FORMATION OF NON-VIABLE SPORES OF DICTYOSTELIUM DISCOIDEUM BY UV-IRRADIATION AND CAFFEINE

Abstract We have characterized the spores formed from amoeboid cells of the wild type strain of Dictyostelium discoideum after UV-irradiation. Cell differentiation in the presence of caffeine after a fluence of 300 J/m² resulted in a population of spores which was 98% non-viable. The UV-irradiation did not affect the conversion of the spores to swollen spores but did affect the conversion of swollen spores to amoeboid cells. When the germination of the spores was done without caffeine, we detected only a small effect on conversion of swollen spores to amoeboid cells and on the beginning of growth. On the other hand, in the presence of caffeine, the spores had a remarkable delay in both. It was also shown that few, if any, pyrimidine dimers exist in the DNA of the non-viable spores. Possible mechanisms of formation of non-viable spores are discussed.     

ESTIMATION OF PHOTOPRODUCT FORMATION IN GERMINATING SPORES OF BACILLUS SUBTILIS

Abstract— Changes in UV sensitivity during spore germination of Bacillus subtilis mutants possessing various defects in DNA repair capacities were analysed in order to estimate the yield of the DNA photoproducts at the transient, UV resistant stage which occurs in the process of germination. It was concluded that the yield of the spore-specific photoproduct (5-thyminyl-5,6-dihydrothymine, TDHT) at the transient stage was only about 3% of that in dormant spores and the yield of the cyclobutane-type pyrimidine dimers at this stage was about 10% (or less) of that in germinated spores.     

CHARACTERIZATION OF ANTIBODIES SPECIFIC FOR UV-DAMAGED DNA BY ELISA

Abstract— The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was abrogated. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with O_sO₄. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.     

VIABILITY OF THE SPORES FORMED AFTER UV IRRADIATION IN DICTYOSTELIUM DISCOIDEUM

Both abilities of germination of spores formed after UV irradiation and of growth of amoeboid cells emerged from the spores were studied on two kinds of Dictyostelium discoideum strains, NC-4 and ys-13.
An inhibition of germination was observed on the spores of ys -13 when formed after UV irradiation, while no inhibition was detected on the ability of germination of spores of NC-4. The amoeboid cells of ys -13 emerged from the spores showed a heavy delay of growth, although no delay of growth was detected even on the amoeboid cells of NC-4 emerged from the spores formed after UV irradiation. The strain of NC-4 must repair UV lesions fully before spore formation, while the spores of ys-13 must keep some UV lesions unrepaired and send them to the next generation of amoeboid cells. The characters of UV lesion inheritable through the spores to the next amoeboid cells in ys-13 were discussed.     

EFFECTS OF CAFFEINE ON DNA REPAIR OF UV-IRRADIATED DICTYOSTELIUM DISCOIDEUM

Abstract— Caffeine enhances the UV-killing of amoeboid cells of NC-4, but UV-irradiated γs-13 is insensitive to caffeine. UV-irradiated NC-4 becomes insensitive to the effect of caffeine during a postirradiation incubation in buffer for about 90 min, but γs-13 remains unchanged in the sensitivity to caffeine throughout the incubation for 180 min. Amoeboid cells of γs-13 can remove pyrimidine dimers as well as NC-4 even in the presence of caffeine. Caffeine inhibits rejoining of strand-breaks of DNA in UV-irradiated NC-4, but the rejoining in γs-13 is insensitive to caffeine.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

PHOTOREVERSIBLE UV-INACTIVATION OF MESSENGER RNA IN AN INSECT EMBRYO (SMITTIA SPEC., CHIRONOMIDAE, DIPTERA)

Smittia embryos were UV-irradiated during intravitelline cleavage. At this stage, nuclei are heavily shielded by yolk-rich cytoplasm, and do not synthesize detectable amounts of RNA. Irradiation at 265, 285 and 295 nm wavelength caused biological inactivation, and pyrimidine dimer formation in maternal RNA as described earlier (Kalthoff, 1976; Jäckie and Kalthoff, 1978). In addition, we observed marked effects on protein synthesis: (1) The overall rate of [³⁵S]-methionine incorporation in vivo was reduced to less than half of the normal rate. (2) Two-dimensional gel electrophoresis revealed quantitative variations in the synthetic rate of some polypeptides, and the appearance of new ones in UV-irradiated embryos. (3) Translation of polyadenylated RNA from Smittia embryos in a cell-free system was inhibited by UV irradiation in vivo. (4) The apparent degradation, during early embryogenesis, of maternal polyadenylated RNA was retarded in UV-irradiated embryos. Exposure to light (400 nm) after UV caused partial photoreversal of all UV effects observed. Both the photoreactivable sector of UV-inactivation, and the photoreactivated portion of UV inhibition of protein synthesis, were correlated with the amounts of pyrimidine dimers generated in maternal RNA by UV irradiation at the three wavelengths used. These correlated effects were produced most efficiently by 295 nm radiation, indicating the involvement of photosensitizing components in the embryos. Our data show, for the first time to our knowledge, that animal mRNA, after UV irradiation, can be photoreactivated in vivo. Moreover, our results strongly suggest that the photorepairable lesions consist of pyrimidine dimers generated in a photosensitized reaction.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor α (TNFα) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V in-creased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNFα expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNFa gene expression in mouse and human cells.     

Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial, Monodelphis domestica

Radioimmunoassays were used to investigate the repair of cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone photoproducts ((6-4] photoproducts) in the epidermis of the South American opossum, Monodelphis domestica. In the absence of photoreactivating light, both types of photodamage were excised with similar kinetics, 50% of the damage remaining 8 h after UV irradiation in vivo. Exposure of UV-irradiated skin to photoreactivating light resulted in removal of most of the cyclobutane dimers and an enhanced rate of (6-4) photoproduct repair. Photoenhanced excision repair of non-dimer damage increases the range of biologically effective lesions removed by in vivo photoreactivation.     

THE RATE OF REMOVAL OF SUNLIGHT-INDUCED PYRIMIDINE DIMERS FROM THE DNA OF CULTURED HUMAN DIPLOID FIBROBLASTS

Abstract The rate of excision of sunlight-induced pyrimidine dimers in DNA of exposed human cells was determined. Two normal excision repair-proficient human diploid fibroblast strains (WS-1 and KD) and a repair-deficient strain (XP12BE, group A) maintained in a nondividing state were exposed to summer noon-time sunlight for times (5 and 20 min) that induced numbers of dimers equivalent to far UV (254 nm) exposures of 1 and 4 J/m². Pyrimidine dimers were quantified in extracted DNA using a U V-endonuclease-alkaline sedimentation assay. The excision rates of these dimers were similar to those observed for the excision of UV-induced pyrimidine dimers. No sunlight-induced inhibition or stimulation of DNA repair was observed in either strain at these low exposures.     

Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro-tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA pho-toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro-sophila, Xenopus and Crotalus.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) I. Photosensitized production and light-dependent disappearance of pyrimidine dimers

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength-dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV-irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono-merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light-dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

IN VIVO EXCISION OF PYRIMIDINE DIMERS IS MEDIATED BY A DNA N-GLYCOSYLASE IN MICROCOCCUS LUTEUS BUT NOT IN HUMAN FIBROBLASTS

Abstract It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro , functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo , we have isolated and examined the excision products of UV-irradiated M. luteus . In addition, we have devised a procedure to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. We find that, in vivo , an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus , but that human fibroblasts do not appear to use this mechanism.     

FORMATION OF 5-HYDROXYMETHYLCYTOSINE-CONTAINING PYRIMIDINE DIMERS IN UV-IRRADIATED BACTERIOPHAGE T4 DNA

Cyclobutyl pyrimidine dimers composed of 5-hydroxymethylcytosine and thymine (5HMC>T dimer for a mutant of T4 ( denV ) that is unable to excise pyrimidine dimers from its DNA. The ability of 5HMC to form dimers suggests that other modified pyrimidines such as 5-methylcytosine can participate in dimer formation, particularly at the UV wavelengths in sunlight likely to be responsible for the induction of skin cancer.     

TRANSITORY UV RESISTANCE DURING GERMINATION OF UV-SENSITIVE SPORES PRODUCED BY A MUTANT OF Bacillus cereus 569

Abstract— A mutant of Bacillus cereus 569, isolated by us and designated 2422 is unable to excise cyclobutane-type dimers and spore-specific photoproducts from the DNA of UV-irradiated vegetative cells and dormant spores. The deficiency in the excision repair mechanism was found to be at the post-incision step in the exonuclease-mediated removal of the photoproducts. During germination, the mutant B. cereus 2422 exhibits UV-resistance and an efficient photoproduct removal which is followed by DNA repair synthesis. The data presented indicate the existence of germinative excision repair in B. cereus 569.     

SENSITIVITIES TO MONOCHROMATIC 254-nm AND 365-nm RADIATION OF CLOSELY RELATED STRAINS OF Saccharomyces cerevisiae WITH DIFFERING REPAIR CAPABILITIES

Abstract— Sensitivity to monochromatic 254- and 365-nm radiation was compared in closely related yeast strains with defects in one or more of the excision-repair ( rad1 ), error-prone repair ( rad18 ), or recombinational-repair ( rad51 ) pathways. At 254 nm, mutants defective in a single repair pathway exhibited slight to moderate UV sensitivity; those defective in two separate pathways were somewhat more UV sensitive, while triple mutants defective in all three pathways exhibited extreme UV sensitivity with a lethal event corresponding to 0.05 J m⁻². Repair defects also rendered mutants sensitive to 365-nm radiation; strains with single defects exhibited slight sensitivity, mutants with two defective pathways were more sensitive, and triple mutants exhibited maximal sensitivity with a lethal event corresponding to 2.4 times 10⁴ J m⁻². In the triple mutant ( rad1, rad18, rad51 ) at both 254 and 365 nm, the dose per lethal event was almost identical with comparable values in a repair-deficient double mutant ( uvrA, recA ) of Escherichia coli. In the E. coli mutant pyrimidine dimers are believed to be the primary cause of lethality at both wavelengths. Evidence for dimer involvement in the yeast mutant was obtained by demonstrating that lethality at both 254 and 365 nm was photoreactivated by light at 405 nm.