Ultraviolet (UV) radiation is one of the major environmental carcinogens and is presumed to cause its harmful effects (mutation, cancer, killing) by producing pyrimidine dimers in DNA. There are several molecular mechanisms to eliminate pyrimidine dimers and prevent cellular damage. Photoreactivation is one of these repair mechanisms and is mediated by the enzyme DNA photolyase. This enzyme binds to pyrimidine dimers and converts visible light (350-600 nm) energy into chemical energy to break the cyclobutane ring joining the two pyrimidines, thus restoring the integrity of the DNA. Photolyase of E. coli, which is encoded by the phr gene, has been purified to homogeneity and shown to contain an FAD cofactor. Our goal is to understand the structure, function, and regulation of this enzyme using genetic and molecular biological techniques. A. structure. E. coli photolyase is a protein of Mr 53,994 which shows considerable homology to yeast photolyase at the NH2- and COOH-terminal regions suggesting that these regions are important for function. To localize the active site we will use recombinant DNA techniques to obtain mutant photolyases deleted internally or at one of the termini and will study their properties. We will also identify the active site by protein-coenzyme and protein-substrate cross-linking, and by site-directed mutagenesis using the cloned phr gene. The enzyme will be crystallized and studied by X-ray diffraction to obtain its tertiary structure. B. Function. The mechanism of photolyase binding will be studied using the nitrocellulose filter binding assay, flash photolysis, and electron microscopy. Enzyme contact sites on DNA will be determined by """"""""footprinting"""""""" techniques as well as alkylation protection and interference experiments. The mechanism of photosensitization will be studied by determining the action spectrum of the enzyme containing FAD or its analogues in various redox states and by determining the relative sensitivity of different dimers by DNA sequencing gels. C. Regulation. The photolyase gene phr is part of an operon that has another gene, orf169, preceding phr. The orf 169 coding region is preceded by a sequence analogous to an """"""""SOS box"""""""". The size of the phr operon will be determined by Northern blotting and whether this operon is induced by DNA damage (SOS response) will be investigated in vivo by gene fusion techniques. Gene fusion will also be used to determine whether photolyase is induced by visible light and whether adenine deficiency (which increases active photolyase) affects transcription. The regulatory sites will be investigated by in vitro and in vivo footprinting techniques.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Radiation Study Section (RAD)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
Zip Code
Krishnaiah, Saikumari Y; Wu, Gang; Altman, Brian J et al. (2017) Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism. Cell Metab 25:961-974.e4
Chiou, Yi-Ying; Yang, Yanyan; Rashid, Naim et al. (2016) Mammalian Period represses and de-represses transcription by displacing CLOCK-BMAL1 from promoters in a Cryptochrome-dependent manner. Proc Natl Acad Sci U S A 113:E6072-E6079
Jang, Hagoon; Lee, Gha Young; Selby, Christopher P et al. (2016) SREBP1c-CRY1 signalling represses hepatic glucose production by promoting FOXO1 degradation during refeeding. Nat Commun 7:12180
Adar, Sheera; Hu, Jinchuan; Lieb, Jason D et al. (2016) Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis. Proc Natl Acad Sci U S A 113:E2124-33
Hu, Jinchuan; Adar, Sheera; Selby, Christopher P et al. (2015) Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution. Genes Dev 29:948-60
Sancar, Aziz; Lindsey-Boltz, Laura A; Gaddameedhi, Shobhan et al. (2015) Circadian clock, cancer, and chemotherapy. Biochemistry 54:110-23
Tan, Chuang; Liu, Zheyun; Li, Jiang et al. (2015) The molecular origin of high DNA-repair efficiency by photolyase. Nat Commun 6:7302
Ozturk, Nuri; Selby, Christopher P; Zhong, Dongping et al. (2014) Mechanism of photosignaling by Drosophila cryptochrome: role of the redox status of the flavin chromophore. J Biol Chem 289:4634-42
Ye, Rui; Selby, Cristopher P; Chiou, Yi-Ying et al. (2014) Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock. Genes Dev 28:1989-98
Annayev, Yunus; Adar, Sheera; Chiou, Yi-Ying et al. (2014) Gene model 129 (Gm129) encodes a novel transcriptional repressor that modulates circadian gene expression. J Biol Chem 289:5013-24

Showing the most recent 10 out of 118 publications