Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.     

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.     

Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay

Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.     

Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.     

Sensitivity of rice to ultraviolet-B radiation

BACKGROUND: Depletion of the stratospheric ozone layer leads to an increase in ultraviolet-B (UVB: 280-320 nm) radiation reaching the earth's surface, and the enhanced solar UVB radiation predicted by atmospheric models will result in reduction of growth and yield of crops in the future. Over the last two decades, extensive studies of the physiological, biochemical and morphological effects of UVB in plants, as well as the mechanisms of UVB resistance, have been carried out. SCOPE: In this review, we describe recent research into the mechanisms of UVB resistance in higher plants, with an emphasis on rice (Oryza sativa), one of the world's most important staple food crops. Recent studies have brought to light the following remarkable findings. UV-absorbing compounds accumulating in the epidermal cell layers have traditionally been considered to function as UV filters, and to play an important role in countering the damaging effects of UVB radiation. Although these compounds are effective in reducing cyclobutane pyrimidine dimer (CPD) induction in plants exposed to a challenge exposure to UVB, certain levels of CPD are maintained constitutively in light conditions containing UVB, regardless of the quantity or presence of visible light. These findings imply that the systems for repairing DNA damage and scavenging reactive oxygen species (ROS) are essential for plants to grow in light conditions containing UVB. CONCLUSION: CPD photolyase activity is a crucial factor determining the differences in UVB sensitivity between rice cultivars. The substitution of one or two bases in the CPD photolyase gene can alter the activity of the enzyme, and the associated resistance of the plant to UVB radiation. These findings open up the possibility, in the near future, of increasing the resistance of rice to UVB radiation, by selective breeding or bioengineering of the genes encoding CPD photolyase.     

Transient induction of photolyase activity in arrested frog cells in response to a short-wave ultraviolet segment of simulated "sunlight"

Induction of photolyase activity was studied in cultured frog cells using clonogenic assays. Exposure of arrested cells to a pre-irradiation (90% survival) of 254 nm ultraviolet light resulted in a transient enhancement of photolyase activity. Cells expressed a decreased level of photolyase activity in response to an equitoxic fluence of simulated "sunlight" wavelengths 280-310 nm. However, no significant increase of enzyme activity was detected in cells following treatment with "sunlight" wavelengths 310-330 nm. In addition, this process depends on newly biosynthesized protein(s).     

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.     

Spontaneously occurring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice

Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering.     

Dependence on Wavelength and Temperature of Growth Inhibition Induced by UV-B Irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumissativus L.) were cultured in vitro at 20C under visible andUV light. Various UV spectra were obtained by using a seriesof cut-off filters that had different transmittance properties.Growth of cotyledons was inhibited by irradiation at wavelengthsshorter than 320 nm and was not affected by irradiation at longerwavelengths. The synthesis of Chl in the cotyledons was stronglyinhibited by irradiation at 280–300 nm and slightly inhibitedby irradiation at 300–320 nm. Inhibition induced by irradiationat 280–300 nm was not affected by temperature, but theextent of inhibition by irradiation at 300–320 nm wasreduced at 25C. This dependence on temperature of the effectof UV light was also observed in the inhibition of photosynthesis.These results indicate that the mode of inhibition by UV lightat 280–300 nm may be different from that induced by UVlight at 300–320 nm and that two or more mechanisms maybe involved in the harmful effects of UV-B radiation. (Received May 10, 1993; Accepted June 15, 1993)     

UVB sensitivity and cyclobutane pyrimidine dimer (CPD) photolyase genotypes in cultivated and wild rice species.

We investigated the UVB-sensitivity in 12 rice strains belonging to two cultivated species (O. sativa and O. glaberrima) and three wild species (O. barthii, O. meridionalis and O. rufipogon) of rice possessing the AA genome, while focusing on the CPD photolyase activity and the genotypes of CPD photolyase. Although the UVB sensitivity, CPD photolyase activity, and CPD photolyase genotype varied widely among these rice species, the sensitivity to UVB radiation depended on the activity of the CPD photolyase, regardless of grass shape, habitat, or species. The rice strains examined here clearly divided into three groups based on the CPD photolyase activity, and the activity of the strains greatly depended on amino acid residues at positions 126 and 296, with the exception of the W1299 strain (O. meridionalis). The amino acid residues 126 and 296 of CPD photolyase in Sasanishiki strain (O. sativa), which showed higher enzymatic activity and more resistance to UVB, were glutamine (Gln) and Gln, respectively. An amino acid change at position 126 from Gln to arginine ("Nori"-type) in the photolyase led to a reduction of enzymatic activity. Additionally, an amino acid change at position 296 from Gln to histidine led to a further reduction in activity. The activity of the W1299 strain, which possesses a "Nori"-type CPD photolyase, was the highest among the strains examined here, and was similar to that of the Sasanishiki. The CPD photolyase of the W1299 contains ten amino acid substitutions, compared to Sasanishiki. The alterations in amino acid residues in the W1299 CPD photolyase compensated for the reduction in activity caused by the amino acid substitutions at positions 126. Knowledge of the activity of different CPD photolyase genotypes will be useful in developing improved rice cultivars.     

qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F(2) plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene.     

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.     

Photogeneration of the superoxide anion oxygen radical and its role in the inactivation of yeast cells by long-wave UV light

Long-wave (320-400 nm) UV-induced oxygen superoxide anion radical (O2-) formation was found in yeast cells. This radical plays an important part in initiation of photodestruction reactions in DNA which serves as a main target of UV irradiation in yeast. The observed cell photoinactivation spectrum at the wavelengths 320-400 nm suggests that NADH can serve as an endogenous sensitizer of O2- formation.     

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.     

Investigation of Real-Time Photorepair Activity on DNA via Surface Plasmon Resonance

The cyclobutane pyrimidine dimer (CPD) and 6-4 lesion formations along with the specific breaks on strands are the most common type of DNA damage caused by Ultraviolet light (UV) irradiation. CPD photolyase I and II construct two subfamilies of flavoproteins, which have recognition and repair capabilities of CPD sites on both single stranded (ssDNA) and double stranded DNA (dsDNA) with the aid of blue light energy. The other types of flavoprotein family consist of cryptochromes (CRY) that act as photoreceptors in plants, or circadian rhythm regulators in animals. Recent findings showed that a specific type of Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (CRY-DASH) has photorepair activity on ssDNA. In this work, real-time interactions between CRY-DASH and ss/dsDNA as well as the interactions between Vibrio cholerae photolyase (VcPHR) and ss/dsDNA were investigated using Surface Plasmon Resonance (SPR). The interactions were then characterized and compared in order to investigate the effect of different types of flavoprotein on UV damaged ss/dsDNA. SPR results confirm the specific binding of VcPHR and CRY-DASH with UV treated DNA. This study is the first instance to quantify the interactions of UV treated and untreated DNA with flavoproteins.     

UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.     

Ultraviolet-B sensitivities in Japanese lowland rice cultivars: cyclobutane pyrimidine dimer photolyase activity and gene mutation

There is a cultivar difference in the response to ultraviolet-B(UVB: 280–320 nm) in rice (Oryza sativa L.). AmongJapanese lowland rice cultivars, Sasanishiki, a leading Japaneserice cultivar, is resistant to the damaging effects of UVB whileNorin 1, a close relative, is less resistant. We found previouslythat Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD)photorepair ability and suggested that the UVB sensitivity inrice depends largely on CPD photorepair ability. In order toverify that suggestion, we examined the correlation betweenUVB sensitivity and CPD photolyase activity in 17 rice cultivarsof progenitors and relatives in breeding of UV-resistant Sasanishikiand UV-sensitive Norin 1. The amino acid at position 126 ofthe deduced amino acid sequence of CPD photolyase in cultivarsincluding such as Norin 1 was found to be arginine, the CPDphotolyase activities of which were lower. The amino acid atthat position in cultivars including such as Sasanishiki wasglutamine. Furthermore, cultivars more resistant to UVB werefound to exhibit higher photolyase activities than less resistantcultivars. These results emphasize that single amino acid alterationfrom glutamine to arginine leads to a deficit of CPD photolyaseactivity and that CPD photolyase activity is one of the mainfactors determining UVB sensitivity in rice. ¹ These authors contributed equally to the paper. ² Corresponding author: E-mail, kumagai{at}ige.tohoku.ac.jp; Fax,+81-22-217-5691.