Solar UV has mutagenic, carcinogenic and lethal effects. With the gradual depletion of atmospheric ozone it is predicted that more UV will reach the earth's surface in the future, with serious consequences for the biosphere. One of the major effects of UV is induction of pyrimidine dimers in DNA. These photoproducts may cause mutation and cancer by miscoding, and cellular death by blocking replication. Pyrimidine dimers are repaired by an enzyme called DNA photolyase. This enzyme uses the energy of a near UV-visible photon to break the cyclobutane ring of a dimer and thus reverse the effect of a photon of higher energy. The goal of our research is to characterize this unusual reaction using the enzyme from E. coli and then to extend our studies to other photolyases in order to develop a general mechanism for photoreactivation. Our studies will focus on three major areas 1. STRUCTURE: (a) Genetic engineering. We will identify the chromophore (flavin and folate) and DNA binding sites by fragment swapping with other photolayses, isolating functional domains, and conducting the specific mutagenesis. (b) Spectroscopy. We will use steady-state and time-resolved absorbance, fluorescence, CD, EPR and NMR spectroscopy to investigate the interactions of the two chromophores with one another and with the apoenzyme. (c) X-ray crystallography. We will prepare enzyme-substrate crystals for detailed analysis of the enzyme and its active site. 2. FUNCTION: (a) Binding. We will measure the affinity of the enzyme to the 4 different photodimers in different sequence context to better define binding determinants. (b) Catalysis. Using time resolved spectroscopy we have discovered a radical ion pair intermediate in the photolysis reaction. We will use various substrates, and enzyme reconstituted with a flavin analog to identify the intermediate and thus determine whether the enzyme donates to, or abstracts an electron from, the photodimer. We will determine the rate and efficiency of energy and electron transfer mediated by the two chromophores. (c) Quantum yield. We will isolate mutants that affect the quantum yield of energy and electron transfer and develop photolyase into a model system for Investigating photoinduced electron transfer. 3. OTHER PHOTOLYASES. We will isolate and characterize a deazaflavin class photolyase and an animal photolyase to find out whether the two chromophore motif is universal, whether there are other second chromophores in addition to the two already identified and whether all photolyases utilize the same basic photochemical mechanism for repair.
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