VISUALIZATION OF ULTRAVIOLET LIGHT-INDUCED THYMINE DIMERS IN DNA BY IMMUNOELECTRON MICROSCOPY

Abstract— To see the damage of DNA due to ultravoilet-B more distinctly, immunoelectron microscopic studies using a monoclonal antibody against cyclobutane-type thymine dimers were performed. As a result, we could detect the existence of thymine dimers on human genomic DNA and _pUC18 plasmid DNA visually. This technique can be useful to locate the photoproducts formed on DNA.     

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.     

REPAIR OF PSORALEN PLUS NEAR ULTRAVIOLET LIGHT DAMAGE IN BACTERIOPHAGE T4

Abstract— The sensitivity of bacteriophage T4 to psoralen plus NUV was investigated using a series of T4 repair defective mutants. The recombinational repair deficient mutants T4x, T4y and T4w were more sensitive than wild-type; while T4v, an endonuclease V mutant, exhibited the same sensitivity as wild-type. However, endonuclease V appears to initiate abortive repair in the absence of the x and presumably y gene products. Host repair gene products were shown not to be involved in the repair of psoralen plus NUV damages in T4.     

REPAIR OF PSORALEN PLUS NEAR ULTRAVIOLET LIGHT DAMAGE IN BACTERIOPHAGES T3 AND T4

Abstract— Repair of T3 and T4 DNA damaged by treatment with 8-methoxypsoralen plus near UV (PNUV) has been investigated. It is shown that the excision repair mechanisms of the host cell can repair a substantial fraction of the psoralen-DNA mono-adducts in T3 DNA, but cannot by themselves repair crosslinks. In contrast neither the excision repair system of the host nor the phage coded v gene endonuclease is involved in the repair of psoralen adducts in T4 DNA. Multiplicity reactivation is effective in the recovery of T4 DNA containing psoralen-DNA mono-adducts, but is ineffective in the recovery of crosslinked phages. Comparisons of the lethality of PNUV treatment and the number of crosslinks induced in T4 DNA show clearly that mono-adducts are lethal to this phage. Both T3 and T4, however, appear to effectively repair many mono-adducts by postreplicational repair.     

INDUCTION OF UMUC+ GENE EXPRESSION IN Escherichia coli IRRADIATED BY NEAR ULTRAVIOLET LIGHT

Abstract— The induction of umu + gene expression caused by irradiation with near ultraviolet light (BLB; black light blue) was studied in Escherichia coli K-12 strains with special reference to the effects of SOS repair deficiencies. The umuC + gene expression was measured as the enzymic activity of (J-galactosidase which is regulated by the promoter of the umuC + operon carried in a plasmid DNA carrying a promoter of umuC* operon, a umuD + gene and a umuC +- lacZ + gene fusion. A high induction of the umuC + gene expression was observed in the uvrA cells in the case of BLB or UV irradiation as compared with the parental wild-type cells. Caffeine inhibited the induction of the umuC* gene expression due to BLB or UV irradiation in both strains. There was very little induction in lexA and recA mutants. In contrast with UV irradiation, there was no killing of cells by BLB irradiation in any strain (wild, uvrA, lexA and recA). Possible implications of the present experimental results were discussed.     

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-DEPENDENT FRACTION OF BIOLOGICAL DAMAGE DUE TO THYMINE DIMERS AND TO OTHER TYPES OF LESION IN ULTRAVIOLET-IRRADIATED DNA

Abstract— The sensitivity of Hemophilus influenzae transforming DNA to monochromatic ultraviolet radiation has been determined with and without maximum photoreactivation. The fraction of ultraviolet damage which is photoreactivable (the photoreactivable sector) is large and varies with the wavelengths of the inactivating radiation, decreasing at the extremes of the ultraviolet spectrum. Equating photoreactivable damage with thymine dimer damage, we may interpret the wavelength dependence of photoreactivability and the spectrum for non-photo-reactivable damage in terms of the absorption spectra of thymidine and cytosine deoxyriboside. The data suggest that cytosine deoxyriboside alteration is important in non-photoreactivable biological damage.     

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.     

DNA REPAIR IN THE VARIABLE PLATYFISH (Xiphophorus variatus) IRRADIATED in vivo WITH ULTRAVIOLET B LIGHT

Abstract— Dark- and light-dependent DNA repair processes were studied in vivo in the variable platyfish, Xiphophorus vuriatus . Excision (dark) repair of the (6–4) photoproduct was more efficient than that of the cyclobutane dimer with ∼ 70% of the (6–4) photoproducts reniovcd by 24 h post-UVB radiation compared to ∼30% of the cyclobutane dimers. Exposure to photoreactivating light resulted in rapid loss of most (>90%) of the cyclobutane dimers and increased excision repair of the (6–4) photoproduct. Preexposure to photoreactivating light 8 h prior to UVB radiation increased the rate of photoreactivation two-fold.     

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.     

RELATIVE INDUCTION OF CYCLOBUTANE DIMERS and CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro and in vivo WITH ULTRAVIOLET-C and ULTRAVIOLET-B LIGHT

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.     

DNA DAMAGE and ITS REPAIR IN Dictyostelium discoideum IRRADIATED BY HEALTH LAMP LIGHT (UV- B)

Irradiation by health lamp (HL) light (280–320 nm) more efficiently induced cell killing and mutation in a radiation sensitive mutant (TW8) of Dictyostelium discoideum as compared with the parental wild-type strain (NC4). This light as well as a germicidal lamp-light (254 nm) produced pyrimidine dimers. The dimers were removed from DNA molecules by excision repair in NC4, but more slowly in TW8. It is suggested that pyrimidine dimers are the main DNA damage caused by HL light in D. discoideum , and that this results in cell killing and induced mutation.     

INDUCED REPAIR IN ESCHERICHIA COLI: INDUCTION BY ULTRAVIOLET LIGHT AT-79°C

Two of the phenomena associated with induced (S.O.S.) repair, namely induced inhibition of post radiation DNA degradation and induced radioresistance have been shown to be elicited by 245 nm radiation applied to E. coli cells in the frozen state at -79°C. The effect of radiation in this condition is considerably less photoreactivable than similar effects produced by exposure in the wet state. Since protein-DNA crosslinks are believed to be formed under these conditions, such consequences of UV radiation appear to be a potent inducer of induced repair.     

EVIDENCE FOR DARK REPAIR OF FAR ULTRAVIOLET LIGHT DAMAGE IN THE BLUE-GREEN ALGA, GLOEOCAPSA ALPICOLA

Abstract— The inactivating effect of far UV light on the unicellular blue-green alga Gloeocapsa alpicola could be totally reversed by exposure to blue light immediately after irradiation. However, if the irradiated cells were held in the dark before exposure to blue light, reversal became progressively less efficient and almost disappeared after 60–80 h holding. Caffeine and acriflavine inhibited loss of photoreversibility, suggesting an involvement of excision functions. Chloramphenicol and rifampicin slightly increased the rate of loss of photoreversibility, indicating that inducible functions play only a minor role. Split UV dose experiments indicated that light-dependent repair remained operational during dark liquid holding. These results provide preliminary evidence for dark repair in G. alpicola .     

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.     

OCCURRENCE OF PYRIMIDINE-RICH TRACTS IN ASCITES TUMOR DNA AND THE FORMATION OF UV-INDUCED THYMINE DIMERS

Abstract— Analysis of the distribution of pyrimidine-rich tracts (up to decanucleotides) in ascites tumor DNA revealed that these tracts occur predominantly in repetitive sequence of DNA. UV irradiation of ascites DNA resulted in preferential formation of thymine dimers in the pyrimidine-rich tracts as compared to other regions of DNA.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

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.     

A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.