The photolyase gene from the plant pathogen Fusarium oxysporum f. sp. lycopersici is induced by visible light and alpha-tomatine from tomato plant

Survival of irradiated spores from Fusarium oxysporum with ultraviolet radiation (UV) was increased following exposition to visible light, indicating that this phytopathogenic fungus has a mechanism of photoreactivation able to counteract the lethal effects of UV. A genomic sequence containing the complete photolyase gene (phr1) from F. oxysporum was isolated by heterologous hybridisation with the Neurospora crassa photolyase gene. The F. oxysporum phr1 cDNA was isolated and expressed in a photolyase deficient Escherichia coli strain. The complementation of the photoreactivation deficiency of this E. coli mutant by phr1 cDNA demonstrated that the photolyase gene from F. oxysporum encodes a functional protein. The F. oxysporum PHR1 protein has a domain characteristic of photolyases from fungi (Trichoderma harziaium, N. crassa, Magnaporthe grisea, Saccharomyces cerevisiae) to bacteria (E. coli), and clusters in the photolyases phylogenetic tree with fungal photolyases. The F. oxysporum phr1 gene was inducible by visible light. The phr1 expression was also detected in presence of alpha-tomatine, a glycoalkaloid from tomato damaging cell membranes, suggesting that phr1 is induced by this cellular stress.     

Evolutionary History of the Photolyase/Cryptochrome Superfamily in Eukaryotes

Background

Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth.

Results

We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6–4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ω = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000–541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth’s geological history.     

PHL1 of Cercospora zeae-maydis encodes a member of the photolyase/cryptochrome family involved in UV protection and fungal development

DNA photolyases harvest light energy to repair genomic lesions induced by UV irradiation, whereas cryptochromes, presumptive descendants of 6-4 DNA photolyases, have evolved in plants and animals as blue-light photoreceptors that function exclusively in signal transduction. Orthologs of 6-4 photolyases are predicted to exist in the genomes of some filamentous fungi, but their function is unknown. In this study, we identified two putative photolyase-encoding genes in the maize foliar pathogen Cercospora zeae-maydis: CPD1, an ortholog of cyclobutane pyrimidine dimer (CPD) photolyases described in other filamentous fungi, and PHL1, a cryptochrome/6-4 photolyase-like gene. Strains disrupted in PHL1 (Deltaphl1) displayed abnormalities in development and secondary metabolism but were unaffected in their ability to infect maize leaves. After exposure to lethal doses of UV light, conidia of Deltaphl1 strains were abolished in photoreactivation and displayed reduced expression of CPD1, as well as RAD2 and RVB2, orthologs of genes involved in nucleotide excision and chromatin remodeling during DNA damage repair. This study presents the first characterization of a 6-4 photolyase ortholog in a filamentous fungus and provides evidence that PHL1 regulates responses to UV irradiation.     

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.     

Cloning and characterization of a class II DNA photolyase from Chlamydomonas

Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases. Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast. Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase. The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency. The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene. Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence. This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase.     

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.     

A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.     

Eukaryotic class II cyclobutane pyrimidine dimer photolyase structure reveals basis for improved ultraviolet tolerance in plants

Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280–315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 Å resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms.     

Molecular Evolution of the Photolyase–Blue-Light Photoreceptor Family

The photolyase–blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes. Received: 23 October 1996 / Accepted: 1 April 1997     

Identification of a Novel Class of Photolyases as Possible Ancestors of Their Family

UV irradiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These two types of lesions can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. Recently, a new class of 6-4 photolyases named iron–sulfur bacterial cryptochromes and photolyases (FeS-BCPs) were found, which were considered as the ancestors of all photolyases and their homologs—cryptochromes. However, a controversy exists regarding 6-4 photoproducts only constituting ∼10–30% of the total UV-induced lesions that primordial organisms would hardly survive without a CPD repair enzyme. By extensive phylogenetic analyses, we identified a novel class of proteins, all from eubacteria. They have relatively high similarity to class I/III CPD photolyases, especially in the putative substrate-binding and FAD-binding regions. However, these proteins are shorter, and they lack the “N-terminal α/β domain” of normal photolyases. Therefore, we named them short photolyase-like. Nevertheless, similar to FeS-BCPs, some of short photolyase-likes also contain four conserved cysteines, which may also coordinate an iron–sulfur cluster as FeS-BCPs. A member from Rhodococcus fascians was cloned and expressed. It was demonstrated that the protein contains a FAD cofactor and an iron–sulfur cluster, and has CPD repair activity. It was speculated that this novel class of photolyases may be the real ancestors of the cryptochrome/photolyase family.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.     

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.     

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.     

Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6-4) photolyase as revealed by resonance Raman spectroscopy: Electron transfer from the FAD cofactor to ultraviolet-damaged DNA

The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm(-1) bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm(-1) band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N5-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N3-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.     

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.