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Proceedings of the National Academy of... Feb 1977DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments...
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
Topics: Adenine; Base Sequence; Biochemical Phenomena; Biochemistry; Cytosine; DNA; DNA Restriction Enzymes; Guanine; Hydrazines; Methods; Nucleic Acid Hybridization; Thymine
PubMed: 265521
DOI: 10.1073/pnas.74.2.560 -
Proceedings of the National Academy of... Feb 2021In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in...
In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in order to detect thymine dimer formation. The thymine dimer formation, as a function of irradiation dose, was determined by Raman spectroscopy. In addition, the formation of a mutagenic (6-4) photoproduct was identified by its synchronous fluorescence spectrum. Our spectroscopic data suggest that the rate of conversion of thymine to thymine dimer decreases after 20 min of UV irradiation, owing to the formation of an equilibrium between the thymine dimers and monomers. However, the formation of the (6-4) photoproduct continued to increase with UV irradiation. In addition, the Raman spectra of nonirradiated and irradiated calf thymus DNA were recorded, and the formation of thymine dimers was detected. The spectroscopic data presented make it possible to determine the mechanism of thymine dimer formation, which is known to be responsible for the inhibition of DNA replication that causes bacteria inactivation.
Topics: Animals; Cattle; DNA; DNA Damage; Pyrimidine Dimers; Spectrometry, Fluorescence; Spectrum Analysis, Raman; Thymine; Ultraviolet Rays
PubMed: 33526704
DOI: 10.1073/pnas.2025263118 -
Nucleic Acids Research Dec 1987Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen... (Review)
Review
Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings.
Topics: Bacterial Proteins; Base Sequence; DNA (Cytosine-5-)-Methyltransferases; DNA, Bacterial; DNA-Binding Proteins; Methylation; Thymine
PubMed: 3320959
DOI: 10.1093/nar/15.23.9975 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2012Substituted toluenyl groups are considered as close isosteres of the thymine residue. They can be recognized by DNA polymerases as if they were thymine. These toluene...
Expanding the horizon of the thymine isostere biochemistry: unique cyclobutane dimers formed by photoreaction between a thymine and a toluene residue in the dinucleotide framework.
Substituted toluenyl groups are considered as close isosteres of the thymine residue. They can be recognized by DNA polymerases as if they were thymine. These toluene derivatives are generally inert toward radical additions, including the [2+2] photo-cycloadditions, due to the stable structure of the aromatic ring and are usually used as solvents for radical reactions. Surprisingly, after incorporating toluene into the dinucleotide framework, we found that the UV excited thymine residue readily dimerizes with the toluenyl moiety through a [2+2] photo-addition reaction. Furthermore, the reaction site on the toluenyl moiety is not the C5=C6 bond, as commonly observed in cyclobutane pyrimidine dimers, but the C4=C5 or C3=C4 instead. Such a reaction pattern suggests that in the stacked structure, it is one of these bonds, not the C5=C6, that is close to the thymine C5=C6 bond. A similar structural feature is found in DNA duplex with a thymine replaced by a 2,4-difluorotoluene. Our results argue that although the substituted toluenyl moieties closely mimic the size and shape of the thymine residue, their more hydrophobic nature determines that they stack on DNA bases differently from the natural thymine residue and likely cause local conformational changes in duplex DNA.
Topics: Cyclobutanes; DNA-Directed DNA Polymerase; Dimerization; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Photochemical Processes; Pyrimidine Dimers; Stereoisomerism; Thymine; Toluene
PubMed: 22588824
DOI: 10.1002/chem.201200816 -
Photochemistry and Photobiology May 20225-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage.... (Review)
Review
5-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage. In DNA strands, the uracil radical generated from 5-halouracil causes DNA damage via a hydrogen-abstraction reaction. We analyzed the photoreaction of 5-halouracil in various DNA structures and revealed that the reaction is DNA structure-dependent. In this review, we summarize the results of the analysis of the reactivity of 5-halouracil in various DNA local structures. Among the 5-halouracil molecules, 5-bromouracil has been used as a probe in the analysis of photoinduced electron transfer through DNA. The analysis of groove-binder/DNA and protein/DNA complexes using a 5-bromouracil-based electron transfer system is also described.
Topics: Bromouracil; DNA; Thymine; Uracil
PubMed: 34543451
DOI: 10.1111/php.13521 -
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 -
Topics in Current Chemistry 2015Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including... (Review)
Review
Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH₂, and CH₃groups to pyrimidine. Results show that H₂O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H₂O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H₂O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine.
Topics: Absorption, Radiation; Cytosine; Extraterrestrial Environment; Ice; Models, Theoretical; Nucleic Acids; Photons; Photosynthesis; Prebiotics; Radiation, Ionizing; Thymine; Ultraviolet Rays; Uracil
PubMed: 24500331
DOI: 10.1007/128_2013_499 -
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 -
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