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.
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