DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

Repair processes on deoxyribonucleic acid

Many cells have the ability to recognize and eliminate damage to their DNA, particularly thymine dimers formed by UV light. The elimination of this damage may be achieved by enzymatic, light-dependent cleavage of the dimers into the monomers (photoreactivation) or more frequently by dark repair, in which the damaged part is completely removed from the, DNA. In this repair process, the DNA is incised by an endonuclease in the immediate vicinity of the thymine dimers. Oligonucleotides containing the thymine dimer are removed hydrolytically from the DNA by the 5→3′ exonuclease activity of DNA polymerase I (Kornberg enzyme). The resulting gaps are immediately closed by a de novo synthesis with the aid of the same DNA polymerase I, the complementary strand serving as a template (excision repair). The final step is the formation of the phosphodiester bond between the newly synthesized DNA fragment and the old DNA strand by a DNA ligase. Xeroderma pigmentosum patients lack the endonuclease as a result of a genetic defect; they therefore cannot eliminate thymine dimers from their DNA, and are extremely sensitive to sunlight. All information so far suggests that genetic recombination and DNA repair are performed by the same enzyme system.     

IDENTIFICATION OF ULTRAVIOLET-INDUCED THYMINE DIMERS IN DNA BY ABSORBANCE MEASUREMENTS

Abstract— The irradiation of native DNA's by ultraviolet radiation of different wave lengths changes their absorption spectra. The changes are similar to those found for the formation of dimers between adjacent thymines in polynucleotide chains. The decreases in absorbance at 270 mµ produced by 280 mµ irradiation are reversed to a large extent by subsequent 239 mµ irradiation. The magnitude of the absorbance changes produced by large doses of 280 mµ correspond to the formation of dimers between approximately 50 per cent of all the TT sequences in the DNA. An incident dose of 100 erg/mm² of 280 mµ radiation forms about one dimer per molecule of calf thymus DNA of molecular weight 6 times 10⁶. The irradiation of heat-denatured DNA produces larger absorbance changes than are observed in native DNA. The absorbance changes in denatured DNA arise in part from a heat-reversible reaction, presumably involving cytidine, part from the formation of thymine dimers, and part from some unknown photoproducts. The reversal of thymine dimers by short wave length irradiation does not pioduce an equivalent change in the melting temperature of the DNA.     

DNA DAMAGE AND REPAIR: INDUCTION AND REMOVAL OF THYMINE DIMERS IN ULTRAVIOLET LIGHT IRRADIATED INTACT WATER PLANTS

Abstract— The production of UV-induced thymine dimers and their fate upon post-irradiation incubation in the dark was studied in DNA of the intact water plants Wolffia microscopica and Spirodela polyrhiza. The results demonstrate production of thymine dimers, and the ability of the plant cells to remove the dimers from their DNA. The rate of removal is rapid during the first few h of post-irradiation incubation in the dark. It continues at a slower rate for the next 24–48 h, at which time it is essentially complete. The disappearance of thymine dimers in light or in the dark is analogous to the well-known processes in other biological systems, namely, photoreactivation and dark excision.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

Use of high-performance liquid chromatography to quantitative 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 quantitaion 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 μg of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a μBondapak C₁₈ column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in wateras 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.     

PHOTOREVERSIBILITY OF UV-INDUCED THYMINE DIMERS AND ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS

Abstract— Irradiation of synchronously dividing 16-cell embryos of a sea-urchin ( Hemicentrotus pul-cherrimus ) with 200 J m⁻² of UV light (254 nm) resulted in the complete inhibition of normal pluteus-larva formation when the embryos were cultured in the dark after UV-irradiation. Illumination of the UV-irradiated embryos with visible light (11 W m⁻²) for 1 h immediately after the UV-irradiation reversed the abnormal morphogenesis. Measurement of thymine dimers indicates that the degree of UV-induced abnormal morphogenesis is greatly correlated with the amount of thymine dimers in the DNA of the embryos. The degree of the photoreversal decreased with an increase in the interval between UV-irradiation and exposure to visible light. Visible light was ineffective as to the reversibility of both thymine dimers and the abnormal morphogenesis at 60 min after the UV-irradiation, when the UV-irradiated 16-cell embryos entered the next cell cycle.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are discussed.     

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS IN DNA BY INDOLE DERIVATIVES AND TRYPTOPHAN-CONTAINING PEPTIDES

Abstract —Indole derivatives, such as serotonin or the oligopeptide Lys-Trp-Lys, are able to photosensitize the splitting of thymine dimers in DNA. These indole derivatives have to be bound to DNA in order to efficiently photosensitize the splitting reaction. Serotonin may also induce the photosensitized formation of thymine-containing dimers in native DNA. In this case, an equilibrium is reached when 5 per cent of the total thymines are dimerized. In both cases (splitting and dimer formation), the formation of electron donor-acceptor complexes between either dimers or two adjacent thymine monomers, and excited indole rings, could be an intermediate step in the reactions. Thymine-dimer splitting would then result from an electron transfer reaction involving the indole ring as the electron donor. These results are discussed with respect to the mechanism of action of the photoreactivating enzyme.     

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.     

The triplet energy of thymine in DNA

Norfloxacin (NFX) photosensitizes formation of thymine dimers (T<>T) in DNA, while its N(4') acetyl derivative (ANFX) does not. This is evident from the observation of single-strand breaks after enzymatic treatment with T4 endonuclease V and subsequent gel electrophoresis. The triplet energies of NFX and ANFX are estimated at 273 and 268 kJ/mol, respectively, on the basis of triplet-triplet energy transfer quenching by a set of biphenyl and naphthalene derivatives. Hence, the triplet energy of thymine in DNA (i.e., the value for a photosensitizer to produce T<>T) can be estimated at 270 kJ/mol.     

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.     

Structure and dynamics of poly(T) single-strand DNA: implications toward CPD formation

The formation of cyclobutane pyrimidine dimers between adjacent thymines by UV radiation is thought to be the first event in a cascade leading to skin cancer. Recent studies showed that thymine dimers are fully formed within 1 ps of UV irradiation, suggesting that the conformation at the moment of excitation is the determining factor in whether a given base pair dimerizes. MD simulations on the 50 ns time scale are used to study the populations of reactive conformers that exist at any given time in T18 single-strand DNA. Trajectory analysis shows that only a small percentage of the conformations fulfill distance and dihedral requirements for thymine dimerization, in line with the experimentally observed quantum yield of 3%. Plots of the pairwise interactions in the structures predict hot spots of DNA damage where dimerization in the ssT18 is predicted to be most favored. The importance of hairpin formation by intra-strand base pairing for distinguishing reactive and unreactive base pairs is discussed in detail. The data presented thus explain the structural origin of the results from the ultrafast studies of thymine dimer formation.     

Expanding the horizon of the thymine isostere biochemistry: unique cyclobutane dimers formed by photoreaction between a thymine and a toluene residue in the dinucleotide framework

Substituted toluenyl groups are considered as close isosteres of the thymine residue. They can be recognized by DNA polymerases as if they were thymine. These toluene derivatives are generally inert toward radical additions, including the [2+2] photo-cycloadditions, due to the stable structure of the aromatic ring and are usually used as solvents for radical reactions. Surprisingly, after incorporating toluene into the dinucleotide framework, we found that the UV excited thymine residue readily dimerizes with the toluenyl moiety through a [2+2] photo-addition reaction. Furthermore, the reaction site on the toluenyl moiety is not the C5=C6 bond, as commonly observed in cyclobutane pyrimidine dimers, but the C4=C5 or C3=C4 instead. Such a reaction pattern suggests that in the stacked structure, it is one of these bonds, not the C5=C6, that is close to the thymine C5=C6 bond. A similar structural feature is found in DNA duplex with a thymine replaced by a 2,4-difluorotoluene. Our results argue that although the substituted toluenyl moieties closely mimic the size and shape of the thymine residue, their more hydrophobic nature determines that they stack on DNA bases differently from the natural thymine residue and likely cause local conformational changes in duplex DNA.     

PYRIMIDINE DIMERS INDUCED IN ESCHERICHIA COLI DNA BY ULTRAVIOLET RADIATION PRESENT IN SUNLIGHT

Abstract— Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine–thymine dimers (C < > T) were more frequent than thymine dimers (T < > T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295–315 nm), C < > T are probably more important than T < > T.     

Optimization of DNA extraction from a scleractinian coral for the detection of thymine dimers by immunoassay

Ultraviolet (UV)-B is known to cause DNA damage, principally by the formation of thymine dimers, but little research has been conducted in coral reef environments where UV doses are high. The majority of tropical reef-dwelling corals form a mutualistic symbiosis with the dinoflagellate Symbiodinium but few studies have been conducted on in situ DNA damage in corals and none have investigated the symbiotic components separately. The aim of this research was to quantify DNA damage in both the coral host and the dinoflagellate symbiont. The first step in this investigation was to optimize the extraction of DNA from the host, Porites astreoides, as well as the symbiont. The optimization was divided into a series of steps: the preservation of the samples, separation of the coral tissue from the skeleton, separation of the host tissue from the algal cells to prevent cross contamination as well as the extraction and purification of genomic DNA from the algae that are located intracellularly within the invertebrate animal tissue. The best preservation method was freezing at low temperatures without ethanol. After scraping with a razor blade, the coral tissue can be divided into host and algal components and the DNA extracted using modifications of published techniques yielding DNA suitable for the quantification of thymine dimer formation using antibodies. Preliminary data suggest that in P. astreoides collected from 1 m depth, thymine dimers form approximately 2.8 times more frequently in the host DNA than in the DNA of its symbionts.     

Role of DNA polymerase II in the tolerance of thymine dimers remaining unexcised in UV-irradiated Escherichia coli exposed to pre-UV nutritional stress.

Nutritional stress applied prior to UV-irradiation to E. coli 15 555-7 reduced thymine dimer excision and inhibited post-UV incorporation of thymidine in polB(+) as well as in polB(-) cells. However, the pre-UV-stressed polB(+) cells were significantly more UV-resistant and after UV synthesized larger DNA molecules than the pre-UV-stressed polB(-) cells. The data suggest that DNA polymerase II is involved in the tolerance of unremoved thymine dimers.     

THE INACTIVATION OF RIBONUCLEIC ACID FROM TOBACCO MOSAIC VIRUS BY ULTRAVIOLET RADIATION AT DIFFERENT WAVE-LENGTHS

Abstract— Although both thymine and uracil can form similar dimers, exposing RNA of tobacco mosaic virus lo ultraviolet radiation of different wavelengths did not reproduce any of the phenomena that implicate dimerization of thymine residues as a major cause of the inactivation of a bacterial transforming DNA. If uracil residues dimerize at all in the irradiated RNA, such dimerization either does not affect infectivity or is not photoreversible in the same way as dirnerization of thymine residues in DNA. Unlike inactivation of the transforming DNA, inactivation of the virus-RNA seems to be a function of the amount of absorbed radiation energy, irrespective of the wave-length within the range 285 to 230 mμ and irrespective of a change in the wave-length during irradiation.     

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.     

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.