daryn Smith, and Silvana Martino
- Subjects
Adult ,medicine.medical_specialty ,Meat ,Adolescent ,DNA damage ,Breast Neoplasms ,medicine.disease_cause ,Oxidative dna damage ,Pentoxyl ,Breast cancer ,Risk Factors ,Vegetables ,medicine ,Humans ,Outpatient clinic ,Food science ,Legume ,Aged ,Univariate analysis ,Nutrition and Dietetics ,business.industry ,food and beverages ,Regression analysis ,DNA ,Middle Aged ,medicine.disease ,Diet ,Surgery ,Oxidative Stress ,Fruit ,Female ,business ,Oxidative stress ,DNA Damage ,Food Science - Abstract
Objective We examined the relationship between intakes of specific foods—namely, meats, vegetables, and fruits—with levels of oxidative DNA damage in women consuming their own usual diet or a diet low in fat. Design Blood was obtained from women who had been assigned randomly to a low-fat or nonintervention diet for 3 to 24 months. Levels of 5-hydroxymethyluracil, a type of oxidative DNA damage, were determined. Diet data were obtained from 3-day food records. Subjects/setting The 21 women were participating in an outpatient clinic. All the women were healthy but had a first-degree relative with breast cancer. Intervention The intervention was a self-selected diet with a goal of 15% of energy from fat. Main outcome measures Existing data on oxidative DNA damage levels were evaluated for possible relationships to foods eaten. Intakes of raw and cooked vegetables were examined separately. Meat intake was examined by type of meat (pork, beef, fish, chicken) and by cooking temperature. Statistical analyses Initial univariate analyses relied on Spearman rank correlations of each food item with DNA damage. Further analyses of the data were performed with univariate and multivariate weighted least squares regression models. Results The model that best explained DNA damage levels was a bivariate regression model that included the intake of cooked vegetables and the sum of beef and pork intake. This model accounted for 85% of the variation in DNA damage levels among women. Preliminary results are suggestive of a positive association of DNA damage with beef and pork intake and a negative association with cooked vegetable intake. Application These observations, if confirmed in larger studies, suggest specific dietary changes to reduce oxidative DNA damage levels and possibly cancer risk. J Am Diet Assoc. 1998;98:524–528 .
- Published
- 1998
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.