RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

THE EFFECTS OF HOST-CELL REACTIVATION ON ASSAY OF U.V.-IRRADIATED HAEMOPHILUS INFLUENZAE TRANSFORMING DNA

Abstract— The host cell reactivation (HCR) mechanism in Haemophilus influenzae cells is inhibited by sub-microgram concentrations of acriflavine (as is already known to be true for Escherichia coli ). Exposure of these cells to similar concentrations of the drug during bacterial transformation increases the apparent ultraviolet light (u.v.) sensitivity of previously irradiated transforming DNA, indicating a repair of this DNA after uptake by the cells under normal conditions. Repair is inhibited by applying acriflavine at any time up to one hour after competent cells contact the irradiated transforming DNA. The fraction of the u.v. damage repaired by HCR is very different for different genetic markers. Those markers which are most u.v. sensitive when assayed in the absence of acriflavine are most poorly repaired, suggesting that this is the reason for their higher sensitivity. For all markers the fraction of the damage repairable by in vitro photoreactivation is approximately constant, and strongly overlaps the damage repairable by HCR. The degree of HCR achieved can be altered by experimental treatment of the H. influenzae DNA prior to transformation. Thus treatment of irradiated DNA with an enzyme from Micrococcus lysodeikticus –known to attack u.v. damaged, but not undamaged DNA–prevents subsequent intracellular repair of the same u.v. lesions whose repair is inhibited by acriflavine. Similarly, partial replacement of the thymine in transforming DNA by 5-bromouracil (BU) strongly inhibits repair of subsequent u.v. damage. As in bacteriophage, the BU effect is relieved if the u.v. exposure occurs in the presence of cysteamine. It is clear that intracellular repair must be considered in interpreting experiments with u.v.-irradiated transforming DNA.     

SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE T4

Abstract— The photoinactivation of an osmotic shock resistant mutant of the bacteriophage T 4 can be sensitized by two cationic derivatives of acetophenone. At least part of the sensitized inactivation appears to be due to the formation of thymine dimers in phage DNA by a mechanism which involves triplet excitation transfer from the sensitizer to thymine in the DNA. The photoreactivable sector of the phage inactivated by sensitized irradiation is about 0.6, almost twice as large as the sector for u.v. irradiated phage.     

THE PHOTOCHEMICAL INTERACTION OF DEOXYRIBONUCLEIC ACID AND PROTEIN IN VIVO AND ITS BIOLOGICAL IMPORTANCE*,†

Summary When bacteria are irradiated with u.v. light there is a dose dependent decrease in the amount of DNA that can subsequently be extracted free of protein with detergent. This appears to be due to the crosslinking of the DNA with protein and the precipitation of the linked DNA when the denatured proteins are precipitated in the procedure used for the isolation of the DNA. The type of linkage between the DNA and the protein is unknown except that it resists the sequential attack of 2% sodium lauryl sulfate and 0.5 M KCI or 55% CsCI. The main evidence that the loss of DNA in vivo is due to the crosslinking of DNA and protein is that the crosslinking of DNA and protein can be demonstrated in vitro . X-rays do not crosslink DNA and protein in vivo , but acridine orange and visible light cause the crosslinking of DNA and protein both in vivo and in vitro .
By pulse labeling the DNA of bacteria with tritated thymine it can be shown that newly synthesized DNA is most sensitive to crosslinking and that this sensitivity shows a cyclic response keyed to the generation time of the bacteria. Under conditions of thymine starvation where the intrinsic sensitivity of the cells to killing by u.v. is markedly increased, there is a parallel increase in the sensitivity of the DNA of these cells to be crosslinked to protein. The similarity in the time sequence of these two events strongly suggests that the crosslinking may play an important role in the loss of viability following u.v. irradiation.     

Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus PHR-1

Abstract— A pyrimidine adduct, 6-4‘-[pyrimidine-2’-one] thymine (PO-T)?, observed in DNA hydrolysates of 254-nm ultraviolet (u.v.) irradiated conidia of Streptomyces coelicolor, increases linearly with u.v. dose up to 2 × 10⁵ ergs/mm². Yields of thymine dimer (T○) and uracil-thymine dimer (U○) level off at much lower doses. Initial relative rates of formation of these u.v. photoproducts are: 1:1.3:4.8 for PO-T, T○ and U○, respectively. Similar results were obtained with a Streptomyces griseus mutant, PHR-1. An equation is derived to estimate the ratio of the amount of PO-T to the total amount of thymine-derived photoproducts at low (biological) u.v. doses. The observed PO-T fractions compare well with the calculated values. Rapid photolysis of the precursor of PO-T was observed by post-u. v. treatment at 313 nm of conidia of S. coelicolor and of S. griseus PHR-1. The photolysis was much slower at 365 nm and did not occur at all at 405 nm. Pyrimidine dimers were not appreciably affected by post-u. v. treatment at the above wavelengths in these Streptomyces strains. Both of these strains are phenotypically photoreactivation-deficient, and the present results indicate that they do not possess active photoreactivating enzyme. In earlier papers[3,4,5], the pyrimidine adduct found in acid hydrolysates of DNA was loosely referred to as “uracil-thymine adduct (U-T adduct)”. Such terminology is not strictly correct. The pyrimidine adduct in acid hydrolysates is PO-T (sometimes called P₂B), which could theoretically result from removal of ammonia from a C-T adduct or removal of water from a U-T adduct (see [6]).     

EFFECTS OF SENSITIZED AND UNSENSITIZED LONGWAVE U.V.-IRRADIATION ON THE SOLUTION PROPERTIES OF DNA

Abstract— Two types of photoreactions occur in DNA irradiated in aqueous systems with longwave u.v.-light (Λ > 295 nm), namely, (a) thymine dimerization, and (b) single- and double-strand breakage of the sugar phosphate backbone; these two reactions are unrelated. The presence of acetophenone as a photosensitizer caused an increase in dimerization by a factor of 16, and an increase in single-strand breaks by a factor of 4. The number of thymine dimers per single-strand break is about 100 in the sensitized and 25 in the unsensitized reaction. The alteration of the radius of gyration of DNA molecules is that expected by the degradation observed. At the same time the change in hyperchromicity is very small. Therefore as far as can be detected by these methods of investigation the gross conformation of the DNA double helix is stable against thymine dimerization.     

THYMINE DIMERIZATION IN L-STRAIN MAMMALIAN CELLS AFTER IRRADIATION WITH ULTRAVIOLET LIGHT AND THE SEARCH FOR REPAIR MECHANISMS

Abstract— The formation of thymine dimers in the DNA of L -strain mammalian cells after irradiation with ultraviolet light has been demonstrated. The amount of dimer formed rises with the dose of u.v. light.
In the course of post-irradiation incubation the thymine dimers remain in the TCA insoluble fraction and diminish as did the other thymidine-H³ derivatives with increasing incubation time. The dimer is not found in the soluble fraction. Thus, dimer excision (i.e. its liberation into the soluble fraction) as an expression of repair of radiation damage analogous to dark repair in E. coli was not found in these experiments.     

Ultraviolet action spectra in perspective: with special reference to mutation

Abstract— Problems of determining action spectra are considered as well as various types of action spectra for U.V. action upon cell activities. U.V. is an effective mutagenic agent producing point mutations and chromosomal changes. U.V. is readily absorbed by superficial layers of cells in tissues; therefore, special experimental procedures are necessary for induction of mutations in animals or plants. U.V. is, however, suitable for mutagenesis in microorganisms because their cells are small, permitting the radiation to reach the nuclei. Action spectrum studies reveal that u.v. mutagenesis results from absorption of the radiation by nucleic acid. The most prominent alteration in DNA following absorption of u.v. is dimerization of pyrimidines, chiefly thymine. Such a change not only retards DNA replication but results in errors (mutations). U.V. mutagenesis therefore depends upon the conditions before, during and after irradiation. Thus immediate post-treatment with visible and long u.v. light splits pyrimidine dimers, thereby reversing impending u.v. mutagenesis. For cells kept in the dark, conditions which prevent DNA replication by interfering with the metabolism of the cell provide time for dark repair of the DNA lesion and so for reversal of the impending mutation.     

槲皮素与尿嘧啶和胸腺嘧啶氢键的结合位点

深入理解药物分子和核酸碱基间的相互作用机制对合理设计研发新型高效药物有重要意义. 本文运用密度泛函理论B3LYP方法对核酸碱基尿嘧啶和胸腺嘧啶与药物分子槲皮素间的氢键相互作用位点进行了研究. 使用B3LYP/6-31G(d)方法优化得到了30个稳定的氢键复合物结构, 使用B3LYP/6-311++G(3df,2p)方法计算了这些复合物的结合能. 研究结果表明, 槲皮素可以使用5个不同的结合位点与尿嘧啶或胸腺嘧啶形成氢键复合物, 尿嘧啶或胸腺嘧啶可以使用3个不同的结合位点与槲皮素形成氢键复合物. 当槲皮素的结合位点固定时, 槲皮素与尿嘧啶的位点u1或胸腺嘧啶的位点t1形成的氢键作用最强, 与位点u2或位点t2形成的氢键强度最弱; 当尿嘧啶或胸腺嘧啶的作用位点固定时, 二者与槲皮素的位点qu1 形成的氢键作用最强, 与位点qu5 作用强度次之, 与位点qu3的作用强度最弱. 分子中原子(AIM)和自然键轨道(NBO)分析计算结果表明, 轨道作用在氢键中起重要作用.     

PHOTOCHEMISTRY OF THYMIDINE AS A THIN SOLID FILM*

Abstract— –Photochemistry of thymidine in the solid state has been investigated. Four isomers of the cyclobutane-type thymidine dimer, two bimolecular addition products with u.v. absorbance maxima above 300 nm, (the formation of which involves the C=C and C=O groups of adjacent thymine residues) and 5-thyminyl-5.6 dihydrothymine (TDHT) (formed by the addition of a thyminyl radical at the C₅ position of a thymyl radical) were isolated. TDHT was found to be the major photoproduct. The results are compared with those for thymidine irradiated in the frozen state.     

DIFFERENTIAL, LETHAL AND MUTAGENIC ACTION OF 254 nm AND 320–400 nm RADIATION ON SEMI-DRIED BACTERIA

Abstract— The effect of culture conditions on the lethal and mutagenic action of 254 nm (u.v.) and 320–400 nm (b.l.) light has been examined. Ten strains of Escherichin coli were used in these investigations. It was found that semi-dehydration in aerosols greatly enhanced the lethal and mutagenic actions of both U.V. and b.l., Mutations induced by U.V. were found to be of a random kind, while those produced by b.l. were specific and of a particular biochemical type depending on the strain of cell and its stage of development. The presence of oxygen during irradiation enhanced b.l. effects but had no effect on U.V. damage while anaerobic growth endowed the cells with added resistance to b.l. and u.v., Stationary phase cells of E. coli B/r were found to be mutated by b.l. specifically at a thymine locus and to be more sensitive than E. coli B to the inhibition by b.l. of respiration. Some mutations induced by b.l. in E. coli B/r were found to hinder the cells ability to carry out the photoreversal of U.V. damage. It is suggested that b.1. affects a specific piece of DNA which is in contact with the cytochrome chain of the cytoplasmic membrane and that this contact point between the cytochrome chain and DNA alters sequentially as the cell proceeds through its life cycle.     

Repair replication in Chinese hamster cells after damage from ultraviolet light

Abstract— After irradiation with u.v. light, Chinese hamster cells perform repair replication of their DNA. Small numbers of nucleosides are inserted into DNA, such that when BrUdR is used there is no detectable change in density. Repair replication begins immediately after irradiation, but it decelerates steadily and at least half is complete within 4 hr. Repair replication saturates above 200 ergs/mm² at a level which represents 0.055 per cent replacement of all thymine sites in 4 hr. Repair replication in mammalian cells, in contrast to that in microorganisms, does not appear to replace pyrimidine dimers excised from DNA in acid soluble form, and neither repair nor semiconservative replication discriminates between BrUdR and TdR.     

Fluorescence quenching study on the interaction of bases of nucleic acid with electron-accepting sensitizer and serum albumin containing electron-rich tryptophan residue

Static and dynamic fluorescence quenchings of electron-accepting sensitizers including positive charged heterocyclics and neutral cyanoaromatics by bases of nucleic acid (NB) have been investigated. It was found that NB could act as effective electron donors to quench the fluorescence of electron-accepting sensitizers. The quenchings by diffusion-controlled rate coincide well with the static and dynamic Stern-Volmer correlation. On the other hand, the diffusion-controlled fluorescence quenchings of the tryptophan (TRP) residue in the protein enzymes, HSA and BSA, by electron-accepting NB reveal that photochemical dual-damage of protein enzyme and thymine (THM) occur upon u.v.-irradiation, which is characteristic of excitation wavelength-dependence. Therefore, the results illustrate that the lesion interactions of NB with electron-deficient sensitizers or electron-rich TRP fluorophore-containing protein enzymes originate mainly from PET-initiated processes in both cases.     

Action of photoreactivating light on pyrimidine heteroadduct in bacteria

Abstract— In stationary-phase Escherichia coli B/r, photoreactivation (PR) at 313 nm of ultraviolet (u.v.) killing is inefficient compared with PR at 405 nm, and can be explained solely by photoenzymatic reversal of pyrimidine dimers. In Staphylococcus epidermidis, PR shows a maximum at 313 nm, suggesting that this organism shows the Type III PR proposed by Jagger et al.[5] for Streptomyces strains. Reversal of pyrimidine dimers is not sufficient to explain this PR. The mechanism of Type III PR remains unknown. With both S. epidermidis and E. coli B/r, the amount of uracil–thymine heteroadduct in DNA hydrolysates decreases if the cells are given a post-u.v. treatment at 313 nm, but no decrease is observed if the post-u.v. treatment is at 405 nm. The biological significance of this adduct and of its removal is not clear. It may play a role in Type III PR.     

GAMMA RAY THERMOLUMINESCENCE IN NUCLEIC ACID CONSTITUENTS

Abstract— The phenomenon of gamma ray thermoluminescence has been studied in DNA, RNA, and their various constituents. Single peaks in the glow curves have been usually observed at 110–120°K except in cytosine which shows two peaks at 135°K and 180°K. Adenine and cytosine show very intense thermoluminescence whereas the intensity of light emitted from thymine, uracil and guanine is very small. The addition of sugar and sugar-phosphate groups to the base-molecules affects only the amount of light emitted. The thermoluminescence emission spectra lie in the same wavelength region as the u.v. induced phosphorescence, suggesting a triplet-singlet transition. The spectral content of DNA thermoluminescence seems to be superposition of contributions from its constituents. An attempt is made to explain the phenomenon as a result of electron-cation recombination.     

Excision of cytosine-thymine adduct from the DNA of ultraviolet-irradiated Micrococcus radiodurans

Abstract— 6-4‘-[pyrimidin-2’-one]-thymine (PO-T), a deamination product of a cytosine–thymine adduct accounted for about 17 per cent of the total detectable thymine-derived photoproducts in u.v.-irradiated Micrococcus radiodurans. On incubation of the irradiated cells, oligonucleotides containing the photoproducts were released into the medium. After various periods of incubation the different photoproducts were isolated both from the cells and from the medium. Analysis of these different photoproducts showed that during the excision process the ratio of PO–T to total thymine-derived products remained constant both in the cells and in the medium. This shows that the precursor to PO–T is excised at the same rate as the other thymine-derived photoproducts by the dark repair mechanisms exhibited by this radiation-resistant organism.     

The self-consistent-field method in the semiempirical approximation for states with unclosed shells for nucleic acid bases

The ionized and triplet -electron states are derived. The order of increasing ionization potential is found to be quanine, cytosine, adenine, uracil, thymine, while the order in electron affinity is adenine, guanine, thymine, uracil, cytosine. It is found that the energies of the lowest triplet states are considerably reduced by reminimization of the wave functions, the reduction for adenine being almost 20% of the transition energy. Nearly equal values are obtained for the energies of transition to the lowest triplet states as found by the method of unclosed shells and by the method of interaction of singly excited configurations.     

Diffusion mechanism of the synergism of u.v.-absorbers and antioxidants

The mechanism of synergism in mixtures of u.v.-absorbers with antioxidants is considered. The synergism is attributed to diffusion of the antioxidant from the deeper-lying layers of polymer film, protected from the action of light by a u.v.-absorber, towards surface layers where the photoreaction takes place. The effect of the antioxidant diffusion process on the duration of the induction period of the unbranched chain oxidation reaction of the polymer is analysed. The experimental investigation of the polybutadiene photo-oxidation process has verified the diffusion mechanism so allowing finding of the optimum relationship between the concentrations of u.v.-absorber and antioxidant and explanation of the observed enhancement of the effect of synergism in thicker polymeric films, for higher antioxidant diffusion coefficients and lower light intensities.     

Direct demonstration of the monomerization of thymine-containing dimers in U.V.-irradiated DNA by yeast photoreactivating enzyme and light

Abstract— Ultraviolet-irradiated E. coli DNA (³H-thymine-labelled) was mixed with un-irradiated E. coli DNA (¹⁴C-thymine-labelled) and exposed to light in the presence of purified yeast photoreactivating enzyme. As the ³H-thymine-containing cyclobutane dimers disappeared during the photoreactivation, there was a stoichiometric increase of monomeric ³H-thymine as determined from the ³H/¹⁴C ratio in thymine. This is the first direct demonstration that thymine-containing dimers in u.v.-irradiated DNA are monomerized by yeast photoreactivating enzyme in the presence of light.