Cryptochromes are proteins related to DNA photolyases and have been shown to function as blue-light photoreceptors and to play important roles in circadian rhythms in both plants and animals. The CPH1 gene from Chlamydomonas reinhardtii was originally predicted to encode a putative cryptochrome protein of 867 amino acids with a predicted molecular mass of 91 kD (Small et al., 1995). However, western blotting with antibodies specific to the CPH1 protein revealed the presence of two proteins that migrate at apparent molecular mass of approximately 126 and 143 kD. A reexamination of the assigned intronexon boundaries has shown that the previously assigned intron 7 is in fact part of exon 7 which leads to a predicted protein of 1,007 amino acids corresponding to a size of 104.6 kD. The two forms of CPH1 that migrate slower on SDS-PAGE presumably result from unknown posttranslational modifications. In C. reinhardtii cells synchronized by light to dark cycles, the two slow migrating forms of CPH1 protein accumulate in the dark and disappear rapidly in the light. Both red and blue light are effective at inducing the degradation of the CPH1 proteins. Proteasomes are implicated because degradation is inhibited by MG132, a proteasome inhibitor. Studies with deletion mutants indicate that the C-terminal region is important for both the posttranslational modification and the protein's stability under both light and dark conditions.The discovery of the first blue light photoreceptor in Arabidopsis (Ahmad and Cashmore, 1993) has led to the subsequent discovery of similar proteins in several other species. Also known as cryptochromes, or CRYs, these include two each in mice (Kobayashi et al., 1998) and humans (Todo et al., 1996;van der Spek et al., 1996), one in Chlamydomonas reinhardtii (Small et al., 1995) and Drosophila melanogaster Stanewsky et al., 1998), and five in fern (Adiantum capillus-veneris; Kanegae and Wada, 1998;Imaizumi et al., 2000). Cryptochromes show significant amino acid similarity to DNA photolyases in their N termini although cryptochromes lack photolyase activity. In addition, cryptochromes contain a C-terminal extension that appears to be unique for each cryptochrome in size and amino acid sequence (Lin and Shalitin, 2003). Recently, a second class of cryptochromes, DASH, has been identified, whose members share similarities to DNA photolyases in DNA binding and recognition (Brudler et al., 2003). These proteins have been hypothesized to have redox activity and act as transcriptional regulators.Cryptochromes in Arabidopsis (AtCRY1 and At-CRY2) belong to the first class of cryptochromes, act as blue light photoreceptors, and have roles in the regulation of flowering time and plant development (Cashmore et al., 1999). The Arabidopsis CRY1 is known to regulate hypocotyl elongation and cotyledon growth in seedlings under high light intensities (Ahmad et al., 1995) while its close relative, AtCRY2, is involved in hypocotyl and cotyledon growth under conditions of low light . The AtCRY1 protein has been show...

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phr1, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.

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.

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