Photolyases are DNA repair enzymes which bind to pyrimidine dimers in DNA and upon exposure to near UV or visible light cleave the cyclobutane ring and restore the pyrimidines to their original structure. Deficiency of photoreactivating activity in certain individuals with the xeroderma pigmentosum syndrome suggests a relationship between photoenzymatic repair and cancer. This proposal describes experiments to study the PHR1 gene and encoded photolyase I in the yeast Saccharomyces cerevisiae. The PHR1 gene from yeast has been cloned. The nucleotide sequence of the gene will be known by the time this project begins. Transcriptional start and polyA addition sites will be determined by S-1 nuclease mapping. The regions 5' and 3' to the gene which are required for expression will be defined by deletional analysis and the length of the primary polyadenylated transcript will be determined by Northern analysis; should evidence for introns be obtained, they will be localized by S-1 mapping. Plasmids which lead to the controlled overproduction of the enzyme will be used to obtain pure photolyase I in mg quantities which will be used in in vitro studies to characterize the DNA binding and photolytic steps of the reaction. The nitrocellulose filter binding assay will be used to determine the effect of substrate topology on binding, to measure the equilibrium and kinetic binding constants for binding to UV-irradiated DNA, and to quantitate the contribution of electrostatic and nonelectrostatic interactions to the free energy of binding. The contact sites made on DNA by photolyase will be determined using enzymatic and chemical protection and interference methods. The chromophore of the enzyme will be identified by absorption and fluorescence spectroscopy, electron spin resonance, and chemical methods. The absolute action spectrum of the photolytic reaction and the photolytic constant will be determined by the flash photolysis assay. The secondary structure of the enzyme will be predicted and compared to that of the E. coli enzyme; structural homologies will be targeted for site-directed mutagenesis to define the DNA-binding and chromophore-binding domains of the enzyme. In vivo studies using PHR1-'lacZ fusions will determine whether the PHR1 gene is inducible by UV or photoreactivating light and whether a specific karyophilic sequence targets the enzyme to the nucleus. A new technique will be developed to determine whether photolyase I repairs dimers in nucleosomal DNA. The contribution of photolyase I to dark repair processes will also be assessed.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Radiation Study Section (RAD)
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University of North Carolina Chapel Hill
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Chapel Hill
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Sancar, G B (2000) Enzymatic photoreactivation: 50 years and counting. Mutat Res 451:25-37
Jang, Y K; Wang, L; Sancar, G B (1999) RPH1 and GIS1 are damage-responsive repressors of PHR1. Mol Cell Biol 19:7630-8
Vande Berg, B J; Sancar, G B (1998) Evidence for dinucleotide flipping by DNA photolyase. J Biol Chem 273:20276-84
Sweet, D H; Jang, Y K; Sancar, G B (1997) Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae. Mol Cell Biol 17:6223-35
Sancar, G B; Ferris, R; Smith, F W et al. (1995) Promoter elements of the PHR1 gene of Saccharomyces cerevisiae and their roles in the response to DNA damage. Nucleic Acids Res 23:4320-8
Baer, M E; Sancar, G B (1993) The role of conserved amino acids in substrate binding and discrimination by photolyase. J Biol Chem 268:16717-24
Malhotra, K; Baer, M; Li, Y F et al. (1992) Identification of chromophore binding domains of yeast DNA photolyase. J Biol Chem 267:2909-14
Sebastian, J; Sancar, G B (1991) A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1. Proc Natl Acad Sci U S A 88:11251-5
Sebastian, J; Kraus, B; Sancar, G B (1990) Expression of the yeast PHR1 gene is induced by DNA-damaging agents. Mol Cell Biol 10:4630-7
Sancar, G B (1990) DNA photolyases: physical properties, action mechanism, and roles in dark repair. Mutat Res 236:147-60

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