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Nucleic Acids Research Feb 2019The sequence dependent structure and flexibility of the DNA double helix is of key importance for gene expression and DNA packing and it can be modulated by DNA...
The sequence dependent structure and flexibility of the DNA double helix is of key importance for gene expression and DNA packing and it can be modulated by DNA modifications. The presence of a C5'-methyl group in thymine or the frequent C5'-methylated-cytosine affects the DNA fine structure, however, the underlying mechanism and steric origins have remained largely unexplained. Employing Molecular Dynamics free energy simulations that allow switching on or off interactions with the methyl groups in several DNA sequences, we systematically identified the physical origin of the coupling between methyl groups and DNA backbone fine structure. Whereas methyl-solvent and methyl-nucleobase interactions were found to be of minor importance, the methyl group interaction with the 5' neighboring sugar was identified as main cause for influencing the population of backbone substates. The sterical methyl sugar clash prevents the formation of unconventional stabilizing hydrogen bonds between nucleobase and backbone. The technique was also used to study the contribution of methyl groups to DNA flexibility and served to explain why the presence of methyl sugar clashes in thymine and methyl-cytosine can result in an overall local increase of DNA flexibility.
Topics: Base Sequence; Cytosine; DNA; Molecular Dynamics Simulation; Nucleic Acid Conformation; Sugars; Thymine
PubMed: 30541032
DOI: 10.1093/nar/gky1237 -
Biochemistry Feb 2020Tandem DNA lesions containing two contiguously damaged nucleotides are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely...
Tandem DNA lesions containing two contiguously damaged nucleotides are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely unknown. Replication of isolated 2-deoxyribonolactone (L), thymine glycol (Tg), and tandem lesion 5'-LTg was examined in human cells. Although nearly 100% of Tg was bypassed in HEK 293T cells, L was a significant replication block. 5'-LTg was an even stronger replication block with 5% TLS efficiency. The mutation frequency (MF) of Tg was 3.4%, which increased to 3.9% and 4.8% in pol ι- and pol κ-deficient cells, respectively. An even greater increase in the MF of Tg (to ∼5.5%) was observed in cells deficient in both pol κ and pol ζ, suggesting that they work together to bypass Tg in an error-free manner. Isolated L bypass generated 12-18% one-base deletions, which increased as much as 60% in TLS polymerase-deficient cells. The fraction of deletion products also increased in TLS polymerase-deficient cells upon 5'-LTg bypass. In full-length products and in all cell types, dA was preferentially incorporated opposite an isolated L as well as when it was part of a tandem lesion. However, misincorporation opposite Tg increased significantly when it was part of a tandem lesion. In wild type cells, targeted mutations increased about 3-fold to 9.7% and to 17.4, 15.9, and 28.8% in pol κ-, pol ζ-, and pol ι-deficient cells, respectively. Overall, Tg is significantly more miscoding as part of a tandem lesion, and error-free Tg replication in HEK 293T cells requires participation of the TLS polymerases.
Topics: DNA; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; HEK293 Cells; Humans; Mutagenesis; Mutagens; Nucleotides; Sugar Acids; Thymine; DNA Polymerase iota
PubMed: 31860280
DOI: 10.1021/acs.biochem.9b01058 -
Neuropharmacology May 2021Alcohol use disorder (AUD) is associated with the dysregulation of brain stress and reward systems, including glucocorticoid receptors (GRs). The mixed...
Alcohol use disorder (AUD) is associated with the dysregulation of brain stress and reward systems, including glucocorticoid receptors (GRs). The mixed glucocorticoid/progesterone receptor antagonist mifepristone and selective GR antagonist CORT113176 have been shown to selectively reduce alcohol consumption in alcohol-dependent rats. Mifepristone has also been shown to decrease alcohol consumption and craving for alcohol in humans with AUD. The present study tested the effects of the GR modulators CORT118335, CORT122928, CORT108297, and CORT125134 on alcohol self-administration in nondependent (air-exposed) and alcohol-dependent (alcohol vapor-exposed) adult male rats. Different GR modulators recruit different GR-associated transcriptional cofactors. Thus, we hypothesized that these GR modulators would vary in their effects on alcohol drinking. CORT118335, CORT122928, and CORT125134 significantly reduced alcohol self-administration in both alcohol-dependent and nondependent rats. CORT108297 had no effect on alcohol self-administration in either group. The present results support the potential of GR modulators for the development of treatments for AUD. Future studies that characterize genomic and nongenomic effects of these GR modulators will elucidate potential molecular mechanisms that underlie alcohol drinking in alcohol-dependent and nondependent states.
Topics: Animals; Aza Compounds; Ethanol; Heterocyclic Compounds, 4 or More Rings; Isoquinolines; Male; Mifepristone; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Glucocorticoid; Self Administration; Thymine
PubMed: 33647278
DOI: 10.1016/j.neuropharm.2021.108510 -
International Journal of Molecular... Jul 2022Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these...
Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized -Orn and -Dab and -Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes.
Topics: Amino Acids; DNA; Peptide Nucleic Acids; Peptides; RNA; Thymine
PubMed: 35955638
DOI: 10.3390/ijms23158504 -
Nature Communications Apr 2022The lack of pyrimidine diversity in meteorites remains a mystery since prebiotic chemical models and laboratory experiments have predicted that these compounds can also...
The lack of pyrimidine diversity in meteorites remains a mystery since prebiotic chemical models and laboratory experiments have predicted that these compounds can also be produced from chemical precursors found in meteorites. Here we report the detection of nucleobases in three carbonaceous meteorites using state-of-the-art analytical techniques optimized for small-scale quantification of nucleobases down to the range of parts per trillion (ppt). In addition to previously detected purine nucleobases in meteorites such as guanine and adenine, we identify various pyrimidine nucleobases such as cytosine, uracil, and thymine, and their structural isomers such as isocytosine, imidazole-4-carboxylic acid, and 6-methyluracil, respectively. Given the similarity in the molecular distribution of pyrimidines in meteorites and those in photon-processed interstellar ice analogues, some of these derivatives could have been generated by photochemical reactions prevailing in the interstellar medium and later incorporated into asteroids during solar system formation. This study demonstrates that a diversity of meteoritic nucleobases could serve as building blocks of DNA and RNA on the early Earth.
Topics: Meteoroids; Purines; Pyrimidines; Thymine
PubMed: 35473908
DOI: 10.1038/s41467-022-29612-x -
Chemical Research in Toxicology Oct 2016DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating...
DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, O-alkylthymidines (O-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific N-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of O-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, were capable of bypassing all O-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol η exhibited high frequencies of misincorporation of dCMP opposite those O-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of O-iPrdT > O-iBudT > O-sBudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.
Topics: DNA Replication; DNA-Directed DNA Polymerase; Humans; In Vitro Techniques; Molecular Structure; Thymine
PubMed: 27611246
DOI: 10.1021/acs.chemrestox.6b00252 -
The Journal of Organic Chemistry Sep 2022Photosensitized thymine<>thymine (Thy<>Thy) formation and repair can be mediated by carbazole (Cbz). The former occurs from the Cbz triplet excited state via energy...
Photosensitized thymine<>thymine (Thy<>Thy) formation and repair can be mediated by carbazole (Cbz). The former occurs from the Cbz triplet excited state via energy transfer, while the latter takes place from the singlet excited state via electron transfer. Here, fundamental insight is provided into the role of the topology and excited state multiplicity, as factors governing the balance between both processes. This has been achieved upon designing and synthesizing different isomers of trifunctional systems containing one Cbz and two Thy units covalently linked to the rigid skeleton of the natural deoxycholic acid. The results shown here prove that the Cbz photosensitized dimerization is not counterbalanced by repair when the latter, instead of operating through-space, has to proceed through-bond.
Topics: Carbazoles; Dimerization; Energy Transfer; Thymine
PubMed: 35980822
DOI: 10.1021/acs.joc.2c00942 -
Proceedings of the National Academy of... Sep 1984Thymine glycol is a DNA damage product of ionizing radiation and other oxidative mutagens. In an attempt to find a noninvasive assay for oxidative DNA damage in... (Comparative Study)
Comparative Study
Thymine glycol is a DNA damage product of ionizing radiation and other oxidative mutagens. In an attempt to find a noninvasive assay for oxidative DNA damage in individuals, we have developed an HPLC assay for free thymine glycol and thymidine glycol in urine. Our results indicate that humans excrete about 32 nmol of the two glycols per day. Rats, which have a higher specific metabolic rate and a shorter life span, excrete about 15 times more thymine glycol plus thymidine glycol per kg of body weight than do humans. We present evidence that thymine glycol and thymidine glycol are likely to be derived from repair of oxidized DNA, rather than from alternative sources such as the diet or bacterial flora. This noninvasive assay of DNA oxidation products may allow the direct testing of current theories which relate oxidative metabolism to the processes of aging and cancer in man.
Topics: Animals; Chromatography, High Pressure Liquid; DNA Replication; Germ-Free Life; Glucose; Humans; Rats; Species Specificity; Spectrophotometry, Ultraviolet; Thymidine; Thymine; Tritium
PubMed: 6592579
DOI: 10.1073/pnas.81.18.5633 -
Scientific Reports Jul 2019GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic...
GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.
Topics: Alanine; Animals; Binding Sites; Cryoelectron Microscopy; Crystallography, X-Ray; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Polysaccharides; Protein Conformation; Rats; Receptors, Kainic Acid; Thymine; GluK3 Kainate Receptor
PubMed: 31311973
DOI: 10.1038/s41598-019-46770-z -
Nucleic Acids Research Sep 2012The 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...
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.
Topics: Animals; Base Pair Mismatch; DNA; DNA Glycosylases; Endodeoxyribonucleases; Mice; Models, Molecular; Pentoxyl; Protein Structure, Tertiary; Thymine
PubMed: 22740654
DOI: 10.1093/nar/gks628