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5 Hydroxymethyluracil

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Ryszard Olinski – 1st expert on this subject based on the ideXlab platform

  • vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
    Free Radical Biology and Medicine, 2016
    Co-Authors: Martyna Modrzejewska, Daniel Gackowski, Marta Starczak, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard Olinski

    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. 5Hydroxymethyluracil 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 5Hydroxymethyluracil 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 5Hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.

  • enigmatic 5 Hydroxymethyluracil oxidatively modified base epigenetic mark or both
    Mutation Research-reviews in Mutation Research, 2016
    Co-Authors: Ryszard Olinski, Marta Starczak, Daniel Gackowski

    Abstract:

    The aim of this review is to describe the reactions which lead to generation of 5Hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5Hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5Hydroxymethyluracil 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 5Hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5Hydroxymethyluracil 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 5Hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5Hydroxymethyluracil is epigenetic mark. The level of 5Hydroxymethyluracil 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 5Hydroxymethyluracil from DNA. 5Hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5Hydroxymethyluracil 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 5Hydroxymethyluracil 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 5Hydroxymethyluracil may be either a regulatory or erroneous compound.

  • tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
    PLOS ONE, 2015
    Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard Olinski

    Abstract:

    Background
    Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.

    Results
    Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively).

    Conclusions
    Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.

Daniel Gackowski – 2nd expert on this subject based on the ideXlab platform

  • vitamin c enhances substantially formation of 5 Hydroxymethyluracil in cellular dna
    Free Radical Biology and Medicine, 2016
    Co-Authors: Martyna Modrzejewska, Daniel Gackowski, Marta Starczak, Maciej Gawronski, Magdalena Skonieczna, Ewelina Zarakowska, Marek Foksinski, Joanna Rzeszowskawolny, Ryszard Olinski

    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. 5Hydroxymethyluracil 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 5Hydroxymethyluracil 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 5Hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.

  • enigmatic 5 Hydroxymethyluracil oxidatively modified base epigenetic mark or both
    Mutation Research-reviews in Mutation Research, 2016
    Co-Authors: Ryszard Olinski, Marta Starczak, Daniel Gackowski

    Abstract:

    The aim of this review is to describe the reactions which lead to generation of 5Hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5Hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5Hydroxymethyluracil 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 5Hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5Hydroxymethyluracil 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 5Hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5Hydroxymethyluracil is epigenetic mark. The level of 5Hydroxymethyluracil 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 5Hydroxymethyluracil from DNA. 5Hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5Hydroxymethyluracil 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 5Hydroxymethyluracil 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 5Hydroxymethyluracil may be either a regulatory or erroneous compound.

  • tissue specific differences in dna modifications 5 hydroxymethylcytosine 5 formylcytosine 5 carboxylcytosine and 5 Hydroxymethyluracil and their interrelationships
    PLOS ONE, 2015
    Co-Authors: Daniel Gackowski, Marta Starczak, Martyna Modrzejewska, Ewelina Zarakowska, Ryszard Olinski

    Abstract:

    Background
    Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5Hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.

    Results
    Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively).

    Conclusions
    Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.

George W Teebor – 3rd expert on this subject based on the ideXlab platform

  • definitive identification of mammalian 5 Hydroxymethyluracil dna n glycosylase activity as smug1
    Journal of Biological Chemistry, 2001
    Co-Authors: Robert J Boorstein, Archie Cummings, Dina R Marenstein, Michael K Chan, Thomas A Neubert, Stuart M Brown, George W Teebor

    Abstract:

    Abstract Purification from calf thymus of a DNAN-glycosylase activity (HMUDG) that released 5Hydroxymethyluracil (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 20× the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.

  • excessive base excision repair of 5 Hydroxymethyluracil from dna induces apoptosis in chinese hamster v79 cells containing mutant p53
    Carcinogenesis, 2001
    Co-Authors: Lijun Mi, George W Teebor, Wenren Chaung, Robert Horowitz, Robert J Boorstein

    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 5Hydroxymethyluracil (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.

  • Oxidative damage to 5-methylcytosine in DNA
    Nucleic Acids Research, 1995
    Co-Authors: Rober J. Boorstein, George W Teebor

    Abstract:

    Abstract
    Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5Hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5Hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2′-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.