The essential role of 7,8-didemethyl-8-hydroxy-5-deazariboflavin for optimal photoreactivation in Chlamydomonas reinhardtii

Jason L. Petersen1-3

1) Avera Research Institute, Sioux Falls SD, USA 2) Sanford School of Medicine of the University of South Dakota, Vermillion SD, USA 3) Sioux Falls VA Medical Center, 2501 W. 22nd St., R&D 151 Sioux Falls SD 57105, USA

DNA photolyases catalyze photoreactivation, the blue light dependent repair of DNA that has been damaged by ultraviolet light. All DNA photolyases non-covalently bind two chromophores. Flavin adenine dinucleotide (FAD) is the catalytic chromophore for all photolyases and is required for photoreactivation. The identity of the second chromophore varies, but is commonly 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). In general, the second chromophore is considered non-essential for photoreactivation since DNA photolyase containing FAD as the sole chromophore efficiently catalyzes photoreactivation. phr1 is a photoreactivation deficient strain of Chlamydomonas generated through chemical mutagenesis. Using molecular-mapping as a means of identifying the PHR1 gene, the phr1 mutation was localized to the right arm of LG XIX. A candidate gene search in this region of LG XIX revealed a gene model, e_gwH.60.32.1, encoding a putative FO synthase. Wild type DNA constructs containing e_gwH.60.32.1 complemented the photoreactivation deficiency of the phr1 strain. The newly identified PHR1 gene was subsequently shown to contain 18 exons and encode a putative protein of 1,106 amino acids with high similarity to known FO synthases. DNA sequencing identified the phr1 mutation as a nonsense mutation in exon 12 of the PHR1 gene. The heterologous expression of PHR1 in E. coli resulted in a functional protein that produced a molecule with characteristics similar to FO as determined by fluorescence, thin layer chromatography, and high performance liquid chromatography. These results indicate that the PHR1 gene of Chlamydomonas encodes the first eukaryotic FO synthase to be characterized.

e-mail address of presenting author: Jason.Petersen@mckennan.org