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 WAVELENGTH DEPENDENCE OF HERPES SIMPLEX VIRUS INACTIVATION BY ULTRAVIOLET RADIATION

The effect of different wavelengths of ultraviolet (UV) radiation on Herpes simplex virus when assayed on mammalian cells (measured by plaque forming ability) was investigated. The wavelength dependence of viral inactivation was obtained for 11 different wavelengths over the region 238–297 nm. The resulting action spectrum does not closely follow the absorption spectrum of either nucleic acid or protein. The most effective wavelengths for viral inactivation are over the region 260–280 nm.     

SINGLE-STRAND BREAKS IN SUPERCOILED DNA INDUCED BY VACUUM-UV RADIATION IN AQUEOUS SOLUTION

We studied the induction of single-strand breaks in the DNA of plasmid pBR 322 by vacuum-UV radiation above 145 nm in aqueous solutions in relation to the production of OH-radicals in water. The similarity and dissimilarity were examined of the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH-radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single-strand breaks.     

WAVELENGTH DEPENDENCE FOR THE INACTIVATION OF ATP IN THE VACUUM-ULTRAVIOLET REGION ABOVE 140 nm

Radiation effects were investigated on the activity and the structure of adenosine triphosphate in the wavelength range from 140 nm to 260 nm, using monochromatized synchrotron radiation from the INS-SOR storage ring. The sample was irradiated as a thin film in vacuum. The activity of adenosine triphosphate decreased sharply below 180 nm as judged by the luminescence in the luciferin-luciferase assay. From the exponential decay of function, the cross-section for inactivation was calculated to be of the order of 10^-21 m²/photon in the range from 140 to 170 nm. No decrease was detected at wavelengths of 190 nm and above. The calculated quantum yield increased as the wavelength became shorter and reached to 0.20 at 150 nm. The release of adenine at 160 nm-irradiation was detected by thin layer chromatography; no adenosine diphosphate or adenosine monophosphate occurred. Only a trace of adenine was found after 190 nm-irradiation. These results indicate that the broad absorption peak for higher excitations attributable to the base moiety around 190 nm does not cause both structural and functional changes, while the absorption by the sugar-phosphate group produces the rupture of N -glycosidic bond, and probably leads to the loss of function.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

WAVELENGTH DEPENDENCE OF DNA INCISION BY A HUMAN ULTRAVIOLET ENDONUCLEASE

The wavelength dependence of an ultraviolet irradiation of the DNA substrate for a human endonuclease was determined. Sites of DNA incision for all UVB and UVC wavelengths examined were at cytosines which were neither cyclobutane pyrimidine dimers nor 6-4'-(pyrimidin-2-one)pyrimidines. The optimal wavelengths for formation of these cytosine photoproducts were between 270 and 295 nm. This human endonuclease therefore has a similar ultraviolet substrate specificity to endonuclease III.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

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.     

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6-4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.     

PHOTOSENSITIZATION OF SINGLE-STRAND BREAKS IN pBR322 DNA BY ROSE BENGAL

Rose bengal photosensitized the formation of frank single-strand breaks (SSBs) in double-stranded, supercoiled pBR322 DNA as measured by neutral agarose electrophoresis. The yield of SSBs followed first order kinetics with respect to light fluence and dye concentration. The efficiency of cleavage was more than 20 times greater in an argon atmosphere than in an oxygen atmosphere. The quantum yield in an air atmosphere was 1.7 (+/- 0.3) X 10(-8). Sodium azide quenched the cleavage more efficiently in an oxygen atmosphere than when the oxygen concentration was reduced. Isopropanol and mannitol were poor quenchers; ribose-5-phosphate and guanosine-5'-monophosphate did not quench the cleavage. Substituting D2O for H2O increased the yield of SSBs in both oxygen and oxygen-depleted atmospheres. The results are consistent with initiation of cleavage by reaction of the triplet state of rose bengal (or a radical derived from it) with DNA. In the presence of oxygen, an additional mechanism is introduced.     

INHIBITION OF DNA REPAIR AND PRODUCTION OF SINGLE-STRAND BREAKS IN ESCHERICHIA COLI BY REDUCTONE

Abstract— Reductone (HOCH₂COCHO), a keto-aldehyde produced by thermal degradation of some sugars, at alkaline pHs, blocks the excision repair of DNA lesions in uv-irradiated wild type Escherichia coli. This probably occurs as a result of inhibition of the exonucleolytic activity of DNA polymerase I. In addition, reductone alone induces DNA single-strand breaks. Repair of this damage is mainly dependent on the polA gene products.     

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.     

THE DOSE-RESPONSE RELATIONSHIP OF TUMORIGENESIS BY ULTRAVIOLET RADIATION OF 254 nm

Abstract— Groups of albino hairless mice, Skh-hrl, were exposed daily to UVC radiation from low pressure Hg arcs (Philips TUV 40W). These lamps emit predominantly radiation of 254 nm. Three groups of animals were used in the experiments, each receiving a different daily dose.
The results were described with the Weibull probability function. As in earlier studies with UVB. the tumor induction time was proportional to a power of the daily dose. The exponent turned out to be as low as -0.2. This implies that the induction time varied only a little with the daily dose. The average number of tumors per animal was proportional to a power of time. A sample of 73 tumors of at least 4 mm in diameter were investigated histologically. The large majority were classified as squamous cell carcinomas.
A comparison was made with the results of an earlier reported experiment with Westinghouse FS40 sunlamps. Throughout the whole range of daily doses used in the present experiment, UVC was less carcinogenic than UVB. An intriguing difference between the two types of radiation was that the tumors induced by UVC appeared much more scattered over the irradiated parts of the animals than the UVB-induced tumors.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The effect of UV radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV-irradiated mammalian virus was tested. An action spectrum for radiation enhanced reactivation (RER) is presented. The shape of the action spectrum points to a combined nucleic acid-protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in RER does not allow us to distinguish which macromolecule is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.     

WAVELENGTH DEPENDENCE AND KINETICS OF UV-INDUCED FREE RADICAL FORMATION IN THE HUMAN CORNEA AND LENS

Abstract— The wavelength dependence of free radical formation in the human lens and cornea was obtained in the ultraviolet-visible (UV-VIS) range with electron paramagnetic resonance. The spectral feature of having their maxima close to 290 nm, negligible intensity above 320 nm, and appreciable intensity toward 260 nm is common to both of these tissues.
The kinetics of free radical formation in the lens and cornea was interpreted in terms of a two component exponential function with characteristic rate constants k _l and k ₂, the latter of which has the same value for both the lens and cornea.     

WAVELENGTH DEPENDENCE (150–290 nm) OF THE FORMATION OF THE CYCLOBUTANE DIMER AND THE (6–4) PHOTOPRODUCT OF THYMINE

The action cross sections for the formation of the cyclobutane dimer and the (6-4) photoproduct of thymine as well as the absorption cross sections of thymine were determined in the wavelength region between 150 and 290 nm. Thymine films sublimed on glass plates were irradiated by monochromatic photons in a vacuum; the induced photoproducts were quantitatively analyzed by high-performance liquid chromatography (HPLC). Under our conditions, two major peaks appeared on the HPLC chromatograms of irradiated samples. The two peaks were identified as being the cis-syn cyclobutane dimer and the (6-4) photoproduct, based on their HPLC retention times, absorption spectra in the effluent, and photochemical reactivity. The fractions of the two photoproducts increased linearly with the fluence at low fluences over the entire wavelength range. Their action cross sections were determined by the slopes of the linear fluence response curve at 10 nm intervals between 150 and 290 nm. The two action spectra showed a similar wavelength dependence and had a maximum at 270 nm as well as two minor peaks at 180 and 220 nm, at which wavelengths the peaks of the absorption spectrum of thymine sublimed on a CaF2 crystal plate appeared. The quantum yields had relatively constant values of around 0.008 for the dimer and 0.013 for the (6-4) photoproduct above 200 nm, decreasing to 0.003 and 0.006, respectively, at 150 nm as the wavelength became shorter.     

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.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract. The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.     

SINGLE-STRAND DNA BREAKS INDUCED BY 365 NM RADIATION IN ESCHERICHIA COLI STRAINS DIFFERING IN SENSITIVITY TO NEAR and FAR UV

The gene mutation nur has been shown specifically to sensitize Escherichia coli stationary phase cells to inactivation by broad spectrum near-UV (NUV) radiation. In the work reported here, E. coli strains RT1. RT2, RT3, and RT4, carrying the 4 possible combinations of recA1, recA⁺, nur , and nur⁺ , were exposed to monochromatic NUV (365 nm). The strains carrying the nur allele (RT1 and RT2) were more sensitive to inactivation by this wavelength and exhibited considerably more single strand break's (SSB's) than the strains carrying the nur⁺ allele (RT3 and RT4). As predicted, following X-irradiation the strains carrying the recA1 allele (RT1 and RT3) were more sensitive than the recA⁺ strains (RT2 and RT4). We conclude that the enhanced SSB's observed in strains RT1 and RT2 following monochromatic NUV irradiation correlated with the nur mutation and are unrelated to the recA1 mutation.