SEQUENCE-SPECIFICITY OF THE ALKALI-SENSITIVE LESIONS INDUCED IN DNA BY HIGH-INTENSITY ULTRAVIOLET LASER RADIATION

The action of high-intensity (10(9)-10(12) W/m2) UV (266 nm) laser radiation pulses (duration ca 10 ns or ca 40 ps) on liquid aqueous solutions of DNA is known to cause not only single- but also two-quantum modification of nucleic bases. The action of hot piperidine on the laser-irradiated DNA results in non-random splitting of polynucleotide chain. Hence, at least some of the modified nucleoside residues are alkali-sensitive lesions (ASLs). The distribution of ASLs along the DNA chain shows that the position of these lesions corresponds with pyrimidines in the PyPy sequences (similar to those formed via single-quantum conversions) as well as with deoxyguanosine residues. The last ASLs result from two-quantum reactions and occur much more efficiently than the direct photo-induced cleavage of the internucleotide (phosphodiester) bond. It has been shown with fragments of plasmids pUC18, pUC19 and pBR322 (total length over 600 base pairs) that the relative efficiency of ASLs at deoxyguanosine sites depends on the primary structure context and can differ by an order of magnitude. The highest efficiency of modification is observed when a purine is 3' neighbour to the 2'-deoxyguanosine, i.e. at 5'-GPu-3' sites. However, considerable variations in the modification efficiency were also found in these sequences.     

ULTRAVIOLET INDUCED BINDING OF DAUNOMYCIN TO DNA

Up to 10–15% of daunomycin present in solutions of DNA-daunomycin remains irreversibly bound to DNA after 2 h of UV irradiation. Three times as much drug is irreversibly bound to DNA when oxygen is present, compared to deoxygenated solutions. Maximal amounts of the irreversibly bound drug are obtained between pH 5.5 and 7.5 and at an ionic strength of 0.76 M. Although the presence of daunomycin inhibits the extent of UV-induced degradation of the DNA, the DNA is still extensively degraded and denatured in the time required for optimal yields of the DNA-daunomycin photoproduct. This degradation of the DNA may impair its use as a lysosomotropic carrier of daunomycin.     

NETROPSIN: INTERACTION WITH ULTRAVIOLET IRRADIATED DNA

Abstract—Ultraviolet irradiation of double-stranded DNA reduces the circular dichroism (i < 300 nm) induced when the basic peptide antibiotic netropsin (Nt) is added to DNA subsequent to thc irradiation compared to the CD induced by the same concentrations of Nt added to unirradiated DNA. Nt is known to bind to A T base pairs in duplex DNA but will not bind to single-stranded DNA. The reduction in the maximum induced CD observed with saturating concentrations of Nt is a linear function of the concentration of pyrimidine dimers which. along with other dinucleotide photoproducts. form short disrupted regions in duplex DNA. The decrease in the CD of Nt bound to irradiated DNA could be due to elimination of potential Nt sites in the vicinity of a dimer. reduction in the average magnitude of the CD of Nt bound near a dimer or various combinations of these effects. In addition there is a reduction in the average binding constant for Nt bound to irradiated DNA compared to unirradiated DNA suggesting that formation of dinucleotide photoproducts either tends to preferentially eliminate the tighter binding sites or that tighter sites are converted to weaker ones. A simple model suggests that no more than one-third to one-half of the pyrimidine dimcrs formed in DNA completely eliminate a Nt site.     

EFFECT OF BASE SEQUENCE ON THE ULTRAVIOLET IRRADIATION PRODUCTS OF DOUBLE-STRANDED POLYNUCLEOTIDES CONTAINING BROMOURACIL AND ADENINE

Abstract —The effects of ultraviolet irradiation of double-stranded synthetic polynucleotides containing BrU and A have been investigated. Homopolymer pairs and alternating copolymers composed of either ribo- or deoxyribo-nucleotides were prepared and were irradiated with either 313 nm or ˜ 285 nm light. Strand separation and a modest amount of strand breakage followed irradiation of the homopolymer pairs. Changes in the ultraviolet absorption spectra of the polymers during irradiation reflected the sum of hyperchromic increases caused by progressive strand separation and loss of absorbance caused by photoproduct formation. Extensive debromination occurred. An RNase digest of irradiated poly(rA)–poly¹⁴C(rBrU), analysed by column chromatography, showed components similar to those found previously upon irradiation of single-stranded poly(rBrU). Little photoproduct was released by RNase digestion as mononucleotides. The major photoproduct was in the dinucleotide fraction, and may be 5,5'-diuracil. Base sequence had a profound effect on the sensitivity of the polynucleotides. Irradiation of alternating copolymers with doses of light comparable to those that produced major photochemical changes in the homopolymer pairs brought about little if any change in the copolymers of alternating base sequence.     

QUANTITATIVE EVALUATION OF ULTRAVIOLET INDUCED ERYTHEMA

Abstract Noninvasive determinations of the amount of cutaneous blood in the superficial plexus are made from skin reflectance measurements after exposure of human skin to UV radiation. The approach considers the skin as consisting of multiple optically distinct layers, and utilizes a previously experimentally verified optical model that relates the optical properties of these skin layers to skin reflectance. When the UV erythema is concurrently graded by visual inspection, our results show that each subjective grade corresponds to a broad range of values of cutaneous blood, and a wide overlap in blood volumes occurs between different grades. The amount of cutaneous blood increases with the UV dose, and follows a sigmoid threshold relationship. Erythema fades with time, but the rate of decrease in cutaneous blood is volume dependent. The sharpest decrease occurs when the amount of cutaneous blood is within 1.5 - 2.5 times the pre-radiation volume. Slower decrease occurs when the volume of blood is outside of this range.     

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.     

ULTRAVIOLET RADIATION INDUCED CHANGES IN MACROPHAGE ACTIVITY

Abstract— The effect of ultraviolet (UV) radiation on macrophage activity was examined. Thioglycollate-stimulated peritoneal exudate cells were collected from adult C57BL/6 mice. Ninety-five per cent of the cells adhering to plastic petri dishes were macrophages as determined by the presence of a non-specific esterase. Adherent cells were exposed to UV radiation of 0.5-13.2 J/m². Viability and phagocytosis were measured at 0, 24, 48, 72 and 96 h after exposure. A statistically significant UV exposure-dependent decrease in macrophage viability and phagocytic capacity was observed. Macrophage viability and phagocytosis also decreased as a function of time after exposure to UV radiation.     

ANGIOTENSIN ALTERATIONS INDUCED BY ULTRAVIOLET RADIATION*

Abstract. Angiotensin is readily inactivated by ultraviolet light presumably due to the photosensitivity of its tyrosyl and phenylalanyl residues. Alkalinity promotes and acidity delays the biological decomposition. Under conditions of oxygen exclusion (i.e. under N₂ or H₂) the rate of decomposition is further depressed and when irradiation is carried out under hydrogen a biologically active product is formed. This altered angiotensin, judging from its chromatographic migration and dialysis rate, represents a polymerized form of angiotensin. Under identical irradiation conditions, angiotensin was coupled with C¹⁴ labelled pheny-lalanine which indicates that this residue might be the photoreactive species. The production of altered forms of angiotensin by ultraviolet irradiation was discussed with respect to possible utility in anti-metabolite, antigen or tracer work.     

SINGLE-STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract— The induction of single-strand breaks (alkali-labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far-UV (below 320 nm) component and a minor near-UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near-UV (above 320 nm) wavelengths faster than the wild-type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali-labile bonds induced by 365-nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali-labile bonds, probably apurinic and apyrimidinic sites.     

ULTRAVIOLET RADIATION - INDUCED MALIGNANT MELANOMA IN Monodelphis domestica

Several lines of evidence support the hypothesis that ultraviolet radiation (UVR) is involved in the etiology of cutaneous melanoma in humans. However, progress in understanding the mechanisms involved in induction of melanotic tumors by UVR has been hindered by lack of a suitable animal model. During the course of multiple exposures (3 times/wk for 70 wk) of the South American opossum, Monodelphis domestica, to UVR, we first observed the appearance of areas of dermal melanocytic hyperplasia (MH) on the exposed skin. Post-UVR exposure to photoreactivating light (320-500 nm) suppressed the occurrence of MH. We also observed at 100 weeks from first exposure that 10 of 46 surviving animals had developed melanotic tumors which arose, presumably, from areas of MH. Tumors on three of the 10 animals have been classified as malignant melanomas based on metastasis to lymph nodes. We conclude from these results that UVR can act as a complete carcinogen for melanoma induction and, based on the photoreactivation of MH induction, that DNA damage is involved in melanoma formation.     

POSSIBLE LONG-TERM CHANGES IN BIOLOGICALLY ACTIVE ULTRAVIOLET RADIATION REACHING THE GROUND

Abstract— Three scenarios for long-term changes in atmospheric ozone over the time period 1960 to 2030 lead to different projections for the ultraviolet radiation flux at the earth's surface. Biologically effective fluxes for damage to DNA and generalized damage to plants vary by a factor of 10 or more with latitude and season irrespective of possible changes in ozone. The natural latitudinal gradient in radiation corresponds to spatial changes in biologically effective fluxes which are large compared to temporal changes expected from trends in ozone over the time period analyzed. In an extreme scenario of ozone change, based on an assumed increase in chlorofluorocarbon release rates of 3% per year after 1980, the annually integrated effective flux for damage to DNA increases by 13.5% at latitude 40°N between 1960 and 2030. With chlorofluorocarbon release rates held fixed at their 1980 values, the corresponding radiation increase is only 2.3%. In a scenario where atmospheric chlorine remains fixed at its 1960 value, trends in atmospheric methane and nitrous oxide imply a decrease in biologically effective flux at 40°N of 5.3% between 1960 and 2030.     

MONOADDITION OF DICTAMNINE TO SYNTHETIC DOUBLE-STRANDED POLYDEOXYRIBONUCLEOTIDES IN UVA AND THE EFFECT OF PHOTOMODIFIED DNA ON TEMPLATE ACTIVITY

Abstract— The photoreactivity of dictamnine, a furoquinoline alkaloid, towards different synthetic DNAs has been studied. The ratio of the photobinding of [₃H]-dictamnine to poly(dA-dT) poly(dA-dT): poly(dG-dC) poly(dG-dC): poly(dA-dU) poly(dA-dU): poly(dA) poly(dT), in relation to that of calf thymus DNA, is 18:1:0.5:0.3. Prior treatment of calf thymus DNA with dictamnine in light inhibits the subsequent incorporation of 8-methoxypsoralen (8-MOP). These results suggest that the sites in DNA for the photobinding of dictamnine are probably identical with those for monoad-ducts of 8-MOP. Furthermore, the template activity of photomodified DNA in the RNA polymerase reaction is considerably inhibited for poly(dA-dT)poly(dA-dT), to a lesser extent for calf thymus DNA, but almost not affected for the linear copolymer, poly(dA)-poly(dT).     

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS-II. PHOSPHORESCENCE AND PHOTODIMERIZATION OF DNA COMPLEXED WITH Ag*

Abstract— We have studied the luminescence (at 77°K) and photochemistry (at 298°K) of a variety of polynucleotides complexed with Ag⁺. In all cases we observed that Ag⁺ induces a 'heavy atom effect' as indicated by quenching of fluorescence, enhancement of phosphorescence, and reduction of triplet lifetimes. The enhancement by Ag⁺ of the phosphorescence intensity of poly (dT) and DNA paralleled a 20-fold enhancement of the rate of thymine dimerization. These results suggest a possible triplet precursor for thymine dimerization in Ag⁺ complexes. Dimers were also made with a variety of triplet sensitizers, and Ag⁺ increased the dimer yield two- to four-fold, depending on the sensitizer used.     

AROMATIC AMINO ACIDS AND ULTRAVIOLET INDUCED PHOTO-ELECTRIC EFFECTS IN BILAYER MEMBRANES

Abstract— Ultraviolet light flashes induced voltage transients across bilayer lipid membranes when aromatic amino acids were adsorbed to one side of the membrane. These photo-effects varied with the chromophore structure, the aqueous solution salt concentration, pH and oxygen partial pressure. These photo-effects are attributed to the migration of electrically charged photochemical intermediates in the membrane, and provide a new method for studying the effects of UV light on membranes.     

NEAR ULTRAVIOLET AND POSTIRRADIATION DNA DEGRADATION: EFFECTS ON THE INDUCIBLE INHIBITOR OF IONIZING RADIATION-INDUCED DNA DEGRADATION

Abstract— Possible effects of near ultraviolet radiation (near UV) on DNA degradation were examined. Postirradiation DNA degradation induced by ionizing radiation in strain B_s-l ( uur-, lex- ) is shown to be inhibited by carbon monoxide (CO) and potassium cyanide (KCN) if the cells are grown on glycerol. Presumably the blockage of respiration by these agents lowers the amount of ATP in the cell. 50 kJ/m² near UV did not simulate the action of CO and KCN, indicating that at this Ruence the supply of ATP remains adequate for postirradiation DNA degradation. Near UV did not, itself, produce DNA degradation. In a strain (B/r) in which an inhibitor of postirradiation DNA degradation can be induced by both UV and ionizing radiation, near UV affects the inhibitor formation, whether administered before or after induction.     

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS—III. INFLUENCE OF Ag + AND Hg 2+ ON THE SENSITIVITY OF PHAGE AND OF TRANSFORMING DNA TO ULTRAVIOLET RADIATION

Abstract— We have studied the influence of the heavy metal ions Ag⁺ and Hg²⁺ on the photoinactivation and photodimerization of transforming DNA and of bacteriophage. The rate of inactivation of Haemophilus influenzae transforming DNA by ultraviolet (UV) radiation was enhanced by a factor of 30 when it was complexed with Ag⁺. This enhancement was correlated with a comparable increase in the rate of thymine dimerization. In contrast, mercuric ions led to a reduction in the rates of both inactivation and dimerization. When we examined the effects of these metal ions on the photobiology of bacteriophage, we again found that Ag⁺ enhanced and Hg²⁺ reduced the rate of ultraviolet inactivation. These results demonstrate that heavy metals may be useful tools for studying the photochemistry and photobiology of nucleic acids.     

REPAIR OF 313-NM INDUCED LESIONS AND PHOTOPROTECTION IN YEAST CANDIDA GUILLIERMONDII

Abstract. The present communication is concerned with the effects of near-UV radiation (300–380 nm) on yeast Candida guilliermondii. It was found that certain doses of 313 nm irradiation caused inactivation of the yeast which was exhibited in a way different from the lethal action of far-UV radiation. It was also found that the cells inactivated by 313 nm are capable of recovering vitality, if incubated for some time in a non-nutrient medium. The yeast inactivated by far-UV radiation also proved to be capable of recovering, though to a lesser degree. Both 334 nm radiation and non-lethal doses at 313 nm induced the photoprotective effect against far-UV damage. The effect was exhibited if there was a certain time interval (2–4 h) between the exposures to photoprotective light and subsequent far-UV radiation. Within this time interval the extent of photoprotection was dependent on temperature.