CHAIN BREAKAGE ACCOMPANYING THE PHOTOLYSIS OF IdUrd-DNA

Abstract DNA isolated from Escherichia coli and substituted with various amounts of iododeoxyuridine (12-95%) was irradiated at either 265. 300 or 313 nm and the frequency of chain breakage measured by sedimentation in either neutral or alkaline sucrose gradients. The wavelength dependence of the photochemical cross section for chain breaks paralleled that for iodine loss, and the average frequency of chain breakage per halogen loss was 0.50 ± 0.07. Approximately 80-90% of the breaks observed in alkali are alkali-labile bonds and are not observed under neutral conditions. The presence of ethanol during irradiation reduced the frequency of chain breakage by more than an order of magnitude. These results are interpreted in terms of a model in which photo-induced deiodination leads to the formation of a uracilyl radical which then abstracts a hydrogen atom from either a nearby sugar moiety or from another hydrogen donor such as ethanol. The resulting modified sugar can then rearrange to form either a clean chain break or an alkali-labile bond.     

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 METABOLIC INHIBITORS TO INVESTIGATE THE EXCISION REPAIR OF PYRIMIDINE DIMERS AND NON-DIMER DNA DAMAGES INDUCED IN HUMAN AND ICR 2A FROG CELLS BY SOLAR ULTRAVIOLET RADIATION

Abstract— ICR 2A frog and normal human skin fibroblasts were exposed to either 5 J/m² of 254 nm UV or 50 kJ/m² of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1–β-D-arabinofuranosyl cytosine (ara C) for 0–20 min (human fibroblasts) or 0–4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-f-old higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV.     

Fluorescence Quenching by Intercalation of a Pyrene Group Tethered to an N4‐modified Cytosine in Duplex DNA

A series of duplex DNA oligomers was prepared that contain a pyrene chromophore linked by a trimethylene chain (‐(CH₂)₃‐) to N⁴ of a cytosine. The pyrene group stabilizes the DNA as evidenced by an increase in melting temperature. The absorption spectrum of the linked pyrene chromophore shows a temperature‐dependent shift and there is also a strong induced circular dichroism spectrum attributed to the pyrene group. The fluorescence of the pyrene chromophore is strongly quenched at room temperature by linkage to the DNA, but it increases above the melting temperature. We attribute these observations to intramolecular intercalation of the pyrene group at a base pair adjacent to its linkage site at cytosine.     

Novel Visible and Ultraviolet Light Photogeneration of Hydroxyl Radicals by 2-Methyl-4-nitro-quinoline-N-oxide (MNO) and 4, 4'-Dinitro-(2, 2') bipyridinyl-N, N'-dioxide (DBD)

Chemicals that upon absorption of light generate hydroxyl radicals (·OH), free of other damaging species under physiological conditions, are useful tools for the study of the biological effects of ·OH radical and for its utilization for analytical purposes. We report the novel property of 2-methyl-4-nitro-quinoline- N -oxide (MNO) and 4, 4'-dinitro-(2, 2') bipyridinyl-N, N'-dioxide (DBD) to act as photogenerators of ·OH with UV and visible light. Upon irradiation with 360–400 nm light MNO and DBD generate free radicals that convert coumarin carboxylic acid (CCA) to fluorescent 7-OH-CCA; the ·OH radical scavengers dimethylsulfoxide (DMSO) and ethanol eliminate the induction of 7-OH-CCA fluorescence. Upon 400 nm illumination in the presence of MNO, supercoiled plasmid DNA is converted to circular and strand break-age is significantly reduced in the presence of DMSO and completely absent in the absence of MNO. The conversion of CCA to 7-OH-CCA and of supercoiled plasmid to circular DNA are also observed in the absence of oxygen. Taken together, these data indicate that MNO and DBD constitute novel ·OH-generating compounds. Because currently known ·OH-photogenerating compounds require UV illumination (< 360 nm) that also damages DNA and cells directly, the property of MNO to generate ·OH upon 400 nm illumination is advantageous when studies on cells, DNA and other biomolecules are conducted.     

CU(II) SENSITIZES pBR322 PLASMID DNA TO INACTIVATION BY UV-B(280–315 nm)

Abstract— Copper(II), in the presence of UV-B radiation(280–315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA. A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold. Concomitant with the damage to the DNA backbone is a loss of transforming activity. Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity. A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II). The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA. These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.     

PROPERTIES OF THYMINE DIMERS

Abstract— The photochemical and chemical properties of the four dimers of thymine have been studied. The extinction coefficients, reversal cross-sections and quantum yields for reversal are presented as a function of wavelength in the range 200–289 nm. At any wavelength, the dimers have different reversal cross-sections but also different extinction coefficients. The quantum yields for reversal are nearly the same for all four dimers, the values ranging from 0·6 to 0·9 over the wavelength range 200–289 nm. Titration curves for the four dimers show that for each dimer, two groups are involved with two pK's at about 10·5 and 12·2. Dimers A and B are stable at alkali and acid pH's. Dimers C and D are stable from pH 1 to 3 and unstable at other pH's, with thymine the main degradation product.     

INDUCTION OF DNA STRAND BREAKS IN NORMAL HUMAN FIBROBLASTS EXPOSED TO MONOCHROMATIC ULTRAVIOLET AND VISIBLE WAVELENGTHS IN THE 240–546 nm RANGE

Abstract— The induction of DNA single-strand breaks in normal human fibroblasts exposed to monochromatic wavelengths from 240–546 nm was measured by the alkaline elution assay. The cells were irradiated at 1°C to prevent both repair of induced breaks and formation of enzymatically induced breaks through excision repair. The cultures were also washed with and irradiated while suspended in phosphate buffered saline to prevent the formation of DNA damaging photoproducts from medium components. The action spectrum for DNA strand breakage was found to exhibit one peak at 265 nm, consistent with DNA absorption, and a second peak at 450 nm. The normalized action spectrum in the visible is similar to the normalized absorption spectrum for riboflavin, a known photosensitizing agent, implicating this molecule as the absorbing chromophore.     

PHOTOLYSIS OF IODODEOXYCYTIDINE IN DNA AND RELATED POLYNUCLEOTIDES: WAVELENGTH DEPENDENCE AND ENERGY TRANSFER*

Abstract— 5'-Iodocytosine (IC) containing denatured DNA and poly(C) were prepared and the photoinduced loss of iodine measured for irradiation at wavelengths between 240 and 313 nm. The following intrinsic quantum yields (Φ_INT) were obtained for irradiation at λ_ex > 300 nm where only IC absorbs: denatured DNA (0.01), poly(C) (0.013), apurinic acid (0.018) and IdCMP (0.026). These results suggest that geometrical or structural restraints in the polymer, which increase with the degree of base stacking, inhibit the loss of iodine from an excited IC residue. The variation in the photochemical cross section for iodine photolysis was measured as a function of the wavelength of irradiation and found to vary in a manner which indicates that absorption by noniodinated residues can lead to iodine photolysis. It is proposed that energy transfer from neighboring bases to an adjacent IC residue takes place, resulting in an action spectrum for iodine photolysis which reflects absorption of excitation energy by noniodinated as well as iodinated residues. The contribution due to energy transfer in denatured DNA was estimated to be from not more than a single base located on either side of an IC residue. The degree of transfer was slightly less in iodinated poly(C) and decreased 4-fold following depurination of the DNA. These results are consistent with a structurally dependent energy transfer process in which IC, because of its lower lying singlet state, can act as an energy trap.     

HOECHST 33258 PHOTOSENSITIZATION OF 5-IODODEOXYURIDINE-SUBSTITUTED DNA TO 365 nm LIGHT: DEPENDENCE OF DEHALOGENATION ON THE DYE-TO-NUCLEOTIDE RATIO

DNA containing 5-iododeoxyuridine substituted at levels ranging from 1 to 24% was irradiated at 365 nm in the presence of various amounts of the Hoechst dye 33258. The subsequent loss of iodine was measured by following the loss of radioactive iodine (125I) from filter immobilized DNA subjected to repeated washings. A plot of the photochemical cross section (sigma B) for this process as a function of r, the molar ratio of dye added per nucleotide present, shows a maximum at r = 0.04 to 0.06, followed by a decrease with increasing dye-to-phosphate ratios to give a minimum at r = 0.5. Increasing the amounts of dye further results in sigma B reaching a second maximum at r greater than or equal to 1, after which it levels off. An attempt was made to interpret these results in terms of the binding models currently employed for analyzing binding of Hoechst dye to DNA. Chain breaks as measured in alkali were also measured and found to be enhanced by the dye; the ratio of breaks per iodine loss was 0.9.     

A study of the iodinated resin column for the determination of iodine in biological fluids by radiochemical neutron activation analysis

The iodinated resin technique for separation of¹²⁸I directly from neutron irradiated biological fluids is attractive due to its rapidity, simplicity and high degree of radiochemical purity; however, some doubts exist about the yields of iodine which can be recovered, particularly from organically bound iodine. In the present study, chemical yields on the resin column were studied for irradiated urine, serum and milk by standard additions of iodide and thyroxine. It was found that yields are not quantitative but lie in the range 80–95%, depending on the matrix, with a variability of up to 6% standard deviation. In addition it was shown that no significant adsorption of iodine occurs on polythene ampoules from irradiated biological fluids of natural pH. Some comparative experiments between the resin technique and the oxygen flask ignition method on fluids before or after drying, respectively, showed satisfactory agreement. It is concluded that the iodinated resin technique is suitable for routine analyses or large scale screening programs.Presented in part at SAC 83, Edinburgh, July 1983.     

Cα-Cβ chromophore bond dissociation in protonated tyrosine-methionine, methionine-tyrosine, tryptophan-methionine, methionine-tryptophan and their sulfoxide analogs

C(α)-C(β) chromophore bond dissociation in some selected methionine-containing dipeptides induced by UV photons is investigated. In methionine containing dipeptides with tryptophan as the UV chromophore, the tryptophan side chain is ejected either as an ion or as a neutral fragment while in dipeptides with tyrosine, the tyrosine side chain is lost only as a neutral fragment. Mechanisms responsible for these fragmentations are proposed based on measured branching ratios and fragmentation times, and on the results of DFT/B3-LYP calculations. It appears that the C(α)-C(β) bond cleavage is a non-statistical dissociation for the peptides containing tyrosine, and occurs after internal conversion for those with tryptophan. The proposed mechanisms are governed by the ionization potential of the aromatic side chain compared to that of the rest of the molecule, and by the proton affinity of the aromatic side chain compared to that of the methionine side chain. In tyrosine-containing peptides, the presence of oxygen on sulfur of methionine presumably reduces the ionization potential of the peptide backbone, facilitating the loss of the side chain as a neutral fragment. In tryptophan-containing peptides, the presence of oxygen on methionyl-sulfur expedites the transfer of the proton from the side chain to the sulfoxide, which facilitates the loss of the neutral side chain.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

SINGLE-STRAND BREAKS INDUCED IN DNA BY VACUUM-ULTRAVIOLET RADIATION

Abstract— The efficiency of vacuum u.v. for producing single-strand breaks in DNA was determined for wavelengths between 58 and 254 nm (corresponding to photon energies of 21·2 and 4·9 eV, respectively) by using the supertwisted RF-DNA of bacteriophage φX174. The cross-section for production of single-strand breaks increases continuously by about 5 orders of magnitude between 5 and 10 eV photon energy, whereas from 11 to 21 eV the number of strand breaks produced per unit of incident radiation energy is approximately constant. Thus, absorption of a 10-eV photon causes DNA strand breaks with maximum efficiency. In addition, the number of electrons liberated from DNA by photons below 10 eV is one or two orders of magnitude higher than the frequency of strand breaks, demonstrating that in this energy range only a small fraction of the ionizations leads to strand breakage in DNA.     

THE SUBUNIT STRUCTURE OF YEAST DNA PHOTOLYASE AND THE PURIFICATION OF A FLUORESCENT ACTIVATOR OF THE ENZYME

Abstract— Yeast DNA photolyase purified twice by affinity chromatography was analyzed by electrophoresis on polyacrylamide gradient gels or by sedimentation velocity through 5–200/, sucrose gradients containing 0.4MKC1. Its molecular weight estimated by both these methods was 130 ,000 and 136 ,000, respectively. However, the enzyme dissociated into two bands having molecular weights of 60 ,000 and 85 ,000 when it was examined by electrophoresis on SDS polyacrylamide gradient gels. The subunit structure of the enzyme was confirmed when two absorption maxima corresponding to polypeptides of 54 ,000 and 82 ,500 daltons were observed in sucrose gradients run in 1.0 M KCI. Upon mixing these two fractions, a time-dependent increase in activity occurred, demonstrating that active enzyme could be reconstituted from these subunits.
The activity of photolyase purified by affinity chromatography is enhanced by a compound (activator III) obtained from yeast by acidification, neutralization, ion exchange chromatography and gel filtration. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that it contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9–11, while the other has a pK of 4–5. Enhancement of photolyase activity by activator III at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator and heat-denatured photolyase suggest that the activator may be the chromophore associated with the enzyme.     

Ring-opening reactions of diarylvinylidenecyclopropanes by iodine and bromine

Diarylvinylidenecyclopropanes undergo a novel ring-opening reaction upon treatment with iodine or bromine at 0-25 °C in 1,2-dichloroethane to give the corresponding iodinated or brominated naphthalene derivatives in good to high yields within 3 h. The further transformation of the corresponding iodinated or brominated naphthalene derivatives has been disclosed.     

Photodecomposition of polystyrene on long-wave ultraviolet irradiation: A possible mechanism of initiation of photooxidation

The long-wave (λ < 3000 Å) photo-oxidation of polystyrene in solution at 25°C has been studied osmometrically. Two types of chain scission have been observed: a purely photo process which occurs completely independently of oxygen and which is attributed to fission of photolabile groups in the polymer, and another process associated with random photolyses of the products of oxidation Scavenger experiments with ¹³¹I₂ have shown that approximately two iodine atoms are incorporated per chain scission when photolysis is carried out in solution (benzene, hexafluorobenzene, methylene chloride) under high vacuum conditions in the presence of ¹³¹I₂. No iodine incorporation or chain scission occurs when ionically prepared polystyrenes are treated similarly. The nature of the photolabile bond has been discussed, and there is some evidence for a peroxidic linkage arising from oxygen copolymerization in the chains. It is suggested that fission of the photolabile groups contributes to the initiation of the long-wave photooxidation of the polymer.     

Photochemical reactions of aromatic ketones with nucleic acids and their components. 3. Chain breakage and thymine dimerization in benzophenone photosensitized DNA

Abstract— Excitation of benzophenone in the presence of calf thymus and E. coli DNA leads to photosensitized damages to the macromolecule. Two main reactions are observed: thymine dimerization and chain break formation. Benzophenone photosensitized chain breaks are also observed in polyadenylic acid. The melting temperature of DNA decreases with the duration of irradiation. Under our experimental conditions, the ratio of the yields of dimers and single-chain breaks produced in DNA is about 1. Photosensitized damage to deoxyribose residues leading to chain breakage is shown to be similar to that produced by X or γ ray irradiation. The oxygen effect upon chain break production is studied and discussed in relation with its effect upon intermediate species. Thymine dimers are formed following energy transfer from benzophenone in its triplet state. In previous flash-photolysis studies we showed that benzophenone in its triplet state reacts with water molecules to give ketyl and OH radicals. Ketyl radicals are not involved in reactions with DNA. It is proposed that OH radicals produced in the above reaction are responsible for the production of single-chain breaks by attack on the deoxyribose residues.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

Determination of iodine in seaweed and table salt by an indirect atomic-absorption method

Decomposition methods based on fusion with alkali are discussed, with respect to the determination of iodine in biological material. It is shown that sodium hydroxide can be used for the decomposition of seaweed without loss of iodine. In spite of the oxidizing conditions, the iodine will be present as iodide in the final ash. The iodide can be determined by an indirect atomic-absorption method, based on the reaction between iodide and mercury(II), with determination of mercury by cold vapour atomic-absorption spectrometry. The basis of the method is discussed, and it is shown that the use of tin(II) as reductant is essential. The effect of the oxidation state of the iodine on the sensitivity of the method is pointed out. High concentrations of chloride interfere, but it is still possible to determine iodide in iodinated table salt.