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.     

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

The Photorepair and Photoisomerization of DNA Lesions in Etiolated Cucumber Cotyledons after Irradiation by UV-B Depends on Wavelength

To examine the light-dependent repair of DNA photo-lesions,such as, cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyriinidonephotoproducts [(6-4)pho-toproducts], we incubated cucumber cotyledonsunder monochromatic light (325-500 nm) after they had been exposedto UV-B irradiation. Irradiation at wavelengths between 375and 425 nm was the most efficient for photo-repair of CPDs.The dependence on wavelength for the removal of (6-4)photoproductsdiffered from that for the photorepair of CPDs. The (6-4)photoproductswere most efficiently removed upon irradiation at 325 nm witha second peak at 400 to 425 nm. An immunological assay usingantibodies specific to Dewar photoproducts revealed that irradiationat 325 nm induced the photoisomerization of (6-4)photoproductsto Dewar photoproducts. Thus, the most efficient wavelengthfor the photorepair of (6-4)photoprod-ucts was between 400 and425 nm. It seems likely that processes similar to those involvedin photorepair of CPDs might be involved in the photorepairof (6-4)photoprod-ucts. (Received October 15, 1997; Accepted April 22, 1998)     

Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are the two main classes of mutagenic DNA damages induced by UVB radiation. Numerous studies have been devoted so far to their formation and repair in human cells and skin. However, the biochemical methods used often lack the specificity that would allow the individual study of each of the four CPDs and 6-4PPs produced at TT, TC, CT and CC dinucleotides. In the present work, we applied an HPLC-mass spectrometry assay to study the formation and repair of CPDs and 6-4PPs photoproducts in primary cultures of human keratinocytes and fibroblasts as well as in whole human skin. We first observed that the yield of dimeric lesions was slightly higher in fibroblasts than in keratinocytes. In contrast, the rate of global repair was higher in the last cell type. Moreover, removal of DNA photoproducts in skin biopsies was found to be slower than in both cultured skin cells. In agreement with previous works, the repair of 6-4PPs was found to be more efficient than that of CPDs in the three types of samples, with no observed difference between the removal of the TT and TC derivatives. In contrast, a significant influence of the nature of the two modified pyrimidines was observed on the repair rate of CPDs. The decreasing order of removal efficiency was the following: C<>T>C<>C>T<>C>T<>T. These data, together with the known intrinsic mutational properties of the lesions, would support the reported UV mutation spectra. A noticeable exception concerns CC dinucleotides that are mutational hotspots with an UV-specific CC to TT tandem mutation, although related bipyrimidine photoproducts are produced in low yields and efficiently repaired.     

Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB-irradiated Arabidopsis leaves

Replication of Arabidopsis nuclear, mitochondrial and chloroplast DNA (ncDNA, mtDNA, cpDNA) was assayed by measuring respective changes in copies per leaf, employing quantitative PCR (QPCR) analysis with genome-specific primer pairs. All three genomes showed parallel increases during growth of cotyledons and 5th leaves in planta, maintaining approximately 13 mtDNA copies and 280 cpDNA copies per haploid nuclear genome. Detached 5th leaves, which showed good growth and DNA replication on agar plates, were irradiated at (DNA-effective) UV-B fluences of 1.3-5.0 kJ m-2 and incubated under blue (photorepair-active) plus gold light or gold light only. Under blue light, replication of all genomes after all UV fluences was approximately as efficient as replication in unirradiated leaves. UV-irradiated leaves showed little growth under gold light only; 5 kJ m-2 stopped replication of all three genomes, 2.5 kJ m-2 stopped only cpDNA replication, and 1.3 kJ m-2 only delayed cpDNA replication. Immunoassays showed that 5 kJ m-2 induced about 1.2 cyclobutane pyrimidine dimers and 0.1 [6-4]photoproducts per kbp of bulk DNA, and that both photoproducts were completely removed during 2-3 days under blue light, suggesting efficient photorepair of at least ncDNA and cpDNA. The evidence for efficient photorepair of organellar DNA contrasts with previous studies of irradiated 5-day-old seedlings, and with the apparent absence of Arabidopsis photolyases bearing transit peptides.     

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant forRAD14, theSaccharomyces cerevisiae homologue of the human gene defective inXPA patients

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in theMAT andHML loci ofSaccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in arad14-2 point mutant and in arad14 deletion mutant. TheRAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III sites (i.e. as wild type), but no removal of CPDs at theMAT andHML loci. Similar experiments were undertaken using the recently createdrad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired inMAT a orHMR a. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that, in the genome overall, repair of 6-4 photoproducts was less impaired than repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that therad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.     

Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli.

We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.     

Changes in UV-B radiation screening effectiveness with leaf age in Rhododendron maximum

We examined how ultraviolet-B radiation (UV-B; 300 nm) screening effectiveness changes with leaf age in Rhododendron maximum growing in a shaded understory by measuring depth of penetration and epidermal transmittance with a fibre-optic microprobe. Depth of penetration (and epidermal transmittance) of UV-B decreased with leaf age in 1- to 4-year-old leaves, averaging 62 (32), 52 (22), 45 (16) and 48 μm (13%), respectively. Epidermal thickness increased with age in 1- to 4-year-old leaves due to a thickening of the cuticle from an average of 20 to 29μm. Ultraviolet-B-absorbing compound concentrations increased with age from 1–3 to 1–5 A₃₀₀ cm^?2 leaf area. Concentrations of UV-B-absorbing compounds (area basis) were a strong predictor of depth of penetration (r²= 0.82) and epidermal transmittance (r²= 0.95) of UV-B in mature (1–4 year-old) foliage. Chlorophyll concentrations (area basis) increased in leaves up to 3 years of age. Current-year leaves (30 d old) were exceptional in that while they were particularly effective at screening UV-B (depth of penetration and epidermal transmittance averaged 39μm and 5%, respectively) they had relatively low concentrations of UV-B-absorbing compounds (1.3 A₃₀₀ cm^?2). Our findings show that UV-B-screening effectiveness is not necessarily related to absorbing compound concentrations on a whole-leaf basis, possibly due to anatomical changes within the epidermis that occur with leaf age.     

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant forRAD14, theSaccharomyces cerevisiae homologue of the human gene defective inXPA patients

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in theMATα andHMLα loci ofSaccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in arad14-2 point mutant and in arad14 deletion mutant. TheRAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III sites (i.e. as wild type), but no removal of CPDs at theMATα andHMLα loci. Similar experiments were undertaken using the recently createdrad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired inMAT a orHMR a. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that, in the genome overall, repair of 6-4 photoproducts was less impaired than repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that therad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.     

Responses to ultraviolet-B radiation (280–315 nm) of pea (Pisum sativum) lines differing in leaf surface wax

To test the hypothesis that leaf surface wax influences plant responses to UV-B, 6 lines of cultivated pea (Pisum sativum L.), selected as having more or less wax, were grown at 0 or 6.5 kJ m^-2 day^-1 plant-weighted UV-B against a background of 850–950 μmol m^-2 s^-1 photosynthetically active radiation. In the 4 lines with least leaf surface wax the amount of wax on adaxial and abaxial leaf surfaces was increased following exposure to 6.5 kJ m^-2 day^-1 UV-B, but UV-B decreased surface wax in Scout, which had the greatest wax deposits. On the adaxial leaf surface, UV-B radiation caused a shift in wax composition from alcohols to esters and hydrocarbons and the ratio of short to long chain length alkyl ester homologues was increased. There was no evidence of a shortening in carbon chain length of hydrocarbons, primary alcohols or fatty acids due to UV-B and no significant correlation between wax amount and UV reflectance from leaves. UV-B induced significant increases in UV-absorbing compounds in the expanded leaves and buds of most lines. UV-B reduced the growth of all lines. Foliage area (leaves plus stipules) declined by 5–30%, plant dry weight by 12–30%, and plant height by 24–38%. Reductions in growth occurred in the absence of any changes in chlorophyll fluorescence or photosynthetic rate. UV-B also had no major effect on carbon allocation patterns. The effects of UV-B on growth appeared to be due to changes in tissue extension and expansion. Indeed, many of the responses to UV-B observed in this study of pea appear more consistent with indirect effects being expressed in developing tissues rather than through the direct action of UV-B on mature tissues. There was no evidence that wax amount or biochemistry was associated with the sensitivity of the lines to UV-B radiation. Furthermore, induction of pigments was not correlated with changes in growth. However, lines with the greatest constitutive amounts of pigments in unexpanded bud tissues were most tolerant of elevated UV-B.     

Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

温度对UV-B诱导的烟草叶圆片DNA损伤的影响

环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4PP)是两种主要的UV-B诱导的DNA光损伤产物。利用单克隆抗体酶联免疫吸附分析法(ELISA),研究了温度对UV-B诱导的烟草叶圆片DNA损伤的影响。室温(24℃)条件下,UV-B处理引起了烟草叶圆片DNA中CPD和6-4PP的积累。0℃条件下,UV-B处理的烟草叶圆片DNA中CPD和6-4PP的积累比室温下分别降低了9.8%和12%。UV-B诱导的DNA损伤曾被认为是纯粹的光化学过程而与不受温度影响,而本实验结果表明,UV-B诱导的烟草叶圆片DNA形成CPD和6-4PP的过程具有温度依赖性。这一特性有利于植物对全球变化的适应,因而具有重要的生态学意义。     

Powerful skin cancer protection by a CPD-photolyase transgene

BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Developmental phase-dependent photosynthetic responses to ultraviolet-B radiation: damage, defence, and adaptation of primary leaves of wheat seedlings

Alterations in photosynthetic capacity of primary leaves of wheat seedlings in response to ultraviolet-B (UV-B; 280–320 nm; 60 μmol m⁻² s⁻¹) exposure alone and in combination with photosynthetically active radiation (PAR; 400–800 nm; 200 μmol m⁻² s⁻¹) during different phases of leaf growth and development were assessed. UV-B exposure resulted in a phase-dependent differential loss in photosynthetic pigments, photochemical potential, photosystem 2 (PS2) quantum yield, and in vivo O₂ evolution. UV-B exposure induced maximum damage to the photosynthetic apparatus during senescence phase of development. The damages were partially alleviated when UV-B exposure was accompanied by PAR. UV-B induced an enhancement in accumulation of flavonoids during all phases of development while it caused a decline in anthocyanin content during senescence. The differential changes in these parameters demonstrated the adaptation ability of leaves to UV-B stress during all phases of development and the ability was modified in UV-B+ PAR exposed samples.