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At least 37 records · Page 2Linked to original sources
PubMed1993

Repair by human cell extracts of single (6-4) and cyclobutane thymine-thymine photoproducts in DNA.

One cis-syn cyclobutane thymine dimer or one (6-4) thymine-thymine photoproduct was built into an identical sequence of a closed-circular M13 duplex DNA, and nucleotide excision repair synthesis carried out by human cell extracts in the area containing each lesion was determined. Extracts from normal cells repaired the (6-4) photoproduct with a patch size of approximately 20-30 nucleotides, but repair was at least 10-fold lower at the cyclobutane dimer. The (6-4) lesion was repaired with comparable efficiency to a single acetylamino-fluorene-guanine adduct in a similar location. Extract from nucleotide excision repair-deficient xeroderma pigmentosum group A cells could not remove any of these adducts but could complete repair of the lesions after incision with Escherichia coli UvrABC proteins. This direct comparison of repair of two UV photoproducts, in an in vitro system where chromatin assembly and transcription are absent, suggests that the more rapid repair of the (6-4) lesion observed in the mammalian cell genome overall is due in part to a significant difference in the ability of the repair complex to locate and incise these lesions in DNA.

Bacteriophage M13↗
PubMed2000

Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta.

The Saccharomyces cerevisiae RAD30 gene functions in error-free replication of UV-damaged DNA. RAD30 encodes a DNA polymerase, Pol eta, which inserts two adenines opposite the two thymines of a cis-syn thymine-thymine (T-T) dimer. Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T-T dimer. Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincorporation of about 10(-2) to 10(-3). These studies support the hypothesis that unlike most DNA polymerases, Pol eta is able to tolerate distortions in DNA resulting from damage, which then enables the polymerase to utilize the intrinsic base pairing ability of the T-T dimer.

Base Sequence↗
PubMed1993

Structure of a thymine-thymine adduct formed by menadione photosensitization.

The structure of one of the dithymine adducts formed by near-UV photosensitization of aqueous oxygenated thymine in the presence of 2-methyl-1,4-naphthoquinone is reported. The compound, 6' beta-hydroxy-5' beta-1-[5-methylpyrimidinyl-2,4-dione]-5'- 6'-dihydrothymine, has one thymine ring (ring I) linked through the 1-nitrogen atom to the C5' atom of a second thymine, which has been hydroxylated at C6' (ring II). Crystals are monoclinic, space group I2/c, and the structure was refined to R(obs) = 0.036 for 2072 unique reflections with intensities I greater than 2.33 sigma(I). Ring I is planar, whereas ring II is not. Ring I and the OH group on ring II are cis to ring II. The planar ring I and the OH group are attached, respectively, in an equatorial and axial manner to ring II (which is in the sofa conformation). The planar rings I of close-lying pairs of molecules stack parallel to each other. The structure is held together by a hydrogen-bonding system consisting of the water molecules, the NH groups, two of the C = O groups and the OH group. The chemical formula and relative configurations at C5' and C6' are established by this analysis.

Hydrogen Bonding↗
PubMed1969

Crystal and molecular structure of a thymine-thymine adduct.

Thymine-thymine adduct is a product isolated from thymine irradiated with ultraviolet light in frozen aqueous solution. This compound is presumably formed through the rearrangement of an initial photoproduct. Single crystal x-ray diffraction analysis has confirmed the molecular formula of the adduct, 5-hydroxy-6-4'-(5'-methylpyrimid-2'-one)-dihydrothymine, except for the possibility of a hydrogen atom on the 3' nitrogren rather than the 1' nitrogen, and has established the stereoconfiguration of the molecule.

Freezing↗
PubMed1978

Changes in cell size and shape in thymine-requiring Escherichia coli associated with growth in low concentrations of thymine.

Thymine auxotrophs of three unrelated strains of Escherichia coli (K-12, B/r, and 15) were grown in media containing various concentrations of thymine. During steady-state growth conditions, cell volume increased as thymine concentration decreased but, in contrast to previous reports, this change was due to an increase in cell length without change in cell diameter.

Cell Count↗
PubMed2002

Determination of laser-induced thymine-thymine dimer in DNA by LC.

A new simple and selective high-performance liquid chromatography (HPLC) method has been developed for the determination of thymine-thymine (T-T) dimer in deoxyribonucleic acid (DNA) for the study of relationship between the yield of T-T dimer and laser irradiation conditions. The HPLC method is not disturbed by other hydrolysate formed in DNA with the good stability and reproducility. The detection of limit of the method is about 8.8 ng of T-T dimer in DNA blank. Three lever of laser intensity to irradiate DNA were selected for calf thymine to test the influence of the pulse number. The mechanism of DNA lesion and repair caused by laser irradiating was studied.

Animals↗
PubMed1993

DNA repair by eukaryotic nucleotide excision nuclease. Removal of thymine dimer and psoralen monoadduct by HeLa cell-free extract and of thymine dimer by Xenopus laevis oocytes.

Using a human cell-free extract, we have recently shown that thymine dimers are removed from DNA in oligonucleotides 27-29 nucleotides in length (Huang, J. C., Svoboda, D. L., Reardon, J. T., and Sancar, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3664-3668). In this study we find that the excision reaction is dependent on ATP, the excised fragments range in length from 27-32 nucleotides, and have 5'-P and 3'-OH termini. We also found that a thymine-psoralen furan side monoadduct is excised from the DNA with a similar incision pattern, indicating that in humans bulky adducts are removed from DNA by the same enzyme system which hydrolyzes mainly the 22-24th and the 5th phosphodiester bonds 5' and 3', respectively, to the lesion. This incision pattern might be common to eukaryotic excision nucleases as thymine dimers were removed from DNA by the same dual incision pattern by Xenopus laevis oocytes.

Adenosine Triphosphate↗
PubMed1984

Radiation-induced formation of thymine-thymine crosslinks.

The formation of dimers upon gamma-irradiation of thymine, thymidine and thymidine-5'-monophosphate in N2O-saturated aqueous solution is reported. A method using capillary gas chromatography-mass spectrometry was developed for their separation and identification. Quantitative measurements reveal that about 50 per cent of OH adduct radicals of thymine undergo dimerization.

Cobalt Radioisotopes↗
PubMed2001

Thymine-metal ion interactions: relevance for thymine quartet structures.

Apart from their function as counter ions for the charge neutralization of nucleic acids, alkali metal ions play important roles in stabilizing particular multistranded nucleic acids, e.g. guanine quartets in telomeres and uracil (U) or thymine (T) quartets. Here X-ray crystal structure determinations of a series of alkali metal ions (Na+, K+, Rb+, Cs+) as well as of Mg2+ and H5O2+ adducts with the model bases 1-methylthymine and 1-ethylthymine are reported, which bear relevance to the question of thymine quartet (T4) geometries. The compounds isolated differ in their stoichiometries (T:M = 4:1, 2:1, 1:1), and the ways the metal ions interact with the bases. The two extremes are exclusive metal coordination to exocyclic oxygen atoms of the T bases and exclusive H bonding between M aqua cations and the bases.

Binding Sites↗
PubMed1994

Contributions of the thymine methyl group to the specific recognition of poly- and mononucleotides: an analysis of the relative free energies of solvation of thymine and uracil.

Experimental results indicate that interactions with the 5-methyl group of thymine often account for around 1 kcal/mol of the total selectivity at A.T base pairs in protein-DNA complexes. The limited ability of methyl groups to form noncovalent interactions of this magnitude has led to the hypothesis that the energy of solvation of this hydrophobic element is responsible for the observed contribution to selectivity; however, it has not been possible to test this experimentally. We report a molecular dynamics perturbation thermodynamics (MD/PT) analysis of the relative free energy of solvation of thymine and uracil, both as the free bases and in the context of double-stranded DNA. The use of MD/PT indicates that the effect of shielding the 5-methyl group from solvent accounts for 0.90 +/- 0.11 kcal/mol of the observed contribution to specificity in protein-DNA complexes. We suggest some implications of these results for the mechanism of sequence-specific DNA recognition, DNA structure, and the evolution of the deoxynucleotide synthesis pathways.

Adenine↗
PubMed1996

Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide.

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.

Amino Acid Sequence↗
PubMed2000

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Aspartate Carbamoyltransferase↗
PubMed2006

Production of cis-syn thymine-thymine cyclobutane dimer oligonucleotide in the presence of acetone photosensitizer.

cis-syn Cyclobutane pyrimidine dimer (CPD) oligonucleotide was produced by UV irradiation in the presence of acetone photosensitizer. Acetone could enhance the productivity but evidently induced the photocleavage of oligonucleotide under a long time irradiation. A statistical approach of orthogonal design was applied to optimize the preparation condition for the production of the modified oligonucleotide. Optimal conditions for maximal cis-syn CPD oligonucleotide productivity were determined based on three factors: acetone concentration, initial oligonucleotide concentration, and irradiation time at several different levels. The optimal modified oligonucleotide that this optimization could produce was 32.7%. Through analysis of 20% polyacrylamide gel electrophoresis, it was found that modified oligonucleotide migrated slightly more slowly than the parent oligonucleotide. The photoreactivation of cis-syn thymine-thymine dimer oligonucleotide displayed the selectivity of the substrate specificity of DNA photolyase with high-performance liquid chromatography (HPLC) analysis.

Acetone↗
PubMed2003

Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta.

DNA polymerase eta (Poleta) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Poleta. Poleta displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.

Base Sequence↗
PubMed1990

In situ localization of the human multidrug-resistance gene mRNA using thymine-thymine dimerized single-stranded cDNA.

In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm. The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗