Thymine lesions produced by ionizing radiation in double-stranded DNA

A DNA glycosylase which catalyzes the release of thymine residues damaged by ring saturation, fragmentation, or ring contraction from double-stranded DNA has been used to characterize such base derivatives in gamma-irradiated DNA. It is shown by chromatographic analysis that irradiation of DNA in neutral solution generates the ring-saturated forms cis-thymine glycol, trans-thymine glycol, and a monohydroxydihydrothymine, probably 6-hydroxy-5,6-dihydrothymine. The latter compound is only observed after irradiation under hypoxic conditions. The ring-contracted thymine derivative 5-hydroxy-5-methylhydantoin is also formed, and it is the major lesion after irradiation of DNA under O2. Ring-fragmented products such as methyltartronylurea were only generated in small quantities. Isolation and analysis of the DNA from gamma-irradiated human cells also revealed the formation of ring-saturated thymine derivatives, but 5-hydroxy-5-methylhydantoin was not found in this case.     

Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro.

Thymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (OsO4). For the formation of urea-containing M13 DNA, OsO4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol- and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.     

The participation of a tryptophan residue in the binding of ferric iron to pyrocatechase

The fast reaction technique of pulse radiolysis was used to produce free radicals in aqueous solution from alcohols, deoxyribose, cytosine, uracil, thymine, dihydrothymine and histidine. The electron transfer reactions from these radicals to p-benzoquinone was observed from the formation kinetics of the semiquinone anion ·BQ^? at 430 nm and the efficiency was found to be as high as 90% or more, with k～5×10⁹ M^?1sec^?1. In acid or neutral solutions in the presence of oxygen the peroxy radicals ·O₂RH formed do not essentially transfer an electron to BQ, and the efficiency is <10%. The significance of these results in the fixation of radiation damage in photobiology and radiation biology are indicated. The reactions of the superoxide ·O₂^? radical with BQ are also presented and discussed.     

Effect of non-volatile scavengers of hydroxyl radicals on thymine radical formation induced by gamma-rays and ultrasound

In order to investigate the mechanism of sonolysis of nucleic acid constituents, the yield of thymine radicals generated by 50 kHz ultrasound in Ar-saturated aqueous solution was compared with that formed by gamma-radiolysis in N2O-saturated solutions in the presence of various non-volatile scavengers, which cannot act in the gas phase of the cavitation bubbles. For comparison of thymine radical yields by sonolysis and gamma radiolysis, the method of spin trapping with 3,5-dibromo-4-nitrosobenzenesulphonate (a water-soluble, non-volatile, aromatic nitroso spin trap) combined with ESR was used. The efficiency of OH radical scavenging is expressed by the reciprocal value of C1/2, the scavenger concentration at which the thymine radical yield is decreased by 50 per cent. In gamma radiolysis the scavenging efficiencies of the solutes depend on their rate constants with OH radicals. For sonolysis the C1/2 values were similar to those obtained for gamma radiolysis except for the hydrophobic 5,5-dimethyl-1-pyrroline-N-oxide. These results suggest that thymine radicals induced by ultrasound are produced in the bulk of the solution as well as in the interfacial region.     

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.     

Repair effect of thymine radical anion by echinocoside using pulse radiolysis

Repair activities of thymine radical anion by echinocoside, isolated from Pedicularis plicata. were studied using pulse radiolysis technique. The thymine radical anion was produced by the reaction of hydrated electron with thymine. Echinocoside. one of the polyphenols of phenylpropanoid glycoside, was added to the thymine aqueous solution saturated with N2. Kinetic analysis by transient absorption spectrum showed that thymine radical anion was formed at first, and then after several decades of microseconds of pulse radiolysis. the spectrum of thymine radical anion was changed to that of echinocoside radical anion. The evidence indicated that thymine radical anion was repaired through one-electron-transfer between the DNA base radical anion and echinocoside. The rate constant of electron transfer by echinocoside was 1.45× 109 dm3 · mol1 · s 1.     

UV-Absorbing Substance in the Red Alga <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) Block Thymine Photodimer Production

The effect of the water-soluble UV-absorbing substance (UVAS) extracted from the marine red alga Porphyra yezoensis Ueda on UV-dependent thymine photodimer production was investigated. The T<>T pyrimidine-pyrimidone 6-4 dimer and the cyclobutane cis-syn T<>T 5-6 dimer produced by UV irradiation with a xenon lamp were analyzed by reverse-phase high-performance liquid chromatography. Although the dimer production was reduced when the irradiation was filtered through a UVAS solution, it decreased more when thymine was mixed with UVAS. Furthermore, UVAS inhibited the degradation of UV-irradiated thymine. The inhibitory effect of UVAS was significantly greater than that of exogenously added adenine or guanine, which forms complementary base pairs with thymine. These data suggest that in addition to its filtering effect against UV radiation, UVAS also protects thymine by a direct molecule-to-molecule energy transfer process. The protective function of UVAS against UV irradiation is advantageous for this alga under strong UV irradiation.     

Changes of fluorescence spectra of thymine in aqueous solution by radiation

Aqueous solution of thymine (5 X 10(-4) M, buffered at pH 7.0) was irradiated with 60Co gamma-rays under four different atmospheric conditions. In the presence of t-BuOH-N2, there was little increase in the fluorescence intensity as was previously reported in the radiolysis of cytosine. Under O2 there was also no significant increase differing from the case of cytosine. The fluorescence intensity was found to increase appreciably under N2O but it was less under N2 indicating that OH radical is mainly responsible for the formation of the highly fluorescent products. However, the fluorescence yields under these conditions were much lower in thymine radiolysis than cytosine radiolysis.     

Adsorption of Adenine and Thymine on Zeolites: FT-IR and EPR Spectroscopy and X-Ray Diffractometry and SEM Studies

The interactions of adenine and thymine with and adsorption on zeolites were studied using different techniques. There were two main findings. First, as shown by X-ray diffractometry, thymine increased the decomposition of the zeolites (Y, ZSM-5) while adenine prevented it. Second, zeolite Y adsorbed almost the same amount of adenine and thymine, thus both nucleic acid bases could be protected from hydrolysis and UV radiation and could be available for molecular evolution. The X-ray diffractometry and SEM showed that artificial seawater almost dissolved zeolite A. The adsorption of adenine on ZSM-5 zeolite was higher than that of thymine (Student-Newman-Keuls test-SNK p<0.05). Adenine was also more greatly adsorbed on ZSM-5 zeolite, when compared to other zeolites (SNK p<0.05). However the adsorption of thymine on different zeolites was not statistically different (SNK p>0.05). The adsorption of adenine and thymine on zeolites did not depend on pore size or Si/Al ratio and it was not explained only by electrostatic forces; rather van der Waals interactions should also be considered.     

γ-Irradiation of Thymine Dimers in Aqueous Solution

Thymine dimers were irradiated in aqueous solution with ⁶⁰Co γ-rays in N₂ or O₂. Thymine and unidentified non-UV-absorbing products appeared. The thymine was identified by spectrophotometry, chromatography, and ability to support the growth of Escherichia coli 15 T^-. Residual dimer was determined by a UV-reversibility assay. The G-values for dimer breakage were approximately equal in N₂ and O₂. At low γ-doses, about two thymines were produced per dimer broken in N₂, whereas only about one thymine appeared per dimer broken in O₂. For dimer irradiated in frozen solution, the yield of thymine was at least 100 times less than in liquid.     

Thymine dimer excision in UV-irradiated and 5-fluorouracil-postincubatedEscherichia coli

The effect of post-irradiation cultivation with 5-fluorouracil on the excision of thymine dimers following UV irradiation was examined inEscherichia coli 15 T-U-his-. It was found that an increase of the number of surviving cells caused by 60-min post-incubation with 5-fluorouracil is not accompanied by any more rapid and complete removal of thymine dimers from the damaged molecule of DNA     

The After Effect of Irradiated Halogenophenol Solutions on the Viability of Escherichia coli

Gamma irradiation has been found to induce a remarkable bactericidal activity in an aqueous soltion of p-bromophenol even at the non-toxic lower concentrations, but not in a crystalline form or under a frozen state. Effects of concentration of reagents, temperature, pH, radiation dose, oxygen and some additives during irradiation on the radiation induction of bactericidal activity in the p-bromophenol solution were investigated. The results suggest that the induced activity is attributable to some long-life products, formed as a result of radiolysis of p-bromophenol and in coupling with radiolysis of water. The processes of formation and destruction of such bactericidal factor(s) were considerably influenced by several conditions during irradiation. Based on the experimental results, relationship between radiolysis process and induction of bactericidal activity was discussed.     

E.S.R. studies of free radicals derived from reaction of OH with uracil and thymine

Short-lived free radicals produced in N2O-saturated aqueous solutions of uracil and thymine have been studied using the in situ radiolysis steady-state e.s.r. method. Radical formed in alkaline aqueous solutions by OH addition to either positions C(5) or (6) were observed. Mechanisms for the formation of transient species were derived. The spin density distribution of the unpaired electron was calculated by means of the INDO method.     

Thymine 7-hydroxylase from Neurospora crassa. Substrate specificity studies.

A partially purified preparation of thymine 7-hydroxylase (thymine, 2-oxoglutarate : oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) from Neurospora crassa was incubated with a number of pyrimidines chemically related to tyymine. 1. Pyrimidines with oxygen or sulfur substituents on atoms Nos. 2 and 4 as well as an alkyl group on atom Nos. 1 or 5 were substrates. 2. Km values were determined for 1-methyluracil, 1-ethyluracil, thymine, 6-azathymine, 1-methylthymine, 1-ethylthymine, 5-formyluracil and 5-hydroxymethyluracil. 3. Uracil was identified as one of the metabolites after incubation with 1-methyluracil. The one-carbon metabolite has not been characterized. 4. Several pyrimidines with polar groups on atoms Nos. 2 and 4 were inhibitory. 5. Addition of 1-methyluracil, 1-methylthymine, 1-ethylthymine or 5-hydroxymethyluracil to incubations with thymine and 2-oxo[1-14C1]glutarate did not result in additional formation of 14CO2, indicating that the same enzyme acts on the different compounds. It has previously been found (Bankel, L., Holme, E., Lindstedt, G. and Lindstedt, S. (1972) FEBS Lett. 21, 135-138) that a mutant strain of N. crassa which is devoid of thymine 7-hydroxylase activity also lacks ability to perform the coupled oxygenation of 2-oxoglutarate and 1-methyluracil, 5-hydroxymethyluracil and 5-formyluracil, respectively. It is concluded that one and the same oxygenase is responsible for the activities studied.     

Thymine glycol lesions terminate chain elongation by DNA polymerase I in vitro.

Single-strand circular DNA from bacteriophage M13mp9 was chemically modified with osmium tetroxide to introduce specifically cis-thymine glycol lesions, a major type of DNA damage produced by ionizing radiation. An oligonucleotide primer was extended on damaged and undamaged templates using either the large fragment of E. coli pol I or T4 DNA polymerase. The reaction products were analysed by electrophoresis alongside a DNA sequence ladder. Synthesis on the damaged templates terminated at positions opposite thymine bases in the template. These results indicate that cis-thymine glycol lesions in single-strand DNA constitute blocks to synthesis by DNA polymerases in vitro. Surprisingly, replication halts after the correct nucleotide, dAMP, is inserted opposite the lesion. These results imply that the primary effect of the thymine glycol lesion is suppression of DNA synthesis and that the lesion is not a potent mutagen.     

Thymine and Thymidine Uptake by Haemophilus influenzae and the Labeling of Deoxyribonucleic Acid

The influence of ribo- and deoxyribonucleosides and ribo- and deoxyribonucleotides on the uptake of radiolabeled thymidine and thymine by Haemophilus influenzae during growth was investigated. A nucleoside-degrading enzyme similar to that reported in Escherichia coli was found to break down thymidine unless other nucleosides were present to divert its action. The presence of other nucleosides permitted a nearly quantitative uptake of even low levels of thymidine. This quantitative uptake of thymidine in the presence of an excess of other nucleosides suggests that the uptake mechanism for thymidine is specific in this organism. Under optimal conditions, as much as 50% of the deoxyribonucleic acid (DNA) thymine was derived from exogenous thymidine. Thymine was not taken up by H. influenzae but, in the presence of purine deoxynucleosides, labeled thymine entered the cells, presumably as thymidine. Ribonucleosides or ribonucleotides inhibited thymine conversion to thymidine, but, as noted above, they were degraded by a cellular enzyme. Thus, unless the ribonucleoside level was excessively high, a cell level of growth was reached at which the inhibiting ribonucleoside was broken down and labeled thymine appeared in the cells at an increasing rate. Twenty-five per cent of the DNA thymine of this organism was labeled with exogenous thymine. The information noted above serves as the basis for isotopically labeling the DNA.     

Antioxidative properties of Martynoside: pulse radiolysis and laser photolysis study

Free radical reactions of Martynoside (MAR), a phenylpropanoid glycoside, with a variety of oxidants were studied in the aqueous solution by laser photolysis and pulse radiolysis techniques. The pK

a

value of MAR in aqueous solution was measured from the pH dependent changes of the UV absorption at 384 nm with value of pK_a=9.2. The phenoxyl radical of MAR which exhibits maximum absorption at 360 nm was generated by one-electron transfer to N3&bull• or Br2• properties of phenoxyl radical such as extinction coefficient, formation and decay rate constants were also determined. The reaction rate constant of O2•la>k=8.5×104 dm3 · mol--1 · s--1, was measured by the method of competition kinetics. By measuring time-resolved luminescence emission at 1270 nm, the quenching rate constant of singlet oxygen by MAR was obtained to be 3.3×10

6

dm

3

· mol

-1

· s

-1

. Reduction potential of the MAR couple (MAR

•

/MAR), determined using rutin as reference compound, gave a value E=0.66 V vs. NHE. The antioxidative properties of MAR were compared with those of some well-known antioxidants.     

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.     

Photoreactions of thymine and thymidine with N-acetyltyrosine.

We report here the photoinduced formation of a thymine-N-acetyltyrosine adduct. Irradiation of dilute solutions of thymine in the presence of N-acetyltyrosine (NAT) leads to the formation of N-acetyl-4-hydroxy-3-(6-hydrothymin-5-yl)phenylalanine (I), isolated as a mixture of the 5R and 5S diastereoisomers; the photoreaction occurs when irradiation is done either at lambda = 254 nm or at wavelengths of lambda > 290 nm. Irradiation of thymidine in the presence of NAT and of thymine in the presence of tyrosine leads to analogous photoadducts. The photoreaction of thymine with NAT is completely quenched by oxygen and cannot be sensitized by acetone. The likely mechanism involves initial photoionization of the amino acid and deprotonation to form the phenoxyl radical. Thymine then probably captures the released aqueous electron, leading to protonation at C6 of the resulting radical anion. Combination of the phenoxyl and 5,6-dihydrothymin-5-yl radicals would then lead to formation of the final products. The quantum yield for production of the thymine-NAT adduct at pH 7.8 was estimated to be about 5.5 x 10(-4), while a value of 2.3 x 10(-3) was estimated for production of corresponding thymidine adduct at pH 8.1. The dependence of the quantum yield for adduct formation on pH has been determined for both the thymine and thymidine reactions with NAT; the maxima in the quantum yield profiles occur at pH 8-8.5, while appreciable values were measured at pH 7.5. We have also demonstrated that a similar reaction occurs when tyrosine is located within a peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III

A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts. The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons. The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites. We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E. coli endonuclease III, that recognizes oxidative DNA damage.