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PubMed1998

Kinetics of the action of thymine DNA glycosylase.

The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.

Base Composition↗

PubMed1990

Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the photoproduct. Poly(dA-[3H]dT).poly(dA-[3H]dT) was UV irradiated and incubated with purified E. coli endonuclease III. 3H-Containing material was released in a manner consistent with Michaelis-Menten kinetics. This 3H-labeled material was determined to be a mixture of thymine hydrates (6-hydroxy-5,6-dihydrothymine), separable from unmodified thymine by chromatography in three independent systems. Both cis-thymine hydrate and trans-thymine hydrate were chemically and photochemically synthesized. These coeluted with the enzyme-released 3H-containing material. No thymine glycol was released from the UV-irradiated polymer. Similar results were obtained with extracts of WI-38 cells as the enzyme source. The release of thymine hydrates by both glycosylase activities was directly proportional to the amount of enzyme and the irradiation dose to the DNA substrate. These results demonstrate the modified thymine residues recognized and excised by endonuclease III and the human enzyme to be a mixture of cis-thymine hydrate and trans-thymine hydrate. The reparability of these thymine hydrates suggests that they are stable in DNA and therefore potentially genotoxic.

Bacterial Proteins↗

PubMed1992

Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.

Chromatography, High Pressure Liquid↗

PubMed1983

Radiation-induced hydroxylation of thymine promoted by electron-affinic compounds.

The effect of 20 electron-affinic compounds including nitroimidazoles, nitrofurans, nitrobenzenes, and quinones on the radiation-induced reaction of thymine in aqueous solution was studied under deaerated and N2O-saturated conditions. The radiolysis of thymine in aerated aqueous solution was also performed for comparison. Thymine decomposition was depressed to some extent by the addition of electron-affinic compounds in both deaerated and N2O-saturated solutions, while promoted in aerated solution. The radiolyses with varying concentration of misonidazole indicated that the depression of thymine decomposition can be attributed to a competition between thymine and electron-affinic compounds for the reactions with .OH. Among the radiolysis products, the formation of thymine glycol was remarkably promoted by the addition of electron-affinic compounds. Irrespective of structures of the electron-affinic compounds, the G-value of thymine glycol increased in sigmoidal form with increasing one-electron reduction potential of the electron-affinic compounds and attained the ultimate values of ca. 1.1 and 1.8 in deaerated and N2O-saturated solutions, respectively. The results are in accord with one-electron oxidation of the hydroxythymyl radical, produced by the reaction of thymine with .OH, to the corresponding cation by electron-affinic compounds. The so-formed hydroxythymine cation undergoes solvolytic substitution to give thymine glycol. Based on the ultimate G-values of thymine glycol, the difference in reactivity between hydroxythymine-5-yl and 6-yl radicals toward electron-affinic compounds is discussed.

Air↗

PubMed1973

Effect of thymine starvation on deoxyribonucleic acid repair systems of Escherichia coli K-12.

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.

Cell Survival↗

PubMed1993

Characterization of the effects of a thymine glycol residue on the structure, dynamics, and stability of duplex DNA by NMR.

A duplex DNA containing a single thymine glycol (5,6-dihydroxy-5,6-dihydrothymidine) has been studied by NMR and other methods. Oxidative stress, ionizing radiation, and other causes can induce the oxidation of thymine to thymine glycol. The presence of thymine glycol is known to have significant biological consequences, and there are repair enzymes for thymine glycol in a wide range of organisms. These studies have been carried out on the DNA duplex of d(C1G2C3A4G5Tg6C7A8G9C10C11) paired with d(G22C21G20T19C18A17G16T15C14G13G12), with Tg indicating thymine glycol. The presence of thymine glycol lowers the thermal stability of duplex DNA. The NMR results indicate that thymine glycol induces a large, localized structural change in duplex DNA with the thymine glycol base being extrahelical as well as the opposing base on the complementary strand. This structural information is consistent with the biological consequences of thymine glycol in DNA.

Base Sequence↗

PubMed1982

Use of high-performance liquid chromatography to quantitate thymine-containing pyrimidine dimers in DNA.

We have developed two high-performance liquid chromatographic systems for the measurement of pyrimidine dimers in hydrolysates of DNA. Normal-phase chromatography on an NH2 column in methanol-ethyl acetate (3.97) at an elution rate of 2.0 ml/min allowed quantitation of thymine-containing (thymine-thymine plus thymine-uracil) pyrimidine dimers at levels as low as 0.1% of the total radioactivity as thymine in DNA. This system was unaffected by the presence of up to 1 mg of contaminating protein (bovine serum albumin) or 40 micrograms of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a muBondapak C18 column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in water as the solvent at a flow-rate of up to 0.6 ml/min, thymine-thymine, thymine-uracil, and uracil-uracil dimers were completely resolved. We were not able to quantitate the latter two dimeric forms, however, owing to the presence of other radioactive components of undefined origin that eluted concomitantly with the uracil-containing dimers.

Chromatography, High Pressure Liquid↗

PubMed1999

Human thymine DNA glycosylase binds to apurinic sites in DNA but is displaced by human apurinic endonuclease 1.

In vitro, following the removal of thymine from a G.T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (koff 1.8 - 3.6 x 10(-5) s-1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C.AP and SMeG.AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C.U and SMeG.T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G.AP sites sufficiently to allow it to remove more than one thymine from G.T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase beta, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.

Base Pair Mismatch↗

PubMed1972

Effect of the folic acid analogue, trimethoprim, on growth, macromolecular synthesis, and incorporation of exogenous thymine in Escherichia coli.

The effect of trimethoprim [2,4-diamino-5(2',4',5'trimethoxybenzyl)-pyrimidine] in the presence of thymine on Escherichia coli B temperature-sensitive and non-temperature-sensitive Thy(') strains and a phosphodeoxyribomutase-negative mutant was studied. The inhibitory effect of 5 mug of trimethoprim per ml on the growth of E. coli B was not overcome by thymine, thymidine, or thymidylate even in the presence of one-carbon metabolites and related metabolites. Deoxyribonucleic acid (DNA) and protein synthesis were more severely inhibited than ribonucleic acid (RNA) synthesis. The inhibition of DNA synthesis was partially reversed by addition of deoxyadenosine to increase the incorporation of exogenous thymine. By contrast, the inhibition of protein was not reversed even with one-carbon metabolites present, in keeping with the requirement for formylmethionyl-transfer RNA(F) for initiation. However, the inhibition of both DNA and protein synthesis in a phosphodeoxyribomutase-negative strain by 1 mug of trimethoprim per ml with thymine present was partially reversed by deoxyadenosine and one-carbon metabolites, and nearly normal growth occurred. 5-Fluorodeoxyuridine added at the time of addition of trimethoprim prevented the inhibition. Sulfadiazine in the presence of thymine inhibited both Thy(+) and Thy(-) strains whereas trimethoprim (with thymine) did not inhibit Thy(-) organisms. The effect of trimethoprim on the incorporation of labeled thymine into DNA was also studied. These experiments support the concept that trimethoprim in conjunction with the action of thymidylate synthetase inhibits the growth of Thy(+) cells because of a depletion of tetrahydrofolate. DNA synthesis is inhibited initially by a limitation of thymine nucleotide precursor, resulting from the indirect inhibition of thymidylate synthetase and the poor incorporation of exogenous thymine.

Anti-Bacterial Agents↗

PubMed1988

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions.

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.

Base Sequence↗

PubMed1983

Monoclonal antibody to DNA containing thymine glycol.

Exposure of DNA to ionizing or near ultraviolet radiation modifies thymine to form ring-saturated products. One of the major products formed is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol). Thymine glycol can also be selectively formed by oxidizing DNA with OsO4. We have isolated hybrids that produce monoclonal antibodies against thymine glycol by fusing mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells from BALB/c mice immunized with OsO4-oxidized poly(dT) complexed with methylated bovine serum albumin. This report describes the characterization of the antibody from one hybridoma using a competitive enzyme-linked immunosorbent assay (ELISA). The antibody reacted with both single- and double-stranded DNA treated with OsO4, and with OsO4-treated poly(dA-dT) and poly(dT); it did not crossreact with unmodified or apurinic DNA. It also reacted with DNA treated with H2O2 or with gamma-rays at doses as low as 250 rad. We were able to detect 2 fmoles of thymine glycol in OsO4-treated DNA and could quantitate 1 thymine glycol per 220 000 thymines. Using the antibody and the ELISA, the formation and removal of thymine glycol was examined in cultures of African green monkey cells irradiated with 25 krad of gamma-rays. The antibody reactive sites produced by irradiation (8.5 per 10(6) thymines) were efficiently removed from the cellular DNA.

Animals↗

PubMed1981

Identification of the cis-thymine glycol moiety in chemically oxidized and gamma-irradiated deoxyribonucleic acid by high-pressure liquid chromatography analysis.

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by chemical oxidants and ionizing radiation. We describe the separation of thymine glycol, 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol), thymine, and thymidine by high-pressure liquid chromatography (HPLC). Enzymatic hydrolysates of chemically oxidized or gamma-irradiated single-stranded DNA were cochromatographed with 14C-containing marker compounds. In chemically oxidized DNA, thymidine glycol was the major derivative formed. In addition, there were four rapidly eluting thymine-derived components. In irradiated DNA, thymidine glycol constituted about 5% of the modified thymines, and the rapidly eluting fractions were proportionately increased. DNA isolated from gamma-irradiated and nonirradiated HeLa cells grown in the presence of [3H]thymidine was subjected to enzymatic hydrolysis and HPLC analysis. In control DNA, 0.3% of the thymines were modified. Thirty-six kilorads of gamma radiation caused a 30% increase in thymine damage. Thus, most of the base damage was due to internal beta radiation from incorporated [3H]thymidine. The chromatographic patterns of irradiated and nonirradiated samples were qualitatively the same, but the yields of some products increased 2-fold, while others remained unchanged. A comparison of the HPLC profiles of hydrolysates of in vitro oxidized and irradiated DNA with those of the cellular DNA revealed one fast eluting peak to be absent in cellular DNA, suggesting that it was formed only in single-stranded DNA. In cellular DNA, the major modified thymine was a more hydrophobic derivative not formed by in vitro radiation nor chemical oxidation. As in in vitro irradiated DNA, thymidine glycol constituted 5% of the modified thymines. The presence of cis-thymidine glycol in hydrolysates was confirmed by chromatography on Sephadex LH-20 using water and borate as eluants.

Chromatography, High Pressure Liquid↗

PubMed1998

Oxidative charge transfer To repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metallointercalators.

Potent oxidants which intercalate in DNA serve as tools to probe DNA-mediated electron-transfer reactions. A photoexcited rhodium intercalator, Rh(phi)2DMB3+ (phi = 9,10-phenanthrenequinone diimine and DMB = 4,4'-dimethyl-2,2'-bipyridine), tethered to DNA, promotes both oxidative damage to 5'-GG-3' doublets in DNA and the repair of thymine dimers from a remote site on the DNA duplex. DNA-mediated repair of a thymine dimer lesion by charge transfer from the tethered rhodium intercalator is quantitative, albeit with low photoefficiency, occurs in an intraduplex reaction over long range (36 A), and requires that the intervening bases be paired. When both oxidative reactions, repair and oxidative damage, are monitored on the same duplex, competition is evident; the presence of both a 5'-GG-3' site and the thymine dimer diminished the dimer repair efficiency by 20-40% and decreased damage at the 5'-GG-3' sites 2-fold compared to similar sequences lacking either the guanine doublet or thymine dimer, respectively. In addition to damage at the 5'-G of 5'-GG-3' sites, we also observe oxidation at the 3'-G of the 5'-GT<>TG-3' tetrad only in the presence of thymine dimer. Overall, the yield of repaired thymine strand was at least 10 times higher than the yield of oxidized guanine in the same sequences. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Dipyridophenanzine (dppz) complexes of ruthenium(III), less potent oxidants which intercalate in DNA, oxidize 5'-GG-3' doublets efficiently but cannot trigger the repair of the thymine dimer lesion. Oxidative damage to DNA from a distance, mediated by the DNA base pair stack, can, however, be utilized to probe the disruption in the base stack generated by the thymine dimer. The presence of the dimer does not diminish oxidation by a Ru(III) intercalator at a distal guanine doublet, suggesting that the disruption caused by the dimer does not block charge transfer through the DNA duplex. DNA-mediated electron-transfer reactions of metallointercalators therefore serve to illustrate important aspects of radical migration and its consequence with respect to reactions at a distance through the DNA base pair stack.

Binding Sites↗

PubMed1997

Structure of a duplex DNA containing a thymine glycol residue in solution.

Oxidative stress, ionizing radiation, and other events can induce the oxidation of the thymine in DNA to thymine glycol. The presence of thymine glycol can have significant biological consequences, and there are specific repair enzymes for thymine glycol in a wide range of organisms. The structure of a duplex DNA containing a single thymine glycol (5,6-dihydroxy-5,6-dihydrothymidine) has been determined by the combined use of NMR and restrained molecular dynamics. The duplex of d(C1G2C3G4A5Tg6A7C8G9C10C11) paired with d(G22C21G20C19T18A17T16G15C14G13G12), with Tg indicating thymine glycol, has been used for these studies. The structure shows that the thymine glycol induces a significant, localized structural change with the thymine glycol largely extrahelical. This structural information is consistent with the biological consequences of thymine glycol in DNA. This structure is compared with that of a DNA duplex with an abasic site in the same sequence context.

Chromatography, High Pressure Liquid↗

PubMed1986

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.

Base Sequence↗

PubMed1967

Thymidine and thymine incorporation into deoxyribonucleic acid: inhibition and repression by uridine of thymidine phosphorylase of Escherichia coli.

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.

DNA, Bacterial↗

PubMed1985

[Metabolism of [2-14C]thymine and [2-14C]thymidine in germinating black gram (Phaseolus mungo) seeds].

The metabolism of [2-14C]thymine and [2-14C]thymidine in the cotyledons and embryonic axes of black gram (Phaseolus mungo) seedlings was investigated. Both [2-14C]thymine and [2-14C]thymidine degraded extensively into [14C]CO2. The rate of release of [14C]CO2 from [2-14C]thymine was much greater than that from [2-14C]thymidine. Radioactivity from both precursors was also observed beta-ureidoisobutyric acid. This indicated that thymine was degraded by the reductive pathway of pyrimidine degradation. Small amounts of [2-14C]thymine and [2-14C]thymidine were salvaged for deoxyribonucleotide and DNA synthesis. The highest incorporation of [2-14C]thymine and [2-14C]thymidine into the DNA fraction was observed in 24 hour-old cotyledons where net DNA synthesis was not observed. These precursors seem to be utilised for DNA synthesis of organelles of the cotyledonary cells, probably mitochondria. In embronic axes, [2-14C]thymine is more effectively salvaged for DNA synthesis than [2-14C]thymine. The incorporation rate increased during the early phase of germination and attained its maximum at 48 h after which it decreased. No thymidine kinase activity was detected in either cotyledons or in the embryonic axes. Thymidine salvage seems to be catalysed by nucleoside phosphotransferase which is present both in the cotyledons and in the embryonic axes. This suggests that, in contrast to other pyrimidine and purine bases and nucleosides, no specific salvage system for thymine and thymidine is present in black gram seedlings.

Carbon Radioisotopes↗

PubMed1989

Processing of ring saturation and fragmentation products of DNA thymine in Escherichia coli.

The biological processing of thymine ring saturation and fragmentation products is summarized in Table 1. The ring saturation product, thymine glycol, is a block to in vitro DNA synthesis, whereas the ring saturation product, dihydrothymine, is not. Both these lesions are recognized in vitro by endonucleases III and VIII. Since thymine glycol is a replicative block, it is a lethal lesion in vivo. The excision repair process for removal of thymine glycols from DNA is initiated in vivo by endonuclease III and is followed by the action of either exonuclease III or endonuclease IV. Thymine glycol is very efficiently bypassed by translesion bypass in both single and double stranded DNA, however, because thymine glycol templates an adenine (A) and retains pairing characteristics, it is at best a weakly mutagenic lesion. The thymine ring fragmentation product, urea, and apurinic/apyrimidinic (AP) sites are both strong blocks to in vitro DNA synthesis. Both are substrates in vitro for endonucleases III, IV, VIII and IX as well as exonuclease III. Both are lethal lesions in single stranded and double stranded phage transfecting DNA. The excision repair of urea residues and AP sites is initiated in vivo by either exonuclease III or endonuclease IV. Neither of these noninstructive lesions are efficiently bypassed by UV-induced translesion bypass, however, when bypass occurs mutations result. beta-ureidoisobutylic acid is also a block to DNA synthesis in vitro. DNA containing this lesion is a substrate for endonucleases VIII and IX. The biological processing of this ring open thymine fragmentation product has yet to be determined. Thus, these ring saturation and fragmentation products of thymine have provided a point of departure for understanding the biological processing of modified bases with altered pairing and/or stacking properties.

DNA Damage↗