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DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.

Ultraviolet-B (UV-B, 280-320 nm) radiation may have severe negative effects on plants including damage to their genetic information. UV protection and DNA-repair mechanisms have evolved to either avoid or repair such damage. Since autotrophic plants are dependent on sunlight for their energy supply, an increase in the amount of UV-B reaching the earth's surface may affect the integrity of their genetic information if DNA damage is not repaired efficiently and rapidly. Here we show that overexpression of cyclobutane pyrimidine dimer (CPD) photolyase (EC 4.1.99.3) in Arabidopsis thaliana (L.), which catalyses the reversion of the major UV-B photoproduct in DNA (CPDs), strongly enhances the repair of CPDs and results in a moderate increase of biomass production under elevated UV-B.

DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.

Proteins from the cryptochrome/photolyase family utilize UV-A, blue or even red light to achieve such diverse functions as repair of DNA lesions by photolyases and signaling by cryptochromes. DASH-type cryptochromes retained the ability to repair cyclobutane pyrimidine dimers ( CPDs) in single-stranded DNA regions in vitro. However, most organisms possess conventional CPD photolyases responsible for repair of these lesions in vivo. Recent work showed that the DASH-type cryptochrome Cry D plays a regulatory role in diverse light-dependent processes in Fusarium fujikuroi. Here, we report our in vitro studies on heterologously expressed Ff Cry D. The purified protein contains N5, N10-methenyltetrahydrofolate and flavin adenine dinucleotide as cofactors. Photoreduction and DNA photorepair experiments confirmed that Ff Cry D is active in light-driven electron transfer processes. However, the protein showed comparable affinities for CPD-comprising and undamaged DNA probes. Surprisingly, after purification, full-length Ff Cry D as well as a truncated version containing only the PHR domain bound RNA which influenced their behavior in vitro. Moreover, binding of Ff Cry D to RNA indicates a putative role in RNA metabolism or in posttranscriptional control of gene expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary 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 α-proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern-day cyanobacteria. [ABSTRACT FROM AUTHOR]

Abstract: Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6–4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FADox, have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADHox, and subsequently FADH− from FADHs in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution. [Copyright &y& Elsevier]

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochromel (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms. [ABSTRACT FROM AUTHOR]

Cryptochromes and photolyases are blue-light photoreceptors and DNA-repair enzymes, respectively, with conserved domains and a common ancestry 1–3. Photolyases use UV-A and blue light to repair lesions in DNA caused by UV radiation, photoreactivation, although cryptochromes have specialized roles ranging from the regulation of photomorphogenesis in plants, to clock function in animals 4–7. A group of cryptochromes (cry-DASH) 8 from bacteria, plants, and animals has been shown to repair in vitro cyclobutane pyrimidine dimers (CPDs) in single-stranded DNA (ssDNA), but not in double-stranded DNA (dsDNA) 9. Cry-DASH are evolutionary related to 6-4 photolyases and animal cryptochromes, but their biological role has remained elusive. The analysis of several crystal structures of members of the cryptochrome and photolyase family (CPF) allowed the identification of structural and functional similarities between photolyases and cryptochromes 8 , 10–12 and led to the proposal that the absence of dsDNA repair activity in cry-DASH is due to the lack of an efficient flipping of the lesion into the catalytic pocket 13. However, in the fungus Phycomyces blakesleeanus , cry-DASH has been shown to be capable of repairing CPD lesions in dsDNA as a bona fide photolyase 14. Here, we show that cry-DASH of a related fungus, Mucor circinelloides , not only repairs CPDs in dsDNA in vitro but is the enzyme responsible for photoreactivation in vivo. A structural model of the M. circinelloides cry-DASH suggests that the capacity to repair lesions in dsDNA is an evolutionary adaptation from an ancestor that only had the capacity to repair lesions in ssDNA. • The cryptochrome DASH from Mucor circinelloides binds the cofactors FAD and 5,10-MTHF • Cryptochrome DASH from Mucor repairs thymine dimers in double-stranded DNA • Cryptochrome DASH from Mucor is responsible for photoreactivation in vivo Navarro et al. show that cry-DASH from the fungus Mucor circinelloides repairs DNA lesions in dsDNA and is the enzyme responsible for light-dependent DNA repair in vivo. The authors suggest that the capacity of Mucor cry-DASH to repair lesions in dsDNA is an evolutionary adaptation from an ancestor that only was able to repair lesions in ssDNA. [ABSTRACT FROM AUTHOR]