DEPTH DISTRIBUTIONS OF DNA DAMAGE IN ANTARCTIC MARINE PHYTO- AND BACTERIOPLANKTON EXPOSED TO SUMMERTIME UV RADIATION

During a survey from January to March 1998, the occurrence of UV-B radiation (UVBR)- induced DNA damage in Antarctic marine phytoplankton and bacterioplankton was investigated. Sampling was done in Ryder Bay, off the British base Rothera Station, 67°S, 68°W (British Antarctic Survey). Samples were taken regularly during the survey period at fixed depths, after which DNA damage was measured in various plankton size fractions (>10, 2–10, and 0.2–2 μm). Incident solar radiation was measured using spectroradiometry, whereas attenuation of biologically effective UVBR was studied using a DNA dosimeter. A diatom bloom was found in the bay during the research period, judging from microscopic observations and HPLC analyses of taxon-specific pigments. The high phytoplankton biomass likely caused strong attenuation of DNA effective UVBR (K_bd-eff). K_bd-eff values ranged from 0.83·m ^{− 1} at the peak of the bloom to 0.47·m ^{− 1} at the end of the season. UVBR-mediated DNA damage, as measured by cyclobutane pyrimidine dimer (CPD) abundance, was detected in all plankton size fractions. Highest levels were found in the smallest size fraction, mainly consisting of heterotrophic bacteria. Clear CPD depth profiles were found during mid-summer (January, beginning of February) with surface levels exceeding 100 CPDs per million nucleotides in the bacterioplankton fraction. At that time, melting of the continuously present shelf ice caused strong salinity gradients in the upper meters, thereby stimulating water column stabilization. At the end of February and beginning of March, this phenomenon was less pronounced or absent. At that time, DNA damage was homogeneously distributed over the first 10 m, ranging between 20 and 30 CPDs per million nucleotides for the smallest size fraction.     

Temperature causes species-specific responses to UV-induced DNA damage in amphibian larvae

Anthropogenic ozone depletion has led to a 2–5% increase in ultraviolet B radiation (UVBR) levels reaching the earth''s surface. Exposure to UVBR causes harmful DNA damage in amphibians, but this is minimized by DNA repair enzymes such as thermally sensitive cyclobutane pyrimidine dimer (CPD)-photolyase, with cool temperatures slowing repair rates. It is unknown whether amphibian species differ in the repair response to a given dose of UVBR across temperatures. We reared larvae of three species (Limnodynastes peronii, Limnodynastes tasmaniensis and Platyplectrum ornatum) at 25°C and acutely exposed them to 80 µW cm⁻² UVBR for 2 h at either 20°C or 30°C. UVBR-mediated DNA damage was measured as larvae repaired damage in photoreactive light at their exposure temperatures. Cool temperatures increased DNA damage in two species and slowed DNA repair rate in P. ornatum. The magnitude of DNA damage incurred from UVBR was species-specific. Platyplectrum ornatum had the lowest CPDs and DNA repair rates, and the depressive effects of low temperature on photorepair were greater in L. tasmaniensis. Considering the susceptibility of most aquatic organisms to UVBR, this research highlighted a need to consider the complexity of species-specific physiology when forecasting the influence of changing UVBR and temperature in aquatic ecosystems.     

DNA DAMAGE AND PHOTOSYNTHETIC PERFORMANCE IN THE ANTARCTIC TERRESTRIAL ALGA PRASIOLA CRISPA SSP. ANTARCTICA (CHLOROPHYTA) UNDER MANIPULATED UV-B RADIATION

The effect of reduced, natural ambient, and enhanced UV-B radiation (UVBR) on photosynthesis and DNA damage in the Antarctic terrestrial alga Prasiola crispa ssp. antarctica (Kützing) Knebel was investigated in two field experiments. Samples of P. crispa were collected underneath snow cover and exposed outside to reduced and natural UVBR in the austral spring. In a second experiment at the end of the austral summer, samples were exposed to ambient and enhanced UVBR. PSII efficiency, net photosynthetic rate (NP), dark respiration rate (DR), UV-absorbing pigments, and cyclobutyl pyrimidine dimer (CPD) formation were measured during the experiments. In October 1998, a spring midday maximum of 2.0 W·m ^{− 2} of UVBR did not significantly affect effective quantum yield (ΔF/F_m′), and a reduction in the ratio of variable to maximal fluorescence (F_v/F_m) in the late afternoon was transient. Exposure to natural ambient UVBR in October increased CPD values significantly. Midday maxima of UVBR during the experiments in October and January were comparable, but Setlow-DNA-weighted UVBR was more than 50% lower in January than in October. In January, 0.5 W·m ^{− 2} additional UVBR during 10 h did not have a negative effect on ΔF/F_m′. The reduction in F_v/F_m was not significant. NP and DR were not affected by supplementation of UVBR. Although photosynthetic activity remained largely unaffected by UVBR treatment, DNA damage was shown to be a sensitive parameter to monitor UVBR effects. Supplementation of additional UVBR did significantly enhance the amounts of CPD in exposed samples and repair took place overnight. It is concluded that PSII and whole-chain photosynthesis of P. crispa is well adapted to ambient and enhanced levels of UVBR but that CPD formation is more sensitive to UVBR than to photosynthesis.     

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet-b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m⁻² ·d⁻¹ (BED_DNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.     

Cooler temperatures slow the repair of DNA damage in tadpoles exposed to ultraviolet radiation: Implications for amphibian declines at high altitude

Ultraviolet B radiation (UVBR) damages the DNA of exposed cells, causing dimers to form between adjacent pyrimidine nucleotides. These dimers block DNA replication, causing mutations and apoptosis. Most organisms utilize biochemical or biophysical DNA repair strategies to restore DNA structure; however, as with most biological reactions, these processes are likely to be thermally sensitive. Tadpoles exposed to elevated UVBR at low environmental temperatures have significantly higher rates of mortality and developmental deformities compared with tadpoles exposed to the same levels of UVBR at higher environmental temperatures. We hypothesized that low environmental temperatures impair the primary enzymatic (photolyase) DNA repair pathway in amphibians, leading to the accumulation of DNA damage. To test this hypothesis, we compared DNA repair rates and photolyase gene expression patterns in Limnodynastes peronii. Tadpoles were acutely exposed to UVBR for 1 hr at either 20 or 30°C, and we measured DNA damage and photolyase expression levels at intervals following this exposure. Temperature had a significant effect on the rate of DNA repair, with repair at 30°C occurring twice as fast as repair at 20°C. Photolyase gene expression (6‐4 PP and CPD) was significantly upregulated by UVBR exposure, with expression levels increasing within 6 hr of UVBR exposure. CPD expression levels were not significantly affected by temperature, but 6‐4 PP expression was significantly higher in tadpoles in the 30°C treatment within 12 hr of UVBR exposure. These data support the hypothesis that DNA repair rates are thermally sensitive in tadpoles and may explain why enigmatic amphibian declines are higher in montane regions where UVBR levels are naturally elevated and environmental temperatures are lower.     

Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet-B-induced CPDs in rice chloroplast and mitochondrial DNA

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet‐B (UV‐B) radiation (280–320 nm) in the process. UV‐B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV‐B‐induced DNA lesions, and are a principal cause of UV‐B‐induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV‐B‐containing sunlight. Nuclear repair of the UV‐B‐induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near‐UV and visible light (300–500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full‐length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV‐B radiation.     

单克隆抗体ELISA测定UVB诱导的DNA损伤(英文)

本文采用单克隆抗体酶联免疫吸附分析法测定了ＵＶＢ诱导ＤＮＡ产生的ＣＰＤ和６４ＰＰ。经０．５ｍＷ／ｃｍ２ＵＶＢ处理１５ｍｉｎ的小牛胸腺和鲱鱼精ＤＮＡ,ＣＰＤ和６４ＰＰ含量显著增加,而未经ＵＶＢ处理的对照ＤＮＡ则没有二聚体形成。     

单克隆抗体ELISA测定UVB诱导的DNA损伤

本文采用单克隆抗体酶联免疫吸附分析法测定了ＵＶ－Ｂ诱导ＤＮＡ产生的ＣＰＤ和６－４ＰＰ。经０．５ｍＷ／ｃｍ＾２ＵＶ－Ｂ处理１５ｍｉｎ的小牛胸物鲱鱼精ＤＮＡ,ＣＰＤ和６－４ＰＰ含量显著增加,而未经ＵＶ－Ｂ处理的对照ＤＮＡ则没有二聚体形成。     

Increased DNA repair in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants overexpressing CPD photolyase

Ultraviolet-B (UV-B, 280–320 nm) radiation may have severe negative effects on plants including damage to their genetic information. UV protection and DNA-repair mechanisms have evolved to either avoid or repair such damage. Since autotrophic plants are dependent on sunlight for their energy supply, an increase in the amount of UV-B reaching the earth’s surface may affect the integrity of their genetic information if DNA damage is not repaired efficiently and rapidly. Here we show that overexpression of cyclobutane pyrimidine dimer (CPD) photolyase (EC 4.1.99.3) in Arabidopsis thaliana (L.), which catalyses the reversion of the major UV-B photoproduct in DNA (CPDs), strongly enhances the repair of CPDs and results in a moderate increase of biomass production under elevated UV-B.     

Effects of UVB radiation on marine phytoplankton communities

The impact of enhanced and reduced UVB radiation (UVBR) on pelagic ecosystems was studied during two mesocosm experiments in May and June/July 1994. The ambient UVBR exposure was either reduced with mylar foil or artificially enhanced with UVB fluorescent tubes. Developments in the phytoplankton communities were followed during 11 and 8 day periods using several structural and functional parameters. In the May experiment, enhanced UVBR significantly stimulated carbon dioxide fixation, activity of alkaline phosphatase and content of fatty acids. In the June-July experiment, the effects induced by changed UVBR were smaller with some indications of decreased algal biomass with enhanced UVBR. Several of the measured parameters indicated that the two experiments represented different stages in the plankton community development. In the May experiment, the community was in a development stage, moving from nutrient-replete to nutrient-depleted conditions, while the community in June/July was depleted of nutrients from the start. The stimulating effects of UVBR in May are suggested to be the secondary effects of a photochemically induced breakdown of dissolved organic matter, resulting in an increase in available nutrients.      

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.     

Effects of ultraviolet radiation on protein content,respiratory electron transport system (ETS) activity and superoxide dismutase (SOD) activity of Antarctic plankton

Depletion of stratospheric ozone causes a significant increase in UV radiation in the Antarctic regions. Its effects include DNA damage, as well as impairment of photosynthesis, respiration, protein synthesis and other metabolic functions. Defence systems of cells are directed against free oxygen radicals liberated through UV radiation. One of their main components of defence systems are superoxide dismutases (SODs). The effects of ultraviolet radiation A and B (UVAR and UVBR) on protein synthesis, respiratory electron transfer (ETS) activity and superoxide dismutase (SOD) activity in Antarctic plankton were examined. Samples were taken in the Gerlache Strait (Antarctica). Three stations were situated in an area, which showed a Cryptomonas bloom. Two stations were located in areas having a bloom of green nanoflagellates. Samples were exposed for 3 h to photosynthetically active radiation (PAR), or to PAR + UVAR or to PAR + UVAR + UVBR, under fixed experimental irradiances. UVBR inhibited protein synthesis and ETS activity, and enhanced SOD activity. UVAR enhanced protein synthesis and ETS activity, and inhibited SOD activity. Samples, which had received more solar radiation prior to experiments showed less inhibition of protein synthesis by experimental UVBR, which suggests acclimation to ambient radiation. Cryptomonas-dominated stations showed less SOD activity than the green flagellate-dominated stations, which might be related to the protection conferred by their phycoerythrin.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

Habitat related variation in UV tolerance of tropical marine red macrophytes is not temperature dependent

Because tropical marine macrophytes experience high ultraviolet-B radiation (UVBR: 280–320 nm) it is assumed that they have high UV tolerance. This was investigated by examining the relative UV sensitivity of five Caribbean red macrophytes. Furthermore, the possibility of temperature dependence of UV effects was examined over a tropical temperature range. Algal fragments of intertidally occurring Gelidiopsis planicaulis (Taylor) Taylor, Wurdemannia miniata (Duby) Feldman and Hamel, and Hypnea spinella (Agardh) Kützing, and the subtidal species Bryothamnion triquetrum (Gmelin) Howe and Heterosiphonia gibbesii (Harvey) Falkenberg were repeatedly subjected to artificial UVBR and ultraviolet-A radiation (UVAR: 320–400 nm) at 22, 26 and 30°C, whereas exposure to photosynthetically active radiation (PAR) served as control. Growth rates, optimal quantum yield of PSII and accumulation of DNA damage were monitored for 10 days, whereas the relative abundance of the D1 reaction centre binding protein and the presence of UV absorbing compounds were investigated in some samples. UVAR and UVBR significantly depressed growth rates of all species. UVBR exposure caused accumulation of DNA damage and resulted in stronger growth reduction than UVAR. UVBR and UVAR caused a depression of optimal quantum yield and a lower D1 abundance. However, the former recovered fast and acclimated to the UV treatments. Some species produced UV absorbing compounds in response to UVAR. UV exposure caused less pronounced effects in intertidally occurring species than in subtidal species. UV effects on growth, the accumulation of DNA damage and UV induced depression of optimal quantum yield were independent of temperature in most species. We conclude that high UVBR in tropical regions may depress in situ growth rates of these intertidal and subtidal red macrophytes.     

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.     

Molecular response of nasal mucosa to therapeutic exposure to broad-band ultraviolet radiation

Ultraviolet radiation (UVR) phototherapy is a promising new treatment for inflammatory airway diseases. However, the potential carcinogenic risks associated with this treatment are not well understood. UV-specific DNA photoproducts were used as biomarkers to address this issue. Radioimmunoassay was used to quantify cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts in DNA purified from two milieus: nasal mucosa samples from subjects exposed to intranasal phototherapy and human airway (EpiAirway™) and human skin (EpiDerm™) tissue models. Immunohistochemistry was used to detect CPD formation and persistence in human nasal biopsies and human tissue models. In subjects exposed to broadband ultraviolet radiation, DNA damage frequencies were determined prior to as well as immediately after treatment and at increasing times post-treatment. We observed significant levels of DNA damage immediately after treatment and efficient removal of the damage within a few days. No residual damage was observed in human subjects exposed to multiple UVB treatments several weeks after the last treatment. To better understand the molecular response of the nasal epithelium to DNA damage, parallel experiments were conducted in EpiAirway and EpiDerm model systems. Repair rates in these two tissues were very similar and comparable to that observed in human skin. The data suggest that the UV-induced DNA damage response of respiratory epithelia is very similar to that of the human epidermis and that nasal mucosa is able to efficiently repair UVB induced DNA damage.     

Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans.

We tested the hypothesis that DNA is a target for solar-simulated ultraviolet radiation (ssUVR)-induced suppression of the reactivation of memory immunity in humans. T4N5 liposomes contain the DNA repair enzyme T4 endonuclease V. This cleaves DNA at the site of ultraviolet radiation (UVR)-induced cyclobutane pyrimidine dimers (CPD), initiating DNA repair. It has previously been used to show that CPDs are a key molecular trigger for UVR-induced immunosuppression in mice. To determine whether CPD formation is involved in UVR immunosuppression in humans, nickel-allergic volunteers were irradiated with a range of doses of ssUVR. T4N5 or empty liposomes were then applied after irradiation. Nickel-induced recall immunity was assessed by reflectance spectrometry. T4N5 liposomes inhibited immunosuppression and prevented ssUVR from reducing the number of epidermal dendritic cells. T4N5 liposomes also reduced macrophage infiltration into irradiated epidermis. These studies show that enhanced removal of CPDs from human skin protects from immunosuppression, hence demonstrating that these photolesions are an important molecular event in ssUVR-induced immunosuppression in humans. CPDs also triggered loss of dendritic cells and infiltration by macrophages. It is possible that these changes to antigen presenting cells contribute to ssUVR induced suppression of recall immunity to nickel in humans.     

Water- and temperature-dependence of DNA damage and repair in the fruticose lichen Cladonia arbuscula ssp. mitis exposed to UV-B radiation

The induction of cyclobutane pyrimidine dimers (CPDs) by ultraviolet‐B radiation (UV‐B, 280–315 nm) and repair mechanisms were studied in the lichen Cladonia arbuscula ssp. mitis exposed to different temperatures and water status conditions. In addition, the development and repair of CPDs were studied in relation to the different developmental stages of the lichen thallus podetial branches. Air‐dried lichen thalli exposed to UV‐B radiation combined with relatively high visible light (HL, 800 μmol m^?2 s^?1; 400–700 nm) for 7 days showed a progressive increase of CPDs with no substantial repair, although HL was present during and after irradiation with UV‐B. Fully hydrated lichen thalli, that had not been previously exposed to UV‐B radiation for 7 days, were given short‐term UV‐B radiation treatment at 25°C, and accumulated DNA lesions in the form of CPDs, with repair occurring when they were exposed to photoreactivating conditions (2 h of 300 μmol m^?2 s^?1, 400–700 nm). A different pattern was observed when fully hydrated thalli were exposed to short‐term UV‐B radiation at 2°C, in comparison with exposure at 25°C. High levels of CPDs were induced at 2°C under UV‐B irradiation, without significant repair under subsequent photoreactivating light. Likewise, when PAR (300 μmol m^?2 s^?1) and UV‐B radiation were given simultaneously, the CPD levels were not lowered. Throughout all experiments the youngest, less differentiated parts of the lichen thallus – namely ‘tips’, according to our arbitrary subdivision – were the parts showing the highest levels of CPD accumulation and the lowest levels of repair in comparison with the older thallus tissue (‘stems’). Thus the experiments showed that Cladonia arbuscula ssp. mitis is sensitive to UV‐B irradiation in the air‐dried state and is not able to completely repair the damage caused by the radiation. Furthermore, temperature plays a role in the DNA damage repairing capacity of this lichen, since even when fully hydrated, C. arbuscula ssp. mitis did not repair DNA damage at the low temperatures.