UV-INDUCED DNA DEGRADATION IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC 6308

Ultraviolet radiation-induced DNA degradation has been demonstrated in the unicellular cyanobacterium Synechocystis PCC 6308. The extent of DNA degradation was greatly reduced by inhibition of DNA replication by preirradiation dark incubation and degradation was completely inhibited by exposure of irradiated cells to photoreactivating wavelengths. DNA degradation was not observed when irradiated cells were incubated in the presence of the excision repair inhibitors caffeine and acriflavin, suggesting that degradation is a manifestation of excision repair of pyrimidine dimers in Synechocystis . Increasing UV fluences resulted in an increase in the final extent of DNA degradation. However, at higher fluences degradation was inhibited, suggesting saturation of the excision repair system. Incubation of irradiated cells under conditions which inhibit protein synthesis greatly increased the extent of DNA degradation and the time over which it occurred. Exposure of cells to a sublethal fluence of UV greatly reduced the extent of DNA degradation produced by a challenge fluence administered after 24 h incubation providing evidence for inducible DNA repair activity in cyanobacteria.     

ULTRAVIOLET-INDUCED CELL DEATH IS INDEPENDENT OF DNA REPLICATION IN RAT KANGAROO CELLS

Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication.     

Excision-repair capacity of UV-irradiated strains of Escherichia coli and Streptococcus pneumoniae, estimated by plasmid recovery

Although the biological role of many bacterial repair genes is known, there is still an interest in evaluating the capacity of repair pyrimidine dimers in some strains. For this purpose, we have developed a rapid assay. Cells bearing a plasmid are UV irradiated and incubated to allow recovery. The plasmid DNA is extracted, purified and treated with UV endonuclease from Micrococcus luteus that specifically produces single strand breaks at the site of pyrimidine dimers. The amount of open circular and covalently closed circular forms of the plasmid DNA after treatment and post-incubation provides an estimate of the repair capability of the host strain. The wild type strain and the uvrA mutant of Escherichia coli were used to adjust the assay. The lexA mutant of E. coli has been tested and its repair capability is equivalent to that of wild-type strain. The assay has been extended to Streptococcus pneumoniae, which is naturally deficient in photoreactivation and SOS-like functions. This strain is efficient in the repair of pyrimidine dimers, formed after UV irradiation.     

PHOTOREACTIVATION OF ULTRAVIOLET IRRADIATED NON-DIVIDING POPULATIONS OF ICR 2A FROG CELLS

Abstract— Ultraviolet (UV) irradiation of non-dividing populations of ICR 2A frog cells led to their detachment from the surface of the culture dish and eventual lysis. Exposure of the cells to photoreactivating light after UV irradiation prevented cell killing and was accompanied by a loss of endonuclease sensitive sites from DNA. This photoreversal did not take place when the cells were exposed at 4°C to photoreactivating light indicating that the reversal was the result of photoenzymatic repair. As the action of photoreactivating enzyme is specific for the repair of pyrimidine dimers in DNA, these results suggest that pyrimidine dimers in DNA are the critical lesions leading to the death of non-dividing populations of UV irradiated 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.     

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.     

IRRADIATION OF CIRCULAR DNA WITH 254 nm RADIATION OR SENSITIZATION IN THE PRESENCE OF Ag+ EVIDENCE FOR UNWINDING BY PHOTOPRODUCTS OTHER THAN PYRIMIDINE DIMERS

Abstract— Structural alterations of DNA irradiated with UV light were analyzed by the agarose gel technique. Relaxed, circular pAT 153 DNA molecules were sensitized by broad band radiation with a maximum at 313 nm in the presence of silver ions or irradiated with 254 nm light in buffer only. In both cases the electrophoretic mobility of DNA topoisomers was altered as a linear function of UV exposure. For DNA irradiated in the sensitized reaction the unwinding angle per site sensitive to Micrococcus luteus pyrimidine dimer endonuclease was found tobe–11.4°. This value is significantly smaller thanthe–14.3° already known for DNA topoisomers irradiated with 254 nm light. The irradiated DNAs were a very good substrate for the Escherichia coli photoreactivating enzyme (PRE). However, the photoenzymic removal of all sites sensitive to the endonuclease specific for pyrimidine dimers was not coupled to a full restoration of the original electrophoretic mobility. Thirty and 23% of the unwinding were still present in the photoreactivated topoisomers and the unwinding angles per pyrimidine dimer were then recalculatedas–10.1°and–8.7° for DNAs irradiated with 254 nm and sensitized, respectively. The limited difference between these two values could result from the different base composition of the pyrimidine dimers generated in the conditions of irradiation used. These results show that the tertiary structure of DNA is measureably altered by UV photodamages other than pyrimidine dimers.     

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.     

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Abstract— Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in vitro. Cells irradiated with ultraviolet (UV)-fluences up to 350 J/m² could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV-irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non-growth conditions did not influence the survival of UV-irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV-inactivation or photoreactivation. Small differences in the rates of UV-inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400-470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8-hydroxy-5-deazaflavin type photoreactivating enzyme.     

SURVIVAL AND PYRIMIDINE DIMERS IN CULTURED FISH CELLS EXPOSED TO CONCURRENT SUN LAMP ULTRAVIOLET AND PHOTOREACTIVATING RADIATIONS

Abstract— Cultured fishcells(RBCF–1 line) were irradiated with filtered sun lamp ultraviolet (SL-UV; > 280 nm) together with or followed by illumination with daylight(DL) radiation (> 350 nm). The colony forming ability of the cells decreased with increasing fluence of SL-UV. Concurrent exposure of cells to SL-UV and DL, however, increased survival relative to exposure to SL-UV alone. The photoreactivable fraction reached 0.52 at22–25_‡C. By using a constant fluence modification factor of 86, the shape of dose-survival curve was found to be almost the same for 254 nm and SL-UV. In parallel with photoreactivation of cell survival, changes in the numbers of pyrimidine dimers in permeabilized cell DNA and in extracted total DNA were determined by measurements of endonuclease-sensitive sites (ESS). The yield of ESS in both DNA's increased almost linearly with increasing SL-UV fluence, although the yield in extracted DNA was about double of that in permeabilized cell DNA. The yield of ESS per unit fluence by 254 nm was about 70-fold greater than SL-UV. The fraction of cells inactivated per ESS was almost the same for 254 UV and SL-UV. In SL-UV-irradiated cells, the photoreactivable fractions in terms of ESS were _‡ 10% higher in extracted DNA than in the DNA of permeabilized cells and also were higher when DL was administered separately after SL-UV-irradiation. When irradiated cells were exposed to DL at 0_‡C, the photoreactivable fractions of both DNAs were appreciably less, indicating that the photoreactivation of ESS was enzymatic. These results support the suggestion that the mechanism for cell killing, mainly formation of pyrimidine dimers, by SL-UV is the same as that by 254 UV.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

Photoreactivity of UV-b damage in bacteriophage phi X174 DNA

Abstract— The fraction of biological damage in isolated single-strand and double-strand forms of bac-teriophage DNA resulting from pyrimidine dimers following exposure to germicidal UV (254 nm) and UV-B (280-320. nm) radiation has been compared. Radiation from a Westinghouse FS-40 sunlamp filtered through a cellulose acetate sheet was used as the UV-B radiation source. Biological damage from pyrimidine dimers was determined by measuring the survival of the viral DNA with and without photoreactivation, an enzymatic process specific for repair of pyrimidine dimers. The same fraction of biological damage in the single strand and double–strand forms of φX174 DNA is repairable by photo-reactivation following exposures to germicidal UV and UV-B radiation.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) II. Evidence for heterogeneous light-dependent repair activities

Abstract. Two biological effects of UV radiation upon Smittia eggs are observed, both of which seem to be associated with the formation of pyrimidine dimers in the RNA (largely ribosomal) of the eggs. While irradiation of the anterior pole region causes the formation of an aberrant segment pattern (double abdomen induction), irradiation of entire eggs leads to an arrest of their development (inactiva-tion). Both UV effects are photoreversible with different action spectra of the photoreactivating light. A dose rate dependence of the photoreactivation can be observed after both UV effects. The saturating dose rate is about 6 W/m² (at 440 nm) after UV induction of double abdomens. Upon UV inactivation, the saturating dose rate level for the photoreactivating light is much higher, and a single light flash causes both a considerable biological reactivation and the disappearance of about 7 × 10⁹ pyrimidine dimers from the total RNA per egg. The results indicate the presence of heterogeneous light-dependent repair activities acting upon UV induced pyrimidine dimers in the RNA of the eggs.     

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light

Abstract—The influence of photoreactivating light on the fate of UV-induced DNA damage has been measured in the epidermis of hairless mice using damage-specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence-dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV-induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.     

SKH-1 hairless mice repair UV-induced pyrimidine dimers in epidermal DNA

The SKH-1 hairless mouse strain has been used extensively as a model for human photocarcinogenesis, photoimmunology and photoaging, but little is known about DNA repair in living mouse skin. Mice were irradiated with UV-B light at doses which produce mild to severe sunburn, and the frequency of pyrimidine dimers in epidermal DNA was measured immediately and 6 h after irradiation using T4 endonuclease V treatment and alkaline agarose gel electrophoresis. The results demonstrate significant removal of pyrimidine dimers in mouse skin in vivo, with a dimer half-life of 7.4 h. These findings are similar to the repair of dimers in human skin in vivo. The SKH-1 hairless mouse is thus a useful model for pyrimidine dimer repair in human skin.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

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.     

REDUCED REPAIR OF NON-DIMER PHOTOPRODUCTS IN A GENE TRANSFECTED INTO XERODERMA PIGMENTOSUM CELLS

Abstract— 4ells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m⁻² of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA.     

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.