Photolyase Activity Research Articles

Crystal structure of cryptochrome 3 from Arabidopsis thaliana and its implications for photolyase activity.

Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Natl Acad Sci USA 103:17696-17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 A resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-binding cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

Photolyases and cryptochrome blue-light photoreceptors are evolutionarily related flavoproteins that perform distinct functions. Photolyases repair UV-damaged DNA in many species from bacteria to plants and animals. Cryptochromes regulate growth and development in plants and the circadian clock in animals. Recently, a new branch of the photolyase/cryptochrome family was identified. Members of this branch exhibited no or trace levels of DNA repair activity in vivo and in vitro and, therefore, were considered to be cryptochromes, and they were named cryptochrome-DASH. Here, we show that Cry-DASH proteins from bacterial, plant, and animal sources actually are photolyases with high degree of specificity for cyclobutane pyrimidine dimers in ssDNA.

Towards Elucidation of the Mechanism of UV1C, a Deoxyribozyme with Photolyase Activity

Among the unexpected chemistries that can be catalyzed by nucleic acid enzymes is photochemistry. We have reported the in vitro selection of a small, cofactor-independent deoxyribozyme, UV1C, capable of repairing thymine dimers in a DNA substrate, most optimally with light at a wavelength of >300 nm. We hypothesized that a guanine quadruplex functioned both as a light antenna and an electron source for the repair of the substrate within the enzyme–substrate complex. Here, we report structural and mechanistic investigations of that hypothesis. Contact-crosslinking and guanosine to inosine mutational studies reveal that the thymine dimer and the guanine quadruplex are positioned close to each other in the deoxyribozyme–substrate complex, and permit us to refine the structure and topology of the folded deoxyribozyme. In exploring the substrate utilization capabilities of UV1C, we find it to be able to repair uracil dimers as well as thymine dimers, as long as they are present in an overall deoxyribonucleotide milieu. Some surprising similarities with bacterial CPD photolyase enzymes are noted.

Photolyase Confers Resistance to UV Light but Does Not Contribute to the Symbiotic Benefit of Bioluminescence in Vibrio fischeri ES114

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark Delta luxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.

Sensitivity of rice to ultraviolet-B radiation.

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. 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. 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.

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

Bioluminescence-mediated stimulation of photoreactivation in bacteria

Although biochemistry and genetics of light emission by cells have been investigated in detail, a biological role for bacterial luminescence has remained obscure for a long time. It was proposed recently that luminescence may stimulate DNA repair, but the specific mechanism of this phenomenon was not investigated. Moreover, experiments showing decreased survival of UV-irradiated lux mutants relative to luminescent cells were performed previously using only one bacterial species, Vibrio harveyi. Here, we demonstrate that dark mutants of various strains of naturally luminescent bacteria ( Photobacterium leiognathi, Photobacterium phosphoreum and Vibrio fischeri) are more sensitive to UV irradiation than wild-type cells. Thus, this phenomenon occurs not only in V. harveyi but also in other bacterial species. Using an artificial system of luminescent Escherichia coli in combination with phr mutants (defective in photolyase functions), we found that bacterial luminescence may stimulate photoreactivation, perhaps by providing photons that are necessary for photolyase activity.

ピリミジン二量体光回復酵素活性の高低が、イネのUVB感受性の程度を決定する主要因である－UVB感受性の異なるイネを用いた遺伝解析－

ピリミジン二量体光回復酵素活性の高低が、イネのUVB感受性の程度を決定する主要因である－UVB感受性の異なるイネを用いた遺伝解析－

(6-4)-Photolyase activity requires a charge shift reaction

A model compound containing a thymine oxetane moiety linked to a flavin chromophore was investigated regarding (6-4)-photolyase activity. The need for a charge shift reaction was demonstrated by a detailed pH-dependent kinetic analysis.

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.). Among Japanese lowland rice cultivars, Sasanishiki, a leading Japanese rice cultivar, is resistant to the damaging effects of UVB while Norin 1, a close relative, is less resistant. We found previously that Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD) photorepair ability and suggested that the UVB sensitivity in rice depends largely on CPD photorepair ability. In order to verify that suggestion, we examined the correlation between UVB sensitivity and CPD photolyase activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki and UV-sensitive Norin 1. The amino acid at position 126 of the deduced amino acid sequence of CPD photolyase in cultivars including such as Norin 1 was found to be arginine, the CPD photolyase activities of which were lower. The amino acid at that position in cultivars including such as Sasanishiki was glutamine. Furthermore, cultivars more resistant to UVB were found to exhibit higher photolyase activities than less resistant cultivars. These results emphasize that single amino acid alteration from glutamine to arginine leads to a deficit of CPD photolyase activity and that CPD photolyase activity is one of the main factors determining UVB sensitivity in rice.

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate

Assay method for Escherichia coli photolyase activity using single-strand cis- syn cyclobutane pyrimidine dimer DNA as substrate

A high-performance liquid chromatography method for the assay of Escherichia coli photolyase activity was developed. When cis- syn cyclobutane pyrimidine dimer was used as substrate, the Michaelis constant ( K m) value for the photolyase activity was 100 nM. The linear range of the calibration curve of the photolyase activity was 0.026–6.64 μU/assay tube. The correlation coefficient for this linearity was 0.998. The limit of detection ( S/ N=3) was 26 nU/assay tube. The photolyase activity was increased 1.6-fold in the presence of 5,10-methenyltetrahydrofolic acid in the enzyme reaction mixture.

Identification of cryptochrome DASH from vertebrates.

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp. PCC 6803, Vibrio cholerae, and Arabidopsis thaliana. The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated. To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases. We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa. We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians. We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences. The proteins encoded by the two genes were purified and characterized. Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity. A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily. This is the first report of the CRY-DASH members from vertebrates and fungi.

An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles.

Cryptochromes (CRYs) are blue/UV-A photoreceptors related to the DNA repair enzyme DNA photolyase. They have been found in plants, animals and most recently in the cyanobacterium Synechocystis. Closely related to the Synechocystis cryptochrome is the Arabidopsis gene At5g24850. Here, we show that the encoded protein of At5g24850 binds flavin adenine dinucleotide (FAD). It has no photolyase activity, and is likely to function as a photoreceptor. We have named it At-cry3 to distinguish it from the other Arbabidopsis cryptochrome homologues At-cry1 and At-cry2. At-cry3 carries an N-terminal sequence, which mediates import into chloroplasts and mitochondria. Furthermore, we show that At-cry3 binds DNA. DNA binding was also demonstrated for the Synechocystis cryptochrome, indicating that both photoreceptors could have similar modes of action. Based on the finding of a new cryptochrome class in bacteria and plants, it has been suggested that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. However, our phylogenetic analyses are also consistent with an alternative explanation that the presence of cryptochromes in the plant nuclear genome is the result of dual horizontal gene transfer. That is, CRY1 and CRY2 genes may originate from an endosymbiotic ancestor of modern-day alpha-proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern-day cyanobacteria.

A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification.

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.

Cleavable Substrate Containing Molecular Beacons for the Quantification of DNA-Photolyase Activity

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U-type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA-photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts.

Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U-type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA-photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts.

Photolyase activity of the embryo and the ultraviolet absorbance of embryo jelly for several Ontario amphibian species

Organisms whose eggs develop at or near the interface between air and water may be particularly vulnerable to damage from ultraviolet radiation. The primary form of ultraviolet B (UV-B) radiation damage to biological systems is the formation of cyclobutane pyrimidine dimers (CBPDs) in DNA. The most common method of repairing this damage is photoenzymatic repair via photolyase, whose actions are specific to CBPDs. We utilized a bacterial-transformation assay to estimate the level of photolyase activity of various tissue types in seven species of amphibians collected in south-central Ontario. In this assay, the photolyase activity of a species is measured as the rate of CBPD removal from UV-B-damaged plasmid DNA by cell-free extracts created from the tissue of the species in question. The depth of oviposition and the UV-B absorbance of the embryo jelly, two variables that alter an embryo's in situ exposure to UV-B radiation, were measured to determine whether the level of photolyase activity was correlated with expected UV-B exposure. In vitro measurements of photolyase activity for the seven species were significantly different (F[6]= 10.31, p < 0.0001) and tended to be positively correlated with expected in vivo exposure to UV-B radiation.

DNA-repair by Photolyase Reveals Dynamic Properties of Nucleosome Positioning in Vivo

Nucleosomes exert a repressive influence on the biological functions of DNA by restricting the access of proteins to DNA. To investigate how intrinsic properties of nucleosomes modulate DNA-accessibility in vivo, we studied DNA repair by photolyase in the yeast URA3 gene. Formation of DNA lesions (cyclobutane pyrimidine dimers, CPDs) and photolyase activity are controlled precisely by light. Preceding work revealed that photolyase repairs nucleosome-free DNA rapidly, while repair of nucleosomes is inhibited severely. The high-resolution data presented here show slow repair in the center of nucleosomes and a gradual increase towards the periphery. This pattern was observed in all nucleosomes and demonstrates that dynamic properties facilitate DNA accessibility. Since the URA3 nucleosomes can occupy alternate positions, the repair data are most consistent with nucleosome mobility that moves CPDs in linker DNA where they are repaired rapidly. A partial and transient unfolding or disruption of nucleosomes, however, may not be excluded. In addition, repair heterogeneity was found between closely spaced sites, indicating that structural properties of nucleosomes contribute to damage processing. Moreover, nucleosome-specific modulation of photolyase was found on the transcribed and non-transcribed strand. This is in contrast to homogeneous repair of the transcribed strand by nucleotide excision repair, and reveals fundamental differences in how both repair systems interact with nucleosomes and transcription.

Ultraviolet damage and counteracting mechanisms in the freshwater copepod Boeckella poppei from the Antarctic Peninsula

The process of ozone depletion over the Antarctic continent has resulted in the increase of incident ultraviolet‐B (UVB) radiation, whose effects may be damaging to living organisms. To counteract the negative effects of ultraviolet radiation (UVR), aquatic organisms may display one or more strategies: (1) avoidance (i.e. deep distribution); (2) photoprotection through the use of “sunscreen” compounds, such as mycosporine‐like amino acids (MAAs); and (3) enzymatic repair of the damage. The effects of UVR were assessed on four populations of the copepod Boeckella poppei from Antarctic lakes using laboratory and field experiments. The results were related to measurements of DNA enzymatic repair activity and MAA concentration. This is the first study that combines these measurements in zooplankton. Boeckella poppei was highly tolerant to UVR (LD50 = 2.2–2.78 J cm−2). However, measurements of photorecovery (comparison of UVB mortality in the presence and absence of photoreactivating light) and dosage of photolyase activity indicated low rates of enzymatic repair, which may be the result of low temperatures typical of Antarctic lakes. Three different MAAs were identified, both in phytoplankton and copepods: porphyra‐334, mycosporine‐glycine, and shinorine. The population of B. poppei from Lake Boeckella had the lowest MAA concentration, as well as the lowest tolerance to artificial and natural UVR. These findings support the idea that UV tolerance in this species is related to the accumulation of MAAs. A comparison of the strategies used to cope with potentially damaging levels of UVR by different species of Boeckella indicates a high degree of plasticity in this genus, which has probably been key for its success to colonize a wide range of UV environments.