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Nature Materials Sep 2020The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel,...
The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.
Topics: DNA; Hydrogen Bonding; Nanostructures; Thymine; Triazines
PubMed: 32661383
DOI: 10.1038/s41563-020-0728-2 -
Physical Chemistry Chemical Physics :... Nov 2020Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the...
Interaction of nucleic acids with light is a scientific question of paramount relevance not only in the understanding of life functioning and evolution, but also in the insurgence of diseases such as malignant skin cancer and in the development of biomarkers and novel light-assisted therapeutic tools. This work shows that the UVA portion of sunlight, not absorbed by canonical DNA nucleobases, can be absorbed by 5-formyluracil (ForU) and 5-formylcytosine (ForC), two ubiquitous oxidatively generated lesions and epigenetic intermediates present in living beings in natural conditions. We measure the strong propensity of these molecules to populate triplet excited states able to transfer the excitation energy to thymine-thymine dyads, inducing the formation of cyclobutane pyrimidine dimers (CPDs). By using steady-state and transient absorption spectroscopy, NMR, HPLC, and theoretical calculations, we quantify the differences in the triplet-triplet energy transfer mediated by ForU and ForC, revealing that the former is much more efficient in delivering the excitation energy and producing the CPD photoproduct. Although significantly slower than ForU, ForC is also able to harm DNA nucleobases and therefore this process has to be taken into account as a viable photosensitization mechanism. The present findings evidence a rich photochemistry crucial to understand DNA damage photobehavior.
Topics: Cytosine; DNA Damage; Dimerization; Epigenesis, Genetic; Oxidation-Reduction; Photochemistry; Sunlight; Thymine; Ultraviolet Rays; Uracil
PubMed: 33169771
DOI: 10.1039/d0cp04557h -
The Journal of Organic Chemistry Jul 2023The quest for simple systems achieving the photoreductive splitting of four-membered ring compounds is a matter of interest not only in organic chemistry but also in...
The quest for simple systems achieving the photoreductive splitting of four-membered ring compounds is a matter of interest not only in organic chemistry but also in biochemistry to mimic the activity of DNA photorepair enzymes. In this context, 8-oxoguanine, the main oxidatively generated lesion of guanine, has been shown to act as an intrinsic photoreductant by transferring an electron to bipyrimidine lesions and provoking their cycloreversion. But, in spite of appropriate photoredox properties, the capacity of guanine to repair cyclobutane pyrimidine dimer is not clearly established. Here, dyads containing the cyclobutane thymine dimer and guanine or 8-oxoguanine are synthesized, and their photoreactivities are compared. In both cases, the splitting of the ring takes place, leading to the formation of thymine, with a quantum yield 3.5 times lower than that for the guanine derivative. This result is in agreement with the more favored thermodynamics determined for the oxidized lesion. In addition, quantum chemistry calculations and molecular dynamics simulations are carried out to rationalize the crucial aspects of the overall cyclobutane thymine dimer photoreductive repair triggered by the nucleobase and its main lesion.
Topics: Pyrimidine Dimers; Cyclobutanes; Thymine; DNA; Guanine
PubMed: 37437138
DOI: 10.1021/acs.joc.3c00930 -
Proceedings of the National Academy of... Aug 2010Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6...
Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5' base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Pol kappa and Pol zeta function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Pol kappa at the insertion step and of Pol zeta at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.
Topics: DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Thymine
PubMed: 20660785
DOI: 10.1073/pnas.1007795107 -
Proceedings of the National Academy of... Jul 2022The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various...
The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.
Topics: Adenine; Base Pairing; DNA; Echinomycin; Magnetic Resonance Spectroscopy; Thymine
PubMed: 35857870
DOI: 10.1073/pnas.2200681119 -
Molecules (Basel, Switzerland) May 2019The clinically widely-used anticancer drug, cisplatin, binds strongly to DNA as a DNA-damaging agent. Herein, we investigated the interaction of cisplatin with a 15-mer...
The clinically widely-used anticancer drug, cisplatin, binds strongly to DNA as a DNA-damaging agent. Herein, we investigated the interaction of cisplatin with a 15-mer single-stranded C,T-rich oligodeoxynucleotide, 5'-CCTTCTTGCTTCTCC-3' (ODN15), using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in conjunction with tandem mass spectrometry (top-down MS). Top-down MS analysis with collision-induced dissociation (CID) fragmentation of the mono-platinated and di-platinated ODN15 provided abundant and informative Pt-containing or Pt-free a/[a - B], w and internal fragments, allowing the unambiguous identification of T, T, C, and T as the platination sites on the cisplatin-ODN15 adducts. These results revealed that, in addition to the well-established guanine site, the unexpected thermodynamic binding of cisplatin to cytosine and thymine bases was also evident at the oligonucleotide level. Furthermore, the binding models of cisplatin with cytosine and thymine bases were built as the Pt coordinated to cytosine-N(3) and thymine-N(3) with displacement of the proton or tautomerization of thymine. These findings contribute to a better understanding of the mechanism of action of cisplatin and its preference for gene loci when the drug binds to cellular DNA, and also demonstrate the great potential and superiority of FT-ICR MS in studying the interactions of metallodrugs with large biomolecules.
Topics: Cisplatin; Cytosine; Models, Molecular; Oligodeoxyribonucleotides; Tandem Mass Spectrometry; Thymine
PubMed: 31091778
DOI: 10.3390/molecules24101852 -
European Journal of Pharmaceutical... Jan 2016The fluoropyrimidine drugs 5-fluorouracil and its oral prodrug capecitabine remain first line therapy for solid tumours of the neck, breast and colon. However,... (Clinical Trial)
Clinical Trial
Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males.
The fluoropyrimidine drugs 5-fluorouracil and its oral prodrug capecitabine remain first line therapy for solid tumours of the neck, breast and colon. However, significant and unpredictable toxicity affects about 10-25% of patients depending upon the mode of 5-fluorouracil delivery. The pharmacokinetics of thymine (5-methyluracil) may provide an approach for screening for 5-fluorouracil toxicity, based on the rationale that thymine is a close structural analogue of 5-fluorouracil and is catabolized by the same enzymatic pathway. Oral thymine loading tests were performed on 12 healthy volunteers. Each subject was given a single oral dose of 250mg thymine in capsule form. Blood, urine and saliva samples were collected pre-dose and up to 5h post-dose. Concentrations of thymine, and its catabolites dihydrothymine and ß-ureidoisobutyrate were analysed by HPLC-tandem mass spectrometry in plasma, urine and saliva. The pharmacokinetic data of healthy volunteers were analysed assuming a non-compartmental model. Thymine peaked quickly (30-45min) in plasma to a maximum concentration of 170±185μg/L (mean±SD). Clearance was high (mean 57.9L/h/kg) exceeding normal human liver blood flow, suggesting low systemic bioavailability; urinary recovery of the thymine dose was low (<1%). Apparent formation rate-limited kinetics were observed for dihydrothymine, and the plasma concentration of dihydrothymine was consistently 10-fold higher than that of thymine. Plasma ß-ureidoisobutyrate concentrations, on the other hand, were similar to that of thymine. Genotyping confirmed that pathological mutations of the DPYD gene were absent. The urinary excretion ratio of thymine/dihydrothymine was informative of the maximum concentration. Saliva thymine was highly variable. These data are potentially useful as a basis for developing of a screening procedure to prospectively identify patients who are at risk of toxicity from fluoropyrimidine drugs.
Topics: Adult; Antimetabolites, Antineoplastic; Fluorouracil; Humans; Male; Middle Aged; Models, Biological; Saliva; Thymine; Urea
PubMed: 26435217
DOI: 10.1016/j.ejps.2015.10.001 -
The EMBO Journal Mar 2021The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication...
The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low-fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading-strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase-polymerase uncoupling a switch from Pol ε, the canonical leading-strand replicase, to the lagging-strand replicase Pol δ, facilitates rapid, efficient and error-free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8-oxoguanine. We propose that replicase switching may promote continued leading-strand synthesis whenever the replisome encounters leading-strand damage that is bypassed more efficiently by Pol δ than by Pol ε.
Topics: DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thymine
PubMed: 33555053
DOI: 10.15252/embj.2020107037 -
Photochemistry and Photobiology Nov 2015Fifty years ago, a new thymine dimer was discovered as the dominant DNA photolesion in UV-irradiated bacterial spores [Donnellan, J. E. & Setlow R. B. (1965) Science,... (Review)
Review
Fifty years ago, a new thymine dimer was discovered as the dominant DNA photolesion in UV-irradiated bacterial spores [Donnellan, J. E. & Setlow R. B. (1965) Science, 149, 308-310], which was later named the spore photoproduct (SP). Formation of SP is due to the unique environment in the spore core that features low hydration levels favoring an A-DNA conformation, high levels of calcium dipicolinate that acts as a photosensitizer, and DNA saturation with small, acid-soluble proteins that alters DNA structure and reduces side reactions. In vitro studies reveal that any of these factors alone can promote SP formation; however, SP formation is usually accompanied by the production of other DNA photolesions. Therefore, the nearly exclusive SP formation in spores is due to the combined effects of these three factors. Spore photoproduct photoreaction is proved to occur via a unique H-atom transfer mechanism between the two involved thymine residues. Successful incorporation of SP into an oligonucleotide has been achieved via organic synthesis, which enables structural studies that reveal minor conformational changes in the SP-containing DNA. Here, we review the progress on SP photochemistry and photobiology in the past 50 years, which indicates a very rich SP photobiology that may exist beyond endospores.
Topics: DNA Repair; Molecular Structure; Photobiology; Photochemistry; Spores, Bacterial; Thymine; Ultraviolet Rays
PubMed: 26265564
DOI: 10.1111/php.12506 -
Journal of Bacteriology Jan 2022Cells that cannot synthesize one of the DNA precursors, dTTP, due to mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of...
Cells that cannot synthesize one of the DNA precursors, dTTP, due to mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the and mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.
Topics: Anti-Bacterial Agents; DNA Replication; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Iron; Microbial Viability; Oxidative Stress; Thymine; Trimethoprim
PubMed: 34633866
DOI: 10.1128/JB.00370-21