A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA.

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair.

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.

Nucleotide excision repair by dual incisions in plants.

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.

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.

Role of the carbonyl group of the (6-4) photoproduct in the (6-4) photolyase reaction.

The (6-4) photoproduct, which is one of the major UV-induced DNA lesions, causes carcinogenesis with high frequency. The (6-4) photolyase is a flavoprotein that can restore this lesion to the original bases, but its repair mechanism has not been elucidated. In this study, we focused on the interaction between the enzyme and the 3' pyrimidone component of the (6-4) photoproduct and prepared a substrate analogue in which the carbonyl group, a hydrogen-bond acceptor, was replaced with an imine, a hydrogen-bond donor, to investigate the involvement of this carbonyl group in the (6-4) photolyase reaction. UV irradiation of oligodeoxyribonucleotides containing a single thymine-5-methylisocytosine site yielded products with absorption bands at wavelengths longer than 300 nm, similar to those obtained from the conversion of the TT site to the (6-4) photoproduct. Nuclease digestion, MALDI-TOF mass spectrometry, and the instability of the products indicated the formation of the 2-iminopyrimidine-type photoproduct. Analyses of the reaction and the binding of the (6-4) photolyase using these oligonucleotides revealed that this imine analogue of the (6-4) photoproduct was not repaired by the (6-4) photolyase, although the enzyme bound to the oligonucleotide with considerable affinity. These results indicate that the carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction. On the basis of these results, we discuss the repair mechanism.

Blue-light-independent activity of Arabidopsis cryptochromes in the regulation of steady-state levels of protein and mRNA expression.

Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and blue-light stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition. However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis, the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light, although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced. These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.

Effects of UV radiation on photolyase and implications with regards to photoreactivation following low- and medium-pressure UV disinfection.

Photolyase activity following exposure to low-pressure (LP) and medium-pressure (MP) UV lamps was evaluated. MP UV irradiation resulted in a greater reduction in photolyase activity than LP UV radiation. The results suggest that oxidation of the flavin adenine dinucleotide in photolyase may have caused the decrease in activity.

CPD photolyase gene from Spinacia oleracea: repair of UV-damaged DNA and expression in plant organs.

The UV-B radiation contained in solar radiation has deleterious effects on plant growth, development and physiology. Specific damage to DNA caused by UV radiation involves the cyclobutyl pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts. CPDs are repaired by CPD photolyase via a UV-A/blue light-dependent mechanism. The gene for the class II CPD photolyase has been cloned from higher plants such as Arabidopsis, cucumbers and rice. We isolated and characterized the cDNA and a genomic clone encoding the spinach class II CPD photolyase. The gene consisted of 3777 bases and 9 exons. The sequence of amino acids predicted from the nucleotide sequence of the cDNA of the gene was highly homologous to that of the higher plants listed above. When a photolyase-deficient Escherichia coli strain was transformed with the cDNA, photoreactivation activity was partially restored, by the illumination with photoreactivating light, resulting in an increased survival and decreased content of CPDs in the Escherichia coli genome. In both the male and female plants, the gene was highly expressed in leaves and flowers under the condition of 14-h light and 10-h dark cycle. The expression in the roots was quite low compared with the other organs.

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling.

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.

Quantitative evaluation of the parameters of bacterial photoreactivation after exposure to ultraviolet light and ionizing radiation.

The purpose was to compare quantitatively the parameters of photoreactivation of an ultraviolet (UV) light hypersensitive strain of Escherichia coli Bs-1 irradiated with UV light and ionizing radiation. In addition, to evaluate the influence of the different physical and chemical factors on the parameters of the photoreactivation kinetics of the bacterial cells exposed to ionizing radiation. Survival curves and kinetics of the photoreactivation were measured in E. coli Bs-1 cells exposed to UV light (254 nm) and ionizing radiations (gamma-rays of 137Cs, gamma-rays of 60Co and 25 MeV pulsed X-rays). A mathematical model describing the process of photoreactivation in terms of a decreasing effective dose was applied to the experimental data obtained here and that published by others to evaluate quantitatively the probability of photoreactivation and the irreversible component of the radiation damage. Both the rate and extent of photoreactivation decreased in the following order of inactivating agents: WUV light, pulsed X-ray beam, gamma-ray of 60Co and gamma-ray of 137Cs. However, the irreversible component of radiation damage increased with the same order of radiations whereas the probability of photoreactivation per unit time was independent of the kind of radiation. After exposure to 6 MeV photons, the parameters of photoreactivation were changed in the presence of caffeine or after irradiation in the presence of the radioprotective agent dithiothreitol. The independence of the probability of photoreactivation on the quality of radiation indicates the cells have the same ability to photoreactivate damage produced by different kinds of radiations and is an additional argument indicating that during ionizing radiation a UV-like damage can be produced. The decrease in the extent and the rate of photoreactivation with radiation quality is explained by the formation of irreversible damage rather than by the impairment of the photorecovery process itself. Chemical and physical factors influencing the relative contribution of ionization and excitation on the ionizing radiation effect could modify both the extent of the photoreactivation and the probability of the recovery per unit time. It is concluded that the mathematical approach used here may be useful to reveal some new relationships between the parameters of photoreactivation.

The efficiency of photolyase and indole complexes to repair DNA containing dimers of pyrimidine: a theoretical analysis of the electron transfer reactions.

We analyze the effects of competing reactions to the efficiency of enzymatic splitting of pyrimidine dimers formed in DNA by the incidence of ultraviolet radiation. This is accomplished with the aid of a formula that expresses the efficiency of the repair in terms of parameters that regulate the reaction rates for primary and for back long-range electron transfers taking place in the process. Comparison of experimental data with estimations on account of this formula supports early conjectures in the literature that attribute the relative high performance of the enzymatic complexes of photolyase to its ability to suppress the back reaction.

Protective role of HSP27 against UVC-induced cell death in human cells.

It is an intriguing problem whether heat shock proteins (HSPs) play a protective role in UVC-induced cell death in human cells, and the problem has not been solved. To search for the HSPs involved in UVC resistance, gene expression profiles using cDNA array were compared between UVC-sensitive human RSa cells and their UVC-resistant variant AP(r)-1 cells. The expression levels of heat shock protein 27 (HSP27) were lower in RSa cells than in AP(r)-1 cells. RSa cells transfected with sense HSP27 cDNA showed slightly lower sensitivity to UVC-induced cell death than the control cells transfected with a vector alone and much lower sensitivity than RSa cells transfected with the antisense HSP27 cDNA. Furthermore, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4)photoproducts) in the cells with the up-regulation of HSP27 were moderately elevated compared with those in the control cells, while those in the cells with down-regulation were remarkably suppressed. These results suggest that HSP27 is involved in the UVC-resistance of human cells, at least those tested, possibly via functioning in nucleotide excision repair.

Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an "inverse" deuterium isotope effect.

We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH(-) and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (k(H)/k(D)=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH(-) (electron transfer time constant tau(et) in the order of 10-50 micros), and one where electron transfer from FADH(-) (tau(et)=25 ms) is followed by reprotonation of the W306 anion.

The significance of the study about the biological effects of solar ultraviolet radiation using the Exposed Facility on the International Space Station.

It is believed that ultraviolet (UV) radiation from the sun participated in events related to the chemical evolution and birth of life on the primitive Earth. Although UV radiation would be also a driving force for the biological evolution of life on Earth, life space of the primitive living organisms would be limited in the UV-shielded place such as in the water at an early stage of the evolution of life. After the formation of stratospheric ozone layer through the production of oxygen by photoautotroph, living organisms were able to expand their domain from water to land. As a result, now, many kinds of living organisms containing human beings are flourishing on the ground. In the near future, increased transmission of harmful solar UV radiation may reach the Earth's surface due to stratospheric ozone layer depletion. In order to learn more about the biological effects of solar UV radiation with or without interruption by the ozone layer, the utilization of an Exposed Facility on the International Space Station is required. Experiments proposed for this facility would provide a tool for the scientific investigation of processes involved in the birth and evolution of life on Earth, and could also demonstrate the importance of protecting the Earth's future environment from future ozone layer depletion.

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation.

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH*) induces an electron transfer over approximately 15 A from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* <-- W382 <-- W359 <-- W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant tau approximately 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (tau approximately 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield ( approximately 0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (tau approximately 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

Repair of UV damage in Halobacterium salinarum.

Halobacterium is one of the few known Archaea that tolerates high levels of sunlight in its natural environment. Photoreactivation is probably its most important strategy for surviving UV irradiation and we have shown that both of the major UV photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, can be very efficiently repaired by photoreactivation in this organism. There are two putative photolyase gene homologues in the published genome sequence of Halobacterium sp. NRC-1. We have made a mutant deleted in one of these, phr2, and confirmed that this gene codes for a CPD photolyase. (6-4) photoproducts are still photoreactivated in the mutant so we are currently establishing whether the other homologue, phr1, codes for a (6-4) photolyase. We have also demonstrated an excision repair capacity that operates in the absence of visible light but the nature of this pathway is not yet known. There is probably a bacteria-type excision-repair mechanism, since homologues of uvrA, uvrB, uvrC and uvrD have been identified in the Halobacterium genome. However, there are also homologues of eukaryotic nucleotide-excision-repair genes ( Saccharomyces cerevisiae RAD3, RAD25 and RAD2 ) so there may be multiple repair mechanisms for UV damage in Halobacterium.

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair.

BACKGROUND/AIMS: Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. METHODS AND RESULTS: Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). CONCLUSIONS: Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.

Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Evasion of UVC-induced apoptosis by photorepair of cyclobutane pyrimidine dimers.

Cyclobutyl pyrimidine dimer (CPD) photolyase is known to reverse pyrimidine dimers specifically under illumination with visible light. OCP13, a Medaka cell line showing a high level expression of the gene for CPD photolyase, completely reversed pyrimidine dimers induced by 20 J/m2 UVC by 1 h of photorepair. When OCP13 cells were irradiated with 20 J/m2 UVC, morphological changes such as shrinkage of cells, distorted nuclear shape, and decrease in the number of nucleoli appeared 2 to 4 h after UVC irradiation. Thereafter, the irradiated cells began to detach from the substratum, and DNA ladders were observed in the DNA extracted from detached cells. Thus, these changes in cells after UVC exposure were used to characterize the progression of UV-induced apoptosis in OCP13 cells. Although formation of DNA ladders and cell detachment were blocked by cycloheximide treatment prior to UVC exposure, the morphological changes were not. With photorepair treatment, even after the morphological changes appeared cells were still able to restore their normal morphological features and remained attached. On the other hand, the cell-cycle progression in UVC-irradiated cells was arrested even after photorepair of pyrimidine dimers. Thus, photorepair can rescue cells from UV-induced apoptosis, although DNA damage other than that of pyrimidine dimers, as well as additional non-DNA damage, possibly remained, and DNA replication was left inhibited. Among the various kinds of damage induced by UVC irradiation, the presence of pyrimidine dimers is proposed to be the major trigger for UVC-induced apoptosis.

Detection of a reduced-flavin triplet state in free flavins and DNA photolyase.

A transient species was observed upon laser excitation (lambda exc = 355 nm) of reduced flavins (cFIH2) under anaerobic conditions. The transient decayed with a lifetime of about 1 microseconds, was quenched by iodide ions and was assigned as the reduced flavin triplet state 3cFIH2. The triplet-state absorption spectrum assumed three forms, depending on the pH of the solution, corresponding to 3cFIH2 and its monoprotonated and monodeprotonated forms. The triplet-state lifetime was not sensitive to the presence of pyrimidine cyclobutane dimers, apparently ruling out its involvement in the dimer splitting reaction observed with model systems of reduced flavins. An analogous species is apparently formed in the reduced form of Escherichia coli DNA photolyase.