UV-C irradiation compromises conidial germination, formation of appressoria, and induces transcription of three putative photolyase genes in the barley powdery mildew fungus, Blumeria graminis f. sp. hordei.

UV-C irradiation is known to compromise germination of Blumeria graminis conidia and to reduce powdery mildew infestation. However, only scarce information is available on the effects of UV-C irradiation on B. graminis appressorium formation. Applying a Formvar® resin-based in vitro system allowed for analyzing B. graminis germination and appressorium formation in absence of plant defense. UV-C irradiation more strongly affected the differentiation of appressoria than conidial germination. In vivo and in vitro, a single dose of 100 J m-2 UV-C was sufficient to reduce germination to less than 20 % and decrease appressorium formation to values below 5 %. UV-C irradiation negatively affected pustule size and conidiation. White light-mediated photoreactivation was most effective immediately after UV-C irradiation, indicating that a prolonged phase of darkness after UV-C treatment increases the efficacy of B. graminis control. UV-C irradiation increased transcript levels of three putative B. graminis photolyase genes, while mere white light or blue light irradiation did not contribute to the transcriptional up-regulation. Thus, UV-C irradiation effectively controls B. graminis infestation and proliferation by restricting prepenetration processes. Nevertheless, photoreactivation plays an important role in UV-C-based powdery mildew control in crops and hence has to be considered for planning specific irradiation schedules.

The first (6-4) photolyase with DNA damage repair activity from the Antarctic microalga Chlamydomonas sp. ICE-L.

The psychrophilic microalga, Chlamydomonas sp. ICE-L, isolated from floating ice in the Antarctic, one of the most highly UV exposed ecosystems on Earth, displays an efficient DNA photorepair capacity. Here, the first known (6-4) photolyase gene (6-4CiPhr) from C. sp. ICE-L was identified. The 6-4CiPhr encoded 559-amino acid polypeptide with a pI of 8.86, and had a predicted Mw of 64.2 kDa. Real-time PCR was carried out to investigate the response of 6-4CiPhr to UVB exposure. The transcription of 6-4CiPhr was up-regulated continuously within 6 h, achieving a maximum of 62.7-fold at 6 h. Expressing 6-4CiPhr in a photolyase-deficient Escherichia coli strain improved survival rate of the strain. In vitro activity assays of purified protein demonstrated that 6-4CiPhr was a photolyase with 6-4PP repair activity. These findings improve understanding of photoreactivation mechanisms of (6-4) photolyase.

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m- 2) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.

UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase.

Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.

The phrA gene of Rhodobacter sphaeroides encodes a photolyase and is regulated by singlet oxygen and peroxide in a sigma(E)-dependent manner.

The genome of the facultatively photosynthetic bacterium Rhodobacter sphaeroides encodes three proteins of the photolyase/cryptochrome family. This paper shows that phrA (RSP2143) encodes a functional photolyase, which is an enzyme that repairs UV radiation-induced DNA damage in a blue light dependent manner. Expression of phrA is upregulated in response to light, with no photoreceptor or the photosynthetic electron transport being involved. The results reveal that singlet oxygen and hydrogen peroxide dependent signals are transmitted by the sigma(E) factor and the anti-sigma(E) factor ChrR affecting phrA expression, while superoxide anions do not stimulate phrA expression. Thus, the sigma(E) regulon is involved not only in the response to singlet oxygen but also in the hydrogen peroxide response.

Induction and inhibition of cyclobutane pyrimidine dimer photolyase in etiolated cucumber (Cucumis sativus) cotyledons after ultraviolet irradiation depends on wavelength.

Under polychromatic ultraviolet (UV) irradiation (maximum energy at 327 nm) the activity of DNA photolyase specific to cyclobutane pyrimidine dimers (CPDs), CPD photolyase, increased by an amount which depended on UV irradiance, and the level of CPD photolyase gene (CsPHR) transcripts temporarily increased before the activity reached a constant level. UV light (>320 nm) was more effective than visible light at increasing CPD photolyase activity. In contrast, monochromatic UV irradiation at wavelengths <300 nm increased the level of CsPHR transcripts similarly to irradiation at wavelengths >320 nm, but reduced CPD photolyase activity compared with the dark control. Exposure of a CPD photolyase solution to UV-C (254 nm) reduced enzyme activity and induced accumulation of H(2)O(2). Addition of H(2)O(2) to the enzyme solution also inactivated CPD photolyase activity. These results suggest the possibility that reactive oxygen species participate in the inactivation of CPD photolyase in cotyledons exposed to UV irradiation of <300 nm.

Rapid accessibility of nucleosomal DNA in yeast on a second time scale.

Packaging DNA in nucleosomes and higher-order chromatin structures restricts its accessibility and constitutes a barrier for all DNA transactions including gene regulation and DNA repair. How and how fast proteins find access to DNA buried in chromatin of living cells is poorly understood. To address this question in a real time in vivo approach, we investigated DNA repair by photolyase in yeast. We show that overexpressed photolyase, a light-dependent DNA-repair enzyme, recognizes and repairs UV-damaged DNA within seconds. Rapid repair was observed in various nucleosomal regions of the genome including inactive and active genes and repressed promoters. About 50% of cyclobutane pyrimidine dimers were removed in 5 s, >80% in 90 s. Heterochromatin was repaired within minutes, centromeres were not repaired. Consistent with fast conformational transitions of nucleosomes observed in vitro, this rapid repair strongly suggests that spontaneous unwrapping of nucleosomes rather than histone dissociation or chromatin remodeling provides DNA access. The data impact our view on the repressive and dynamic nature of chromatin and illustrate how proteins like photolyase can access DNA in structurally and functionally diverse chromatin regions.

Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo.

Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification. The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.

BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride.

In fungi, phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. Due to its apparent simplicity, this response was chosen for pursuing photoreceptor isolation. Two genes were cloned, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, respectively. The BLR-1 protein has all the characteristics of a blue-light photoreceptor, whereas the structure of the deduced BLR-2 protein suggests that it interacts with BLR-1 through PAS domains to form a complex. Disruption of the corresponding genes demonstrated that they are essential for blue-light-induced conidiation. blr-1 and blr-2 were also shown to be essential for the light-induced expression of the photolyase-encoding gene (phr-1). Mechanical injury of mycelia was found to trigger conidiation of T. atroviride, a response not described previously. This response was not altered in the mutants. A novel effect of both red and blue light on mycelial growth was found involving another light receptor, which is compensated by the BLR proteins.

Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis.

The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two-dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three-fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post-translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild-type and the deletion mutants, CAMB43 (deltapra1) and CAS10 (deltaphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1-deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post-translational level.

Induction of photolyase activity in wood frog (Rana sylvatica) embryos.

Rising ultraviolet-B (UVB, 280-320 nm) radiation has been proposed as a factor which may explain nonnormal amphibian population declines. Accordingly research has been directed toward estimating the photolyase activity of several amphibian species in order to predict a species' resilience to UV damage. Unfortunately, in spite of published research which demonstrated that the activity of one of the principal photorepair enzymes, photolyase, can be induced, these estimates did not address the potential for in vivo induction by environmental factors present in situ. We show here that wood frog (Rana sylvatica) embryos exposed to periods of ambient solar radiation (1) displayed significantly different photolyase activities from embryos exposed to equivalent periods of dark; and (2) were positively correlated with the UVB fluence received in vivo. Such results suggest that previous conclusions regarding the relationship between photorepair and population decline must be reevaluated. Estimating amphibian photorepair is a complicated process, and caution must be exercised when interpreting such data.

The TRAP-like SplA protein is a trans-acting negative regulator of spore photoproduct lyase synthesis during Bacillus subtilis sporulation.

UV resistance of bacterial endospores derives from a unique DNA photochemistry in which the major UV photoproduct is the thymine dimer 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]) instead of cyclobutane pyrimidine dimers. Repair of SP during spore germination is due in large part to the activity of the enzyme SP lyase encoded by splB, the second cistron of the splAB operon. Expression of the splAB operon in Bacillus subtilis is transcriptionally activated by the Esigma(G) form of RNA polymerase during morphological stage III in the developing forespore compartment, and SP lyase is packaged into the dormant spore. In addition to temporal and compartmental control of splAB expression, a second regulatory circuit which modulates the level of expression of splB-lacZ fusions without altering their developmental timing or compartmentalization is reported here. This second regulatory circuit involves the negative action of the splA gene product, a 79-amino-acid protein with approximately 50% similarity and 17% identity to TRAP, the tryptophan RNA-binding attenuation protein from B. subtilis and Bacillus pumilus.

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase.

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.

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.

Influence of phage proteins on formation of specific UV DNA photoproducts in phage T7.

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.

Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.

Photoreactivating enzyme for (6-4) photoproducts in cultured goldfish cells.

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6-4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6-4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6-4) photoproducts and its dissociation from (6-4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of protein(s) to (6-4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA photoreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6-4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6-4) photolyase in cultured goldfish cells as in Drosophila, Xenopus and Crotalus.

Induction of cyclobutane pyrimidine dimer photolyase in cultured fish cells by UVA and blue light.

The cyclobutane pyrimidine dimer (CPD) photolyase in fish cells is known to be regulated by environmental factors, such as light, hydrogen peroxide and growth inhibition. The induction of CPD photolyase by light in cultured goldfish cells was dependent on the wavelength of the light, and UVA and blue light had high inductive activity. The spectrum for CPD photolyase activity was different from that for the induction. Treatment with blue or yellow light for a short time, which did not induce any CPD photolyase, induced high CPD photolyase activity in the presence of the photosensitizers, TPPS (monosulfonated meso-tetraphenyl porphine) and ALPS (aluminum phthalocyanine tetrasulfonate), respectively. These results suggest that the induction of CPD photolyase might be triggered by active oxygen produced by light and cellular photosensitizers. We also found that immediately after treatment with UVA, blue light or a photosensitizer in combination with light, cellular attachment to the substratum was enhanced, as was the CPD photolyase activity. Pretreatment with a flavonoid, quercetin, inhibited both photoinduction of CPD photolyase and enhancement of cellular attachment. Vitamin E inhibited only photoinduction of CPD photolyase activity. Treatment with H7, a strong inhibitor for protein kinase C, after light treatment inhibited photoinduction of CPD photolyase activity, but an analogue of H7, Ha1004, which is a weak inhibitor of protein kinase C, did not have such an effect.

Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors.

Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases. Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis. In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1. Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.

Overexpression of Medaka (Oryzias latipes) photolyase gene in Medaka cultured cells and early embryos.

To study the role and the regulation of the photolyase gene in the Medaka (small teleost), we constructed a eukaryotic expression plasmid of the Medaka photolyase gene and introduced it into Medaka cells in vivo and in vivo. The expression plasmid contains a cytomegalovirus enhancer and a thymidine kinase promoter to overexpress the photolyase gene of the Medaka. First, we transfected this construct into cultured Medaka cells and established several lines of transfectant. Every transfectant showed enhanced ability of pyrimidine dimer repair in the presence of fluorescent light. In the transfectant that showed the most enhanced ability of photorepair, the augmented transcription of photolyase gene was observed compared with that of progenitor OL32 cells. In this transfectant, we also observed an enhanced rate of UV survival with 20 min of fluorescent light treatment after irradiation with a 400 J/m2 UV sunlamp. Next, the expression construct was microinjected into the embryos of the Medaka at the one cell stage. Compared with the nontreated counterparts, the overexpression of a photolyase gene was detected in the microinjected embryos, but we failed to detect a significant increase in photo-reactivability of death at the midblastula stage.