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At least 19 recordsLinked to original sources
PubMed1996

Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells.

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.

Animals↗
PubMed1991

Synthesis of 1-(3-azido-2,3-dideoxy-beta-D-allofuranosyl)thymine, 1-(2,3-dideoxy-beta-D-allofuranosyl)thymine, and 1-(2,3-dideoxy-beta-D-erythro-hex-2-enofuranosyl)thymine.

1-(2-O-Acetyl-3,5,6-tri-O-benzoyl-beta-D-glucofuranosyl)thymine (1) was converted into the 2,2'-anhydro derivative 4 by selective deacetylation, mesylation, and treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene. Cleavage of the 2,2'-anhydro ring in 4 with hydrogen bromide or hydrogen chloride led to the 2'-bromo (5) or 2'-chloro (6) derivative, respectively. Dehalogenation of 6 with tributylstannane and then debenzoylation gave 1-(2-deoxy-beta-D-arabino-hexofuranosyl)thymine (8). Isopropylidenation of 8 followed by mesylation, azide displacement, and deprotection gave 1-(3-azido-2,3-dideoxy-beta-D-ribo-hexofuranosyl)thymine (12). Oxidation of 12 with Dowex 1 (IO4-) resin followed by reduction with Dowex 1 (BH4-) resin gave 1-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)thymine (AZT). Catalytic hydrogenation of 5 afforded a mixture of 1-(5,6-di-O-benzoyl-2,3-dideoxy-beta-D-erythro-hexofuranosyl)thymi ne (13) and 1-(3,5,6-tri-O-benzoyl-2-deoxy-beta-D-arabino-hexofuranosyl)thymin e (7). Reaction of 5 with a Cu/Zn couple gave 1-(5,6-di-O-benzoyl-2,3-dideoxy-beta-D-erythro-hex-2-enofuranosyl) thymine (15). 1-(2,3-Dideoxy-beta-D-erythro-hexofuranosyl)thymine (14) and 1-(2,3-dideoxy-beta-D-erythro-hex-2-enofuranosyl)thymine (16) were obtained by debenzoylation.

Antiviral Agents↗
PubMed1968

Thymine-thymine adduct as a photoproduct of thymine.

A product isolated from thymine irradiated with ultraviolet light in frozen aqueous solution undergoes dehydration on heating with acids. As judged by elemental analysis, mass, ultraviolet, infrared, and nuclear magnetic resonance spectra, the most probable structures for this compound and its dehydration product, respectively, are 5-hydroxy-6-4'-[5''-methylpyrimidin-2'-one]-dihydrothymine and 6-4'-[5'-methylpyrimidin-2'-one]-thymine. Apparently, this compound is a thymine-thymine adduct and presumably is formed through the rearrangement of an initial photoproduct. Both compounds are closely related to 6-4'-[pyrimidin-2'-one]-thymine which has been isolated from acid hydrolyzates of ultraviolet-irradiated DNA and supposedly is derived from cytosine-thymine adduct. Formation of such adducts between pyrimidine bases is apparently a common photoreaction and may be important to the study of the photochemistry and photobiology of nucleic acids.

Chromatography, Paper↗
PubMed1991

The thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3' thymine-to-cytosine transitions in Escherichia coli.

We have constructed single-stranded, M13-based vectors that contain a specifically located thymine-thymine pyrimidine-pyrimidone(6-4) UV photoproduct and have used these to estimate the frequency and accuracy of DNA replication past this adduct in uvrA6 cells of Escherichia coli. Both the normal and the Dewar valence photoisomer of the (6-4) adduct were studied. In the absence of SOS induction, vectors carrying the photoproducts were rarely replicated; relative to the lesion-free control, 1.9% of vectors carrying the normal (6-4) isomer produced plaques, and with the Dewar valence isomer the proportion was 0.4%. In SOS-induced cells, these frequencies rose to 22.1% and 12.3%, respectively. The error frequency of replication past the normal isomer in SOS-induced cells was high; in a random sample of 185 progeny phage analyzed, 169 (91%) contained mutations, all of which were targeted. Equally striking, a high proportion of the mutations (158/169; 93%) were of only one type, namely 3' T----C transitions. Both the error frequency and the specificity were much reduced with the Dewar valence isomer; overall, 74/140 (53%) of the phage analyzed were mutant, and of these only 34 (46%) entailed the 3' T----C transition. We speculate that the high error frequency and specificity arise from the formation of a stable T-G base pair, involving hydrogen bonds at O-2 and N-3 in the pyrimidone ring. Potential hydrogen bonds at these sites are coplanar in the normal but not in the Dewar isomer, perhaps explaining the reduced specificity of mutagenesis with the latter adduct.

Base Sequence↗
PubMed2002

The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine.

Metabolites of vinyl chloride react with cytosine in DNA to form 3,N(4)-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG.(epsilon)C-DNA) was by far the best substrate having a specificity constant (k(2)/K(d)) of 25.1 x 10(6) m(-1) s(-1). The next best substrates were DNA duplexes containing TpG.(epsilon)C, GpG.(epsilon)C, and CpG.T. These had specificity constants 45-130 times smaller than CpG.(epsilon)C-DNA. The worst substrates were DNA duplexes containing ApG.(epsilon)C and TpG.T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG.(epsilon)C-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G.T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G.(epsilon)C base pair more easily than it can flip out thymine from a G.T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.

5-Methylcytosine↗
PubMed2003

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair.

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.

5-Methylcytosine↗
PubMed1999

Derivatization of thymine and thymine photodimers with 4-bromomethyl-7-methoxycoumarin for fluorescence detection in high-performance liquid chromatography.

Exposure of DNA to utraviolet radiation results in the formation of a number of photoproducts, including thymine photodimers. A sensitive and selective analytical method based on high-performance liquid chromatography (HPLC) and fluorescent labeling with 4-bromomethyl-7-methoxycoumarin has been developed to quantify both thymine and thymine photodimers. The identity of the thymine and thymine dimer derivatives were determined by HPLC-electrospray ionization mass spectrometry. The derivatization reaction yield was maximized by optimizing several reaction variables. The limit of detection for HPLC method was 1.0 pmol thymine and 0.4 pmol thymine dimer for S/N = 3.

Calibration↗
PubMed1980

X-ray structural and conformational studies of [5'-(tryptaminocarbonyl)-2'-deoxyribofuranosyl]thymine, a model compound for the study of the interaction between indole and thymine rings.

The crystal structure of [5'-(tryptaminocarbonyl)-2'-deoxyribofuranosyl]-thymine was determined by an X-ray method in order to elucidate a fundamental feature of interaction between indole and thymine rings. Refinement resulted in a conventional R value of 0.043. The bond lengths and angles are in good agreement with those of the related compounds. The deoxyribose unit has a C2'-endo envelope conformation, and orientation of the thymine base with respect to the sugar is anti. In the crystal, the double layers of the thymidine moieties are formed by the hydrogen bonds around a two-fold screw axis and a single layer of the indole moieties is held together by van der Waals contact, and they are alternately packed parallel to the bc-plane. The conformation of the molecule is an extended form, and the intra- and intermolecular interactions between the indole and thymine rings are not observed in the crystal because the dihedral angle of both rings is approximately a right angle (95.8 degrees), and there is no hydrogen bond or short contact between these rings. It was, however, suggested from the ultraviolet spectroscopic study in dilute solution and from the conformational analysis by energy calculation that the most stable conformation is a folded form with the intramolecular stacking interaction between indole and thymine rings. Therefore, the extended form observed in the crystal might be caused by the packing interaction between the neighboring molecules.

Chemical Phenomena↗
PubMed2006

Preferential formation of (5S,6R)-thymine glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to thymine glycol-containing DNA.

We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.

Anti-Bacterial Agents↗
PubMed1992

Splitting of cis-syn cyclobutane thymine-thymine dimers by radiolysis and its relevance to enzymatic photoreactivation.

The 137Cs-gamma-irradiation of cis-syn thymine-thymine cyclobutane type dimers has been studied in aqueous solution. The mechanism of thymine dimer cleavage by eaq-, CO2.-, OH.,SO4.-,Br2.- and isopropanol radicals was studied using high pressure liquid chromatography (HPLC). Evidence that the one-electron reductants studied induce dimer cleavage partially by a chain reaction is presented. Approximate values for the one-electron reduction potential of thymine-thymine are obtained and thermodynamic calculations are presented in order to predict the direction of electron transfer in the case of enzymatic photoreactivation.

Cesium Radioisotopes↗
PubMed2006

MercuryII-mediated formation of thymine-HgII-thymine base pairs in DNA duplexes.

The very specific binding of the HgII ion unexpectedly and significantly stabilizes naturally occurring thymine-thymine base mispairing in DNA duplexes. Following this finding, we prepared DNA duplexes containing metal-mediated base pairs at the desired sites, as well as novel double helical architectures consisting only of thymine-HgII-thymine pairs.

Base Pairing↗
PubMed1997

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.

Circular Dichroism↗
PubMed1972

R factor elimination during thymine starvation: effects of inhibition of protein synthesis and readdition of thymine.

R factor 1818 is shown to be eliminated from a thymineless strain of Escherichia coli J6-2 (R-1818) during thymine starvation. Readdition of thymine to the starved cultures produces a partial recovery in viable count but does not affect the proportion of R(-) cells. The R factor is not cured from exponential- or stationary-phase cultures which are starved of required amino acids as well as thymine, nor from cells which are deprived of thymine in the presence of chloramphenicol. However, in both of these cases, the extent of thymineless death is reduced. It is suggested that protein synthesis is a requirement for R-1818 elimination, and the possible nature of this protein is discussed.

Amino Acids↗
PubMed2000

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota.

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

Base Pair Mismatch↗
PubMed1999

Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta.

The RAD30 gene of the yeast Saccharomyces cerevisiae is required for the error-free postreplicational repair of DNA that has been damaged by ultraviolet irradiation. Here, RAD30 is shown to encode a DNA polymerase that can replicate efficiently past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally blocks DNA polymerases. When incubated in vitro with all four nucleotides, Rad30 incorporates two adenines opposite the thymine-thymine dimer. Rad30 is the seventh eukaryotic DNA polymerase to be described and hence is named DNA polymerase eta.

Adenine↗
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↗