In this work, thePHR1gene ofChlamydomonaswas cloned through molecular mapping and shown to encode a protein much like known FO synthases. results indicate that theChlamydomonas PHR1gene encodes an FO synthase and that optimal Senkyunolide H photoreactivation inChlamydomonasrequires FO, a molecule known to serve as a second chromophore for DNA photolyases. Keywords:DNA Damage; DNA Enzymes; DNA Repair; Flavin; Genetics; 7,8-Didemethyl-8-hydroxy-5-deazariboflavin; Cyclobutane Pyrimidine Dimers; Photoreactivation; Ultraviolet Light == Introduction == Cyclobutane pyrimidine dimers (CPDs)2and 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. These types of DNA damage are hazardous to cells by potentially stalling DNA replication and introducing mutation (13). Photoreactivation is a blue light-dependent DNA repair process catalyzed by enzymes known as DNA photolyases. There are different classes of DNA photolyases with unique repair specificities. For instance, you will find DNA photolyases responsible for repairing CPDs and ones specific for repairing (6-4) photoproducts. During photoreactivation, DNA photolyase binds to the UV-induced lesion specific for its class and uses blue light as a cosubstrate to return the DNA to its undamaged state (4). Each DNA photolyase binds two chromophores noncovalently. The first, FAD, is usually common to all photolyases. It is the catalytic chromophore, and as such, it is essential for photoreactivation (5). The identity of the second chromophore is usually Senkyunolide H variable, but 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) serves this role for a number of photolyases (610). During photoreactivation, the second chromophore acts as a photoantenna, becoming excited by blue light energy and transferring this energy to FADH. The excited FADHreversibly transfers an electron to the damaged DNA allowing the DNA to return to its initial undamaged Rabbit Polyclonal to ZADH2 form. Even though antenna chromophore increases the repair rate of DNA photolyase 10100-fold under limited-light conditions, it is generally considered nonessential for photoreactivation under standard light conditions (4,11,12) because DNA photolyase bound to FADHalone is sufficient to catalyze the repair of UV-induced DNA damage (5,1315). FO is usually generated from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, an intermediate in riboflavin biosynthesis, and 4-hydroxyphenylpyruvate, a precursor to tyrosine. The reaction is usually catalyzed by an enzyme known as FO synthase (EC 2.5.1.77), a Senkyunolide H member of the radicalS-adenosylmethionine (AdoMet) superfamily of proteins (16). Members of this family catalyze many diverse reactions, but all use a unique [4Fe-4S] cluster to bind AdoMet as a ligand. The AdoMet is usually split to form a 5-deoxyadenosyl radical that abstracts a hydrogen from your substrate initiating the reaction mechanism (17,18). FO synthases have been partially characterized from mycobacteria andMethanocaldococcus jannaschii(16,19). BLASTP searches have recognized FO synthase Senkyunolide H homologs in Archaea, some classes of Bacteria, and in the Chlorophyta of the eukaryotes (10,16,19). FO is a biosynthetic precursor for the hydride carrier coenzyme F420. Accordingly, FO synthase homologs have been identified in all organisms known to produce F420, including the methanogens and the actinomycetes. However, you will find organisms, including the cyanobacteria, that do not produce F420but still generate FO as an apparent end product (16). Thephr1strain of the unicellular green algaChlamydomonas reinhardtiiwas isolated following chemical mutagenesis and shown to have a defect in the photoreactivation of CPDs (20). In an attempt to identify thePHR1gene, a homology-based method was used to clone a gene ofChlamydomonasthat encoded a CPD-specific DNA photolyase. However, the newly cloned gene was not closely linked to thephr1mutation and was thus namedPHR2(21). Moreover, further work revealed that PHR2p requires PHR1p for full activity, indicating that photoreactivation inChlamydomonasrequires the products of two genes (22). This discovery markedphr1as an unequaled photoreactivation mutant. In this work, we statement Senkyunolide H the identification and cloning of thePHR1gene ofC. reinhardtii, which encodes the first characterized eukaryotic FO synthase. Furthermore, we show that FO, a known second chromophore for DNA photolyases, is critical for photoreactivation inChlamydomonas. == EXPERIMENTAL PROCEDURES == == Molecular Mapping == phr1was mated with S1-D2 (cc-2290), a polymorphic field isolate ofChlamydomonas(23). Progeny from this cross that were photoreactivation-deficient, and thus experienced inherited thephr1mutation, were used as the mapping populace. Genomic DNA was isolated from each member of the mapping populace and used as the template for PCRs with primers from theChlamydomonasmolecular mapping kit (24). The mapping kit uses PCR to uncover polymorphisms in the mapping populace, providing a means for measuring the cosegregation frequency of molecular markers and the mutation of interest. Thephr1mutation was localized to LG XIX using the Zys1B marker from your molecular mapping kit. The location of thephr1mutation was further refined using primers for the FLA10 marker.
Categories:CYP