-
Proceedings of the National Academy of... Aug 2020Utilizing an ionic liquid strategy, we report crystal structures of salts of free anionic nucleobases and base pairs previously studied only computationally and in the...
Utilizing an ionic liquid strategy, we report crystal structures of salts of free anionic nucleobases and base pairs previously studied only computationally and in the gas phase. Reaction of tetrabutylammonium ([N]) or tetrabutylphosphonium ([P]) hydroxide with adenine (HAd) and thymine (HThy) led to hydrated salts of deprotonated adenine, [N][Ad]·2HO, and thymine, [P][Thy]·2HO, as well as the double salt cocrystal, [P][Ad][Thy]·3HO·2HThy. The cocrystal includes the anionic [Ad(HThy)] base pair which is a stable formation in the solid state that has previously not even been suggested. It exhibits Watson-Crick connectivity as found in DNA but which is unusual for the free neutral base pairs. The stability of the observed anionic bases and their supramolecular formations and hydrates has also been examined by electronic structure calculations, contributing to more insight into how base pairs can bind when a proton is removed and highlighting mechanisms of stabilization or chemical transformation in the DNA chains.
Topics: Adenine; Anions; Base Pairing; Crystallography; Hydrogen Bonding; Models, Molecular; Molecular Structure; Nucleic Acid Conformation; Thymine
PubMed: 32680959
DOI: 10.1073/pnas.2008379117 -
Nature Feb 2018Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here,...
Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (G•T/UG•T/U; population less than 0.4%) and one anionic species (G•T/U; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10 to 10-fold discrimination against misincorporation, which proceeds primarily via tautomeric dG•dT and dG•dT, with contributions from anionic dG•dT dominant at pH 8.4 and above or for some mutagenic nucleotides.
Topics: Animals; Anions; Base Pair Mismatch; DNA; DNA Replication; DNA-Directed DNA Polymerase; Guanine; Humans; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Mutagenesis; Probability; Rats; Thymine
PubMed: 29420478
DOI: 10.1038/nature25487 -
Genes Apr 2019Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair....
Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from , and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.
Topics: Cell Cycle; DNA; DNA Glycosylases; DNA Replication; DNA, Single-Stranded; Humans; Models, Genetic; N-Glycosyl Hydrolases; Thymine; Uracil
PubMed: 31018584
DOI: 10.3390/genes10040315 -
The Journal of Chemical Physics Apr 2016Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly...
Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.
Topics: Evolution, Chemical; Origin of Life; Quantum Theory; Thymine
PubMed: 27083722
DOI: 10.1063/1.4945745 -
PLoS Genetics Oct 2022Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and...
Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD. Here, we used a diverse set of unbiased approaches to systematically determine the genetic and regulatory underpinnings of TLD in Escherichia coli. In addition to discovering novel genes in previously implicated pathways, our studies revealed a critical and previously unknown role for intracellular acidification in TLD. We observed that a decrease in cytoplasmic pH is a robust early event in TLD across different genetic backgrounds. Furthermore, we show that acidification is a causal event in the death process, as chemical and genetic perturbations that increase intracellular pH substantially reduce killing. We also observe a decrease in intracellular pH in response to exposure to the antibiotic gentamicin, suggesting that intracellular acidification may be a common mechanistic step in the bactericidal effects of other antibiotics.
Topics: Escherichia coli; DNA, Bacterial; Microbial Viability; Thymine; Recombination, Genetic; Hydrogen-Ion Concentration
PubMed: 36279294
DOI: 10.1371/journal.pgen.1010456 -
The Biochemical Journal Dec 1958
Topics: Adenine; RNA; Thymine
PubMed: 13607422
DOI: 10.1042/bj0700642 -
Hydroxymethyluracil modifications enhance the flexibility and hydrophilicity of double-stranded DNA.Nucleic Acids Research Mar 2016Oxidation of a DNA thymine to 5-hydroxymethyluracil is one of several recently discovered epigenetic modifications. Here, we report the results of nanopore translocation...
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.
Topics: DNA; DNA-Binding Proteins; Epigenesis, Genetic; Hydrophobic and Hydrophilic Interactions; Molecular Dynamics Simulation; Nanopores; Nucleic Acid Denaturation; Oxidation-Reduction; Pentoxyl; Protein Binding; Surface Properties; Thymine
PubMed: 26578595
DOI: 10.1093/nar/gkv1199 -
PloS One 2023Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory...
Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5'-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, and cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37°C) where NP1 does not form the oral apparatus. We found that phagocytosis was not required for pyrimidine analog entry and that all tested pyrimidine analogs inhibited growth except for cytarabine. IC50 values did not significantly differ between CU428 and NP1 for the same analog at either room temperature or 37°C. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, and 5-methyluridine) to determine whether the inhibitory effects from the pyrimidine analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5'-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently in Tetrahymena.
Topics: Tetrahymena thermophila; Floxuridine; Nucleosides; Thymine; Antimetabolites; Gemcitabine; Pyrimidines; Uracil; Fluorouracil; Cytarabine
PubMed: 37708236
DOI: 10.1371/journal.pone.0284309 -
Nucleic Acids Research Jun 2016G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological...
G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological processes including transcription, replication, translation and telomere maintenance. Recent studies have demonstrated the existence of GQ DNA in live mammalian cells and a significant number of potential GQ forming sequences in the human genome. We present a systematic and quantitative analysis of GQ folding propensity on a large set of 438 GQ forming sequences in double-stranded DNA by integrating fluorescence measurement, single-molecule imaging and computational modeling. We find that short minimum loop length and the thymine base are two main factors that lead to high GQ folding propensity. Linear and Gaussian process regression models further validate that the GQ folding potential can be predicted with high accuracy based on the loop length distribution and the nucleotide content of the loop sequences. Our study provides important new parameters that can inform the evaluation and classification of putative GQ sequences in the human genome.
Topics: Base Composition; Base Sequence; DNA; G-Quadruplexes; Linear Models; Thymine
PubMed: 27095201
DOI: 10.1093/nar/gkw272 -
Chemistry (Weinheim An Der Bergstrasse,... Dec 2022Short oligonucleotides incorporating either 1-mercuri-6-phenylcarbazole, 8-mercuri-6-phenylcarbazole, or 1,8-dimercuri-6-phenylcarbazole C-nucleoside in the middle of...
Short oligonucleotides incorporating either 1-mercuri-6-phenylcarbazole, 8-mercuri-6-phenylcarbazole, or 1,8-dimercuri-6-phenylcarbazole C-nucleoside in the middle of the chain have been synthesized and studied for their potential as hybridization probes for sequences containing thiopyrimidine nucleobases. All of these oligonucleotides formed very stable duplexes with complementary sequences pairing the organometallic moiety with either 2- or 4-thiothymine. The isomeric monomercurated oligonucleotides were also able to discriminate between 2- and 4-thiothymine based on the different melting temperatures of the respective duplexes. DFT-optimized structures of the most stable mononuclear Hg -mediated base pairs featured a coordinated covalent bond between Hg and either S2 or S4 and a hydrogen bond between the carbazole nitrogen and N3. The dinuclear Hg -mediated base pairs, in turn, were geometrically very similar to the one previously reported to form between 1,8-dimercuri-6-phenylcarbazole and thymine and had one Hg ion coordinated to a thio and the other one to an oxo substituent.
Topics: Oligonucleotides; Base Pairing; Nucleic Acid Hybridization; Thymine; Hydrogen Bonding; Mercury
PubMed: 36108095
DOI: 10.1002/chem.202202530