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.     

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, <Emphasis Type="Italic">Euphlyctis cyanophlyctis</Emphasis>

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32°C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32°C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32°C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32°C) during stages 7–42 produced male-biased sex ratio. Rearing of tadpoles at 32°C during stages 25–42 produced male-biased sex ratio, while exposure during stages 7–25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22°C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.     

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.     

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.     

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.     

The native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.     

Eukaryotic class II cyclobutane pyrimidine dimer photolyase structure reveals basis for improved ultraviolet tolerance in plants

Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280–315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 Å resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms.     

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.     

Diel patterns of UVBR-induced DNA damage in picoplankton size fractions from the Gulf of Aqaba,Red Sea

This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation: 280 to 315 nm) on bacterio- and phytoplankton (<10 mm) from the Gulf of Aqaba, Red Sea. Incident biologically effective doses (BEDs) and attenuation of biologically effective radiation in the water column were measured using a DNA biodosimeter. UVBR-induced DNA damage was measured as cyclobutane pyrimidine dimers (CPDs), using an antibody directed to CPDs followed by chemiluminescent detection. Depth profiles of DNA damage were determined in two plankton size fractions (0.2 to 0.8 mm and 0.8 to 10 mm) collected down to 50 m depth. Furthermore, accumulation and removal of CPDs were monitored in surface plankton samples during several daily cycles. Small plankton (plankton <10 mm) composition was determined by flow cytometry. The plankton community in the Gulf of Aqaba was dominated by nonphototrophic bacteria and the free-living prochlorophyte Prochlorococcus spp. (<0.8 mm). In general, no DNA damage could be detected in dosimeter DNA below 15 m. In contrast, DNA damage (up to 124 CPD Mnucl-1) could be detected in all bacterio- and phytoplankton samples. DNA damage accumulated throughout the day, indicating that plankton in the Gulf of Aqaba undergo UVBR stress via CPD induction. Although the numbers of CPDs decreased during darkness, both size fractions showed some residual DNA damage at the end of the night. This suggests that dark repair processes did not remove all CPDs, or that part of the plankton community was incapable of repair at all. CPD levels in the two size fractions showed no significant differences in situ. During full solar radiation exposures (samples incubated in bags), more CPDs were detected in the smaller (0.2 to 0.8 mm) size fraction as compared to the larger (0.8 to 10 mm) size fraction. In these experiments, initial plankton composition was significantly different from the field samples. This implies that a shift in the population structure or irradiance conditions can lead to a significant change in UVBR sensitivity. In conclusion, the results show that the picoplankton-dominated phyto- and bacterioplankton communities in the clear surface waters from the Gulf of Aqaba undergo UVBR stress. Repair pathways are not sufficient to eliminate damage during or after UVBR exposure hours, suggesting photomortality as a potential loss parameter of the plankton community.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

Functional acclimation to solar UV-B radiation in Gunnera magellanica, a native plant species of southernmost Patagonia

The ecosystems of Tierra del Fuego (in southern Patagonia, Argentina) are seasonally exposed to elevated levels of ultraviolet‐B radiation (UV‐B: 280–315 nm), due to the passage of the ‘ozone hole’ over this region. In the experiments reported in this article the effects of solar UV‐B and UV‐A (315–400 nm) on two UV‐B defence‐related processes: the accumulation of protective UV‐absorbing compounds and DNA repair, were tested. It was found that the accumulation of UV‐absorbing sunscreens in Gunnera magellanica leaves was not affected by plant exposure to ambient UV radiation. Photorepair was the predominant mechanism of cyclobutane‐pyrimidine dimer (CPD) removal in G. magellanica. Plants exposed to solar UV had higher CPD repair capacity under optimal conditions of temperature (25 °C) than plants grown under attenuated UV. There was no measurable repair at 8 °C. The rates of CPD repair in G. magellanica plants were modest in comparison with other species and, under equivalent conditions, were about 50% lower than the repair rates of Arabidopsis thaliana (Ler ecotype). Collectively our results suggest that the susceptibility of G. magellanica plants to current ambient levels of solar UV‐B in southern Patagonia may be related to a low DNA repair capacity.     

Divergence in DNA photorepair efficiency among genotypes from contrasting UV radiation environments in nature

Populations of organisms routinely face abiotic selection pressures, and a central goal of evolutionary biology is to understand the mechanistic underpinnings of adaptive phenotypes. Ultraviolet radiation (UVR) is one of earth's most pervasive environmental stressors, potentially damaging DNA in any organism exposed to solar radiation. We explored mechanisms underlying differential survival following UVR exposure in genotypes of the water flea Daphnia melanica derived from natural ponds of differing UVR intensity. The UVR tolerance of a D. melanica genotype from a high‐UVR habitat depended on the presence of visible and UV‐A light wavelengths necessary for photoenzymatic repair of DNA damage, a repair pathway widely shared across the tree of life. We then measured the acquisition and repair of cyclobutane pyrimidine dimers, the primary form of UVR‐caused DNA damage, in D. melanica DNA following experimental UVR exposure. We demonstrate that genotypes from high‐UVR habitats repair DNA damage faster than genotypes from low‐UVR habitats in the presence of visible and UV‐A radiation necessary for photoenzymatic repair, but not in dark treatments. Because differences in repair rate only occurred in the presence of visible and UV‐A radiation, we conclude that differing rates of DNA repair, and therefore differential UVR tolerance, are a consequence of variation in photoenzymatic repair efficiency. We then rule out a simple gene expression hypothesis for the molecular basis of differing repair efficiency, as expression of the CPD photolyase gene photorepair did not differ among D. melanica lineages, in both the presence and absence of UVR.     

Thermal acclimation of locomotor performance in tadpoles and adults of the aquatic frog Xenopus laevis

Among amphibians, the ability to compensate for the effects of temperature on the locomotor system by thermal acclimation has only been reported in larvae of a single species of anuran. All other analyses have examined predominantly terrestrial adult life stages of amphibians and found no evidence of thermal acclimatory capacity. We examined the ability of both tadpoles and adults of the fully aquatic amphibian Xenopus laevis to acclimate their locomotor system to different temperatures. Tadpoles were acclimated to either 12 °C or 30 °C for 4 weeks and their burst swimming performance was assessed at four temperatures between 5 °C and 30 °C. Adult X. laevis were acclimated to either 10 °C or 25 °C for 6 weeks and their burst swimming performance and isolated muscle performance was determined at six temperatures between 5 °C and 30 °C. Maximum swimming performance of cold-acclimated X. laevis tadpoles was greater at cool temperatures and lower at the highest temperature in comparison with the warm-acclimated animals. At the test temperature of 12 °C, maximum swimming velocity of tadpoles acclimated to 12 °C was 38% higher than the 30 °C-acclimation group, while at 30 °C, maximum swimming velocity of the 30 °C-acclimation group was 41% faster than the 12 °C-acclimation group. Maximum swimming performance of adult X. laevis acclimated to 10 °C was also higher at the lower temperatures than the 25 °C acclimated animals, but there was no difference between the treatment groups at higher temperatures. When tested at 10 °C, maximum swimming velocity of the 10 °C-acclimation group was 67% faster than the 25 °C group. Isolated gastrocnemius muscle fibres from adult X. laevis acclimated to 10 °C produced higher relative tetanic tensions and decreased relaxation times at 10 °C in comparison with animals acclimated to 25 °C. This is only the second species of amphibian, and the first adult life stage, reported to have the capacity to thermally acclimate locomotor performance. Accepted: 28 October 1999     

Influence of a cis,syn‐cyclobutane pyrimidine dimer damage on DNA conformation studied by molecular dynamics simulations

The photo‐induced formation of cis‐syn‐cyclobutane pyrimidine dimers (CPD) is a highly mutagenic and cancerogenic DNA lesion. In bacteria photolyases can efficiently reverse the dimer formation employing a light‐driven reaction after looping out the CPD damaged bases into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The CPD damage may alter the DNA structure and dynamics already in the absence of the repair enzyme which can facilitate the initial binding of a photolyase repair enzyme. To characterize the effect of a CPD damage, extensive comparative molecular dynamics (MD) simulations on duplex DNA with central regular or CPD damaged nucleotides were performed supplemented with simulations of the DNA‐photolyase complex. Although no spontaneous flipping out transitions of the damaged bases were observed, the simulations showed significant differences in the conformational states of regular and CPD damage DNA. The isolated damaged DNA adopted transient conformations which resembled the global shape of the repair enzyme bound conformation more closely compared to regular B‐DNA. In particular, these conformational changes were observed in most of helical and structural parameters where the protein bound DNA differs drastically from regular B‐DNA. It is likely that the transient overlap of isolated DNA with the enzyme bound DNA conformation plays a decisive role for the specific and rapid initial recognition by the repair enzyme prior to the looping out process of the damaged DNA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 215–222, 2015.     

Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis

Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.     

MONITORING ULTRAVIOLET-B-INDUCED DNA DAMAGE IN INDIVIDUAL DIATOM CELLS BY IMMUNOFLUORESCENT THYMINE DIMER DETECTION1

We developed a method to investigate the effect of ultraviolet-B radiation (UVBR) on the formation of thy-mine dimers in microalgal DNA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer-specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance levels. Thy-mine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR-induced DNA damage on a cell-to-cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.     

Echinostome metacercariae cyst elimination in Rana clamitans (green frog) tadpoles is age-dependent

Host response to parasite exposure is an important determinant of the outcome of host-parasite interactions. Factors such as host body condition and age can strongly influence host response to parasites and infection. This study followed Echinostoma revolutum infection levels in larval green frogs (Rana clamitans) exposed at 2 different ages. Tadpoles at early developmental stages are more susceptible to the adverse effects of echinostomes. Green frog tadpoles approximately 2 wk apart in age and of the same developmental stage can exhibit dramatically different responses to echinostome infection, with the younger tadpoles having high rates of mortality and the older tadpoles experiencing no mortality. The goal of the present study was to begin to explore the mechanism underlying the striking age-dependent response of tadpoles to echinostomes. I conducted 2 controlled infection laboratory experiments in which tadpoles were preserved at 6 time points ranging from 4 hr to 1 wk postinfection (PI). Tadpoles infected at the younger age did not eliminate echinostome metacercariae. However, tadpoles that were 13 days older at the time of echinostome exposure steadily eliminated metacercariae during the hours and days PI. The absence of echinostome cyst elimination in the younger tadpoles likely contributes to their elevated, infection-induced mortality rates.     

Cis-syn thymidine dimer repair by DNA photolyase in real time

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.