Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY’s catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine–pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.

Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii. This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UVvisible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 s after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6 -4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mM injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner.Cryptochromes represent a group of diverse sensory photoreceptors present in all kingdoms of life (1, 2). Together with the UV-light-dependent DNA repair enzymes, the photolyases (3), they constitute the cryptochrome/photolyase family. Members of this family share a highly conserved photolyase homology region (PHR) 2 , which comprises ϳ500 amino acids and carries a non-covalently bound flavin adenine dinucleotide (FAD) as a chromophore. The C-terminal extension (CCT) present in many cryptochromes and photolyases is strongly variable in amino acid composition and length and has been shown to be crucial for signal transduction in the Arabidopsis cryptochrome AtCRY1 (4). The diverse subfamilies of cryptochromes comprise proteins acting as central blue light sensors in bacteria, fungi, plants, and insects (animal type I CRY) (1). Moreover, CRYs are also found as the light-independent, central part of the oscillator of the biological clock in mammals (animal type II CRY) (5) and as a mediator for light-dependent magnetosensitivity in flies (6). Opposed to these findings, DASH cryptochromes have been found to repair lesions in single-stranded DNA and double-stranded loop-structured DNA in vitro (7,8). Therefore, DASH cryptochromes are more similar in terms of functionality to photolyases than cryptochromes. Among the photolyases, two different types are separated dependin...

Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga ( throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in pCRY is down-regulated in pregametes and gametes, and in the mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in that is involved in both circadian timing and life cycle progression.

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Cryptochromes are known as flavin-binding blue light receptors in bacteria, fungi, plants, and insects. The animal-like cryptochrome (aCRY) of the green alga has extended our view on cryptochromes, because it responds also to other wavelengths of the visible spectrum, including red light. Here, we have investigated if aCRY is involved in the regulation of the sexual life cycle of, which is controlled by blue and red light at the steps of gametogenesis along with its restoration and germination. We show that aCRY is differentially expressed not only during the life cycle but also within the cell as part of the soluble and/or membrane-associated protein fraction. Moreover, localization of aCRY within the algal cell body varies between vegetative cells and the different cell types of gametogenesis. aCRY is significantly (early day) or to a small extent (late night) enriched in the nucleus in vegetative cells. In pregametes, gametes and dark-inactivated gametes, aCRY is localized over the cell body. aCRY plays an important role in the sexual life cycle of : It controls the germination of the alga, under which the zygote undergoes meiosis, in a positive manner, similar to the regulation by the blue light receptors phototropin and plant cryptochrome (pCRY). However, aCRY acts in combination with pCRY as a negative regulator for mating ability as well as for mating maintenance, opposite to the function of phototropin in these processes.