Evolution of Proteins of the DNA Photolyase/Cryptochrome Family.

Proteins of the cryptochrome/DNA photolyase family (CPF) are phylogenetically related and structurally conserved flavoproteins that perform various functions. DNA photolyases repair DNA damage caused by UV-B radiation by exposure to UV-A/blue light simultaneously or subsequently. Cryptochromes are photoreceptor proteins regulating circadian clock, morphogenesis, phototaxis, and other responses to UV and blue light in various organisms. The review describes the structure and functions of CPF proteins, their evolutionary relationship, and possible functions of the CPF ancestor protein.

Phylogenetic and Functional Classification of the Photolyase/Cryptochrome Family.

The photolyase/cryptochrome (PHR/CRY) family is a large group of proteins with similar structure but very diverge functions such as DNA repair, circadian clock resetting and regulation of transcription. As a result of advances in the biochemistry of the CRY/PHR family and identification of new members, several adjustments have been made to the classification of this protein family. For example, a new class of PHRs, Class III, has been proposed. Furthermore, CRYs have been suggested to function as photosensory proteins in the primordial eye of sponge larvae. Additionally, a magnetosensory function has been attributed to certain CRYs. Recent advances in the field enabled us to propose a comprehensive classification scheme and nomenclatural system for this family. This review focuses on the computational and biochemical classifications of the PHR/CRY family. Several examples show that computational analysis can give a hinge about the function of newly discovered members before performing any biochemical study.

Bifurcating electron-transfer pathways in DNA photolyases determine the repair quantum yield.

Photolyase is a blue-light-activated enzyme that repairs ultraviolet-induced DNA damage that occurs in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts. Previous studies on microbial photolyases have revealed an electron-tunneling pathway that is critical for the repair mechanism. In this study, we used femtosecond spectroscopy to deconvolute seven electron-transfer reactions in 10 elementary steps in all classes of CPD photolyases. We report a unified electron-transfer pathway through a conserved structural configuration that bifurcates to favor direct tunneling in prokaryotes and a two-step hopping mechanism in eukaryotes. Both bifurcation routes are operative, but their relative contributions, dictated by the reduction potentials of the flavin cofactor and the substrate, determine the overall quantum yield of repair.

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.

First characterisation of a CPD-class I photolyase from a UV-resistant extremophile isolated from High-Altitude Andean Lakes.

UV-resistant Acinetobacter sp. Ver3 isolated from High-Altitude Andean Lakes (HAAL) in Argentinean Puna, one of the highest UV exposed ecosystems on Earth, showed efficient DNA photorepairing ability, coupled to highly efficient antioxidant enzyme activities in response to UV-B stress. We herein present the cloning, expression, and functional characterization of a cyclobutane pyrimidine dimer (CPD)-class I photolyase (Ver3Phr) from this extremophile to prove its involvement in the previously noted survival capability. Spectroscopy of the overexpressed and purified protein identified flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate (MTHF) as chromophore and antenna molecules, respectively. All functional analyses were performed in parallel with the ortholog E. coli photolyase. Whereas the E. coli enzyme showed the FAD chromophore as a mixture of oxidised and reduced states, the Ver3 chromophore always remained partly (including the semiquinone state) or fully reduced under all experimental conditions tested. Functional complementation of Ver3Phr in Phr(-)-RecA E. coli strains was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) detection. The results identified strong photoreactivation ability in vivo of Ver3Phr while its nonphotoreactivation function, probably related with the stimulation of nucleotide excision repair (NER), was not as manifest as for EcPhr. Whether this is a question of the approach using an exogenous photolyase incorporated in a non-genuine host or a fundamental different behaviour of a novel enzyme from an exotic environment will need further studies.

Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA.

Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA. We solved crystal structures of Mm0852, the first one for a class II photolyase, alone and in complex with CPD lesion-containing duplex DNA. The lesion-binding mode differs from other photolyases by a larger DNA-binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophane dyad as electron transfer pathway to the catalytic FAD cofactor.

The cryptochromes: blue light photoreceptors in plants and animals.

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Molecular and functional analysis of the (6-4) photolyase from the hexactinellid Aphrocallistes vastus.

The hexactinellid sponges (phylum Porifera) represent the phylogenetically oldest metazoans that evolved 570-750 million years ago. At this period exposure to ultraviolet (UV) light exceeded that of today and it may be assumed that this old taxon has developed a specific protection system against UV-caused DNA damage. A cDNA was isolated from the hexactinellid Aphrocallistes vastus which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases. Subsequently functional studies were performed. It could be shown that the sponge gene, after transfection into mutated Escherichia coli, causes resistance of the bacteria against UV light. Recombinant sponge photolyase was prepared to demonstrate that this protein binds to DNA treated with UV light (causing the formation of thymine dimers). Finally, it is shown that the photolyase gene is strongly expressed in the upper part of the animals and not in their middle part or their base. It is concluded that sponges not only have an excision DNA repair system, as has been described earlier by us, but also a photolyase-based photo-reactivating system.

Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA.

Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of alpha/beta domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered alpha/beta folds with the generic "Rossmann-like" topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15-18 distinct alpha/beta ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world.

A gene required for the novel activation of a class II DNA photolyase in Chlamydomonas.

DNA photolyases catalyze the blue light-dependent repair of UV light-induced damage in DNA. DNA photolyases are specific for either cyclobutane-type pyrimidine dimers or (6-4) photoproducts. PHR2 is a gene that in Chlamydomonas reinhardtii encodes a class II DNA photolyase which catalyzes the photorepair of cyclobutane-type pyrimidine dimers. Based on amino acid sequence analysis of PHR2, which indicates the presence of a chloroplast targeting sequence, PHR2 was predicted to encode the chloroplast photolyase of Chlamydomonas. Using a sensitive gene-specific in vivo repair assay, we found that overexpression of PHR2 in Chlamydomonas results in targeting of the protein to not only the chloroplast, but also to the nucleus. Overexpression of PHR2 photolyase in a photoreactivation-deficient mutant, phr1, results in a largely inactive product. The phr1 mutant was found to be deficient in both photorepair of a chloroplast gene, rbcL, and a nuclear gene, rDNA. These results suggest that PHR2 is the structural gene for the photolyase targeted to both the chloroplast and the nucleus, and that the PHR1 gene product is necessary for full activity of PHR2 protein. To our knowledge, the requirement for a second gene for full activity of a DNA photolyase is novel.

A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae.

Plants have evolved a number of adaptive responses to cope with growth in conditions of limited phosphate (Pi) supply involving biochemical, metabolic, and developmental changes. We prepared an EMS-mutagenized M(2) population of an Arabidopsis thaliana transgenic line harboring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS), and screened for mutants altered in Pi starvation regulation. One of the mutants, phr1 (phosphate starvation response 1), displayed reduced response of AtIPS1::GUS to Pi starvation, and also had a broad range of Pi starvation responses impaired, including the responsiveness of various other Pi starvation-induced genes and metabolic responses, such as the increase in anthocyanin accumulation. PHR1 was positionally cloned and shown be related to the PHOSPHORUS STARVATION RESPONSE 1 (PSR1) gene from Chlamydomonas reinhardtii. A GFP::PHR1 protein fusion was localized in the nucleus independently of Pi status, as is the case for PSR1. PHR1 is expressed in Pi sufficient conditions and, in contrast to PSR1, is only weakly responsive to Pi starvation. PHR1, PSR1, and other members of the protein family share a MYB domain and a predicted coiled-coil (CC) domain, defining a subtype within the MYB superfamily, the MYB-CC family. Therefore, PHR1 was found to bind as a dimer to an imperfect palindromic sequence. PHR1-binding sequences are present in the promoter of Pi starvation-responsive structural genes, indicating that this protein acts downstream in the Pi starvation signaling pathway.

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.

A new class of DNA photolyases present in various organisms including aplacental mammals.

DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.

DNA photorepair: chromophore composition and function in two classes of DNA photolyases.

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.