Abstract
DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.
Highlights
Cyclobutane pyrimidine dimers (CPDs)2 and 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts are the primary forms of DNA damage induced by UV light
Expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. These results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases
Cloning and Characterizing the PHR1 Gene—The phr1 strain of Chlamydomonas has a defect in photoreactivation, a blue light-dependent repair mechanism for DNA damaged by UV light [20]
Summary
The phr strain of the unicellular green alga Chlamydomonas reinhardtii was isolated following chemical mutagenesis and shown to have a defect in the photoreactivation of CPDs [20]. In an attempt to identify the PHR1 gene, a homologybased method was used to clone a gene of Chlamydomonas that encoded a CPD-specific DNA photolyase. Further work revealed that PHR2p requires PHR1p for full activity, indicating that photoreactivation in Chlamydomonas requires the products of two genes [22]. This discovery marked phr as an unmatched photoreactivation mutant. We report the identification and cloning of the PHR1 gene of C. reinhardtii, which encodes the first characterized eukaryotic FO synthase. We show that FO, a known second chromophore for DNA photolyases, is critical for photoreactivation in Chlamydomonas
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