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.     

AN ACTION SPECTRUM FOR PHOTOTAXIS BY PSEUDOPLASMODIA OF Dictyostelium discoideum

Abstract— The sensitivity of phototactic orientation of pseudoplasmodia (slugs) of the cellular slime mold Dictyosrelium discoideum has been measured for white light and monochromatic light using computer aided directional statistics. The zero threshold for white light was found at about 10^-5 Ix. An action spectrum for positive phototaxis has been calculated from fluence rate-response curves; it shows two major maxima at about 420 and 440 nm and secondary peaks at 560 and 610 nm. This action spectrum is significantly different from the one for phototactic orientation in Dictyostelium amoebae.     

REPAIR OF ULTRAVIOLET-IRRADIATED TRANSFORMING DNA IN A recA MUTANT OF Haemophilus influenzae

—Ultraviolet-irradiated transforming DNA was assayed on a wild-type strain of Haemophilus influenzae strain Rd, on an excision repair-deficient ( uvr-2 ) mutant, on a recombination repair-deficient ( recA4 ) mutant, and on a strain carrying both mutations. The donor DNA had a point mutation genetic marker ( strAl ) and a long nonhomologous plasmid-derived DNA segment inserted in the HP1 prophage. The shape of the inactivation curves suggested that only recombination was responsible for the inverse square root kinetics observed with excision repair-proficient recipients.     

POSTREPLICATION REPAIR IN AN EXCISION-DEFECTIVE MUTANT OF ESCHERICHIA COLI: ULTRAVIOLET LIGHT-INDUCED INCORPORATION OF BROMODEOXYURIDINE INTO PARENTAL DNA*

Abstract— Ultraviolet (UV) light-induced incorporation of bromodeoxyuridine (BrdUrd) into parental DNA of an excision-defective mutant of Escherichia coli has been observed by selective photolysis of bromouracil (BrUra)-containing regions in the parental DNA. It appears that the BrUra-containing regions occur only in that DNA which has served as a template for normal semiconservative replication. After an exposure at 254 nm which results in one pyrimidine dimer per 45times 10⁶ daltons, incubation in BrdUrd resulted in BrUra–containing regions ˜ 1.5 times 10⁴ nucleotides in length at intervals of ˜ 55 times 10⁶ daltons in the parental DNA. Thus approximately one BrUra-containing region has occurred for every 1.2 pyrimidine dimers in the parental DNA. The observed incorporation of BrdUrd is interpreted in terms of a proposed model for postreplication repair in which genetic exchanges produce single-strand gaps in the parental DNA.     

EFFECTS OF UV-B IRRADIATION ON SOROCARP DEVELOPMENT OF Dictyostelium discoideum

Abstract— The effect of UV-B irradiation on the development of Dictyostelium discoideum from amoebae to mature sorocarps was studied. Radiation with wavelength ≤ 310 nm was very efficient in retarding and inhibiting the development especially when the organisms were exposed during the first 12 h. At a wavelength of 280 nm an irradiation of 1 h at an irradiance of < 0.2 W m^-2 was sufficient to completely inhibit sorocarp development. The fluence-dependence shows as well that the development of D. discoideum is a very sensitive system to indicate UV-B irradiation. Furthermore, since the sorocarp development is concluded within 48 h it can serve as a fast bioassay for hazardous levels of increased UV-B irradiation which have been predicted as a result of the ozone reduction in the stratosphere due to the manmade production and emission of chlorofluoromethanes.     

NEGATIVE PHOTOTAXIS OF Dictyostelium discoideum PSEUDOPLASMODIA IN UV RADIATION

Abstract— Pseudoplasmodia of the cellular slime mold Dictyostelium discoideum show negative phototactic orientation in lateral ultraviolet radiation. The action spectrum has a peak in the UV-B band near 280 nm. The absorption spectrum shows a prominent peak in the same wavelength range. Thus, negative phototaxis can be easily explained by the assumption that the lens effect, by which D. discoideum slugs detect the light direction, is defeated by the high internal absorption in this wavelength range.     

PHOTOMOVEMENT IN AN "EYELESS" MUTANT OF Chlamydomonas

The function of the stigma ("eyespot") in the green flagellate Chlamydomonas reinhardtii was investigated by comparing the photomovement of the wild-type alga with that of an "eyeless" mutant ( ey 627). Movements of individual cells in response to a blue-green stimulus light were recorded using a videomicroscope system and were analyzed using vectorial methods. Cells of the "eyeless" mutant were phototactic; at a high stimulus fluence rate, their swimming paths were directed away from the light source. Although the orientation of the mutant was not as strongly directional as that of the wild type, it was statistically significant. However, the swimming paths of the mutant cells were very erratic in the presence of the stimulus beam, undergoing frequent changes of direction. Despite the differences in their phototactic orientation, cells of mutant and wild type all showed a distinct step-up photophobic response at the onset of stimulation. These results are consistent with the hypothesis that the stigma plays an accessory role in phototaxis, either by shading the photoreceptor or by acting as a quarter-wave reflector.     

INHIBITION OF DNA REPAIR SYNTHESIS BY SUNLIGHT

Abstract— DNA repair synthesis as determined by thymidine incorporation in the presence of hydroxyurea reached a much lower maximum level after solar compared with UVC exposure in five human melanoma cell lines, in HeLa cells, and in two human fibroblast strains. This finding was confirmed by determination of unscheduled DNA synthesis where both the number of labelled nuclei and grain count per nucleus were lower in sun-exposed cells. In a cloned human melanoma line (MM253cl), glass-filtered sunlight inhibited UVC repair synthesis, and solar UVB alone induced a higher level of repair synthesis than either complete sun or solar UVA plus solar UVB. The fluence response of filtered sunlight for inhibition of UVB (sunlamp) and UVC showed that most inhibition was obtained at low fluences (5-10 min), further exposure giving a plateau at 40% of the original level. Ultraviolet C and sunlight inactivated adenovirus 5 giving F ₀ values for virus survival 40-fold higher than for cell survival. Replication of either UVC- or solar-irradiated virus was not affected by prior irradiation of cells with glass-filtered sunlight. Stathmokinetic analysis of cell cycle progression by DNA flow cytometry showed that UVC and sunlamp UVB retarded cell movement from the G1 and S phases whereas equitoxic sunlight and glass-filtered sunlight (nontoxic) had no effect. These results indicate that solar UVA at low environmental fluences partially inhibits UVB repair synthesis in a range of human cell types but does not affect the replication of a UVB- or UVC-damaged virus when applied to the genome alone or to the host cell.     

OXYGEN EFFECT IN SURVIVAL OF Dictyostelium discoideum TREATED WITH NEAR ULTRAVIOLET RADIATION

Abstract— Fluence-response survival curves have been measured for the cellular slime mold Dictyostelium discoideum exposed to near ultraviolet radiation. Data were obtained for a wild type strain and three UV-sensitive mutant strains in exponential growth phase. Fluences for 10% survival (F₁₀) are about 1 MJ m⁻² for cells irradiated in saline solution saturated with nitrogen. When air is bubbled through the saline, the Fm values are only one third as large. Strain HPS50, which is the strain most sensitive to gamma radiation and to 254 nm UV, also exhibits the greatest sensitivity to near UV. However, the difference in sensitivity to near UV between wild type and mutant strains is small compared to other physical and chemical agents known to damage DNA.     

SATURATION OF DNA REPAIR IN MAMMALIAN CELLS*

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m². We measured excision repair in normal human fibroblasts up to 80J/m². The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus , and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m² doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.     

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.     

EFFECTS OF SOLAR RADIATION ON DEVELOPMENT IN THE CELLULAR SLIME MOLD, Dictyostelium discoideum

–The effect of solar radiation on development was studied in two strains (wild type N04 and axenic AX2) of the cellular slime mold, Dictyostelium discoideum. In both strains early development was retarded by solar radiation and amoebae exposed for >200 min never developed into mature sorocarps. Overheating can be excluded as a mechanism for inhibition since the organisms were exposed in a temperature-controlled growth chamber. The most effective component seems to be UV-B radiation (while UV-A has a smaller inhibitory effect) as shown by cutting off shorter wavelength radiation using WG filters.     

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 ).     

SITES OF PHOTODYNAMICALLY INDUCED DNA REPAIR IN HUMAN CELLS

Abstract Human REH cells were incubated with the photosensitizers meso -tetra(4-sulfonatophenyl)porphyrin (TSPP=TPPS₄) or meso -tetra(3-hydroxyphenyl)porphyrin (3-THPP). The relatively hydrophilic TSPP was partly found in the cytoplasm and partly in the nuclei, whereas the lipophilic 3-THPP was found apparently in membranes and not inside the nuclei. After illumination, sites of DNA repair were labeled by means of a monoclonal antibody against proliferating cell nuclear antigen (PCNA) bound in the nuclei. The amount of bound PCNA in non-S-phase cells was proportional to the light dose. The bound PCNA was homogeneously distributed in the nuclei 0.5 h after photodynamic treatment (PDT) with TSPP. In contrast, for cells given PDT with 3-THPP, the periphery of the nuclei was selectively labeled, indicating that the initial DNA damage was localized close to the sensitizer at the nuclear membrane.     

REPAIR OF UV-DAMAGED INCOMING PLASMID DNA IN Saccharomyces cerevisiae

A whole-cell transformation assay was used for the repair of UV-damaged plasmid DNA in highly transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. Six rad alleles were selected from the three epistasis groups: rad 1-1 and rad2-1 from the RAD3 group, rad6-1 and rad18-2 from the RAD6 group, and rad52-1 and rad54-1 from the RAD52 group. Cells carrying single, double and triple rad alleles were transformed to uracil prototrophy by centromeric plasmid DNA (YCp19) modified in vitro with UV (254 nm). Surviving fractions were calculated as the number of transformants at each fluence relative to the number of transformants with unirradiated plasmid DNA. The sensitivity of incoming DNA in single rad mutants shows that most repair is carried out by excision repair and a RAD18-dependent process. In the rad52-1 host, the sensitivity of incoming DNA was intermediate between those found in RAD+ and rad2-1 hosts, suggesting the involvement of a recombinational repair process. Non-epistatic interactions were observed between rad alleles belonging to different epistasis groups. This provides validation for the classification of the three epistasis groups concerning the repair of chromosomal DNA for UV-incoming DNA. In both rad1-1 rad6-1 and rad1-1 rad18-2 rad54-1 hosts, the mean fluence for one lethal event corresponds approximately to one pyrimidine dimer per plasmid molecule, indicating that they are absolute repairless hosts for incoming DNA. A comparison between cell and plasmid survival reveals that there are differences in the repairability of both chromosomal and incoming DNA. The large effect of rad6-1 mutation on cell survival and the small effect on incoming DNA suggest that, in the RAD+ strain, the RAD6 product may be essential for the repair processes which act on chromosomal DNA, but not for those which act on incoming DNA. It is proposed that in yeasts postreplication repair of incoming DNA is limited to supercoiled molecules with 1-2 pyrimidine dimers that can initiate replication.     

SUBSTRATE DEPENDENCE OF THE ACTION SPECTRUM FOR PHOTOENZYMATIC REPAIR OF DNA

Abstract— The absolute action spectrum has been determined for photoenzymatic splitting of cyclobutadipyrimidines ("pyrimidine dimers") from natural DNA, and from the synthetic polydeoxyribonucleotides poly(dA)·poly(dT) (forming only cyclobutadithymine) and poly(dG)·poly(dC) (forming only cyclobutadicytosine). These action spectra differ strikingly from each other, even when using the same enzyme preparations. On the other hand, the action spectrum for splitting cyclobutadithymine in natural DNA containing "dimers" of only this one type closely resembles the action spectrum for splitting the total mixture of "dimer" types in natural DNA, and is entirely different from the spectrum for splitting of the same photoproduct from poly(dA)·poly(dT). These results mean that the action spectrum is not simply the absorption spectrum of a chromophore carried by the photoreactivating enzyme, nor is it solely determined by the nature of the substrate photoproduct. It is at least partly determined by the over-all polynueleotide structure (viz. exact helical dimensions, pattern of neighboring bases to the "dimers," or both), affecting a ground state interaction between the enzyme and substrate in the enzyme-substrate complex.