Photodamage to DNA results in mutagenesis and cell death. In humans this damage can result in skin cancer. Repair of DNA photolesions is essential for the survival of living systems in sunlight and involves either an excision or in situ repair of the lesion. In this proposal, studies designed to elucidate the enzymatic mechanism of the in situ repair of the cyclobutane pyrimidine photodimer and the spore product are described. The major DNA damage caused by UV irradiation of bacteria is the cyclobutane thymine dimer. DNA photolyase catalyzes the in situ repair of this lesion in a light-dependent reaction. To conclude our mechanistic studies on this enzyme, we propose to further explore the use of iodomethyl substituted photodimers as probes for radical intermediates and to overexpress the Salmonella typhimurium photolyase for crystallographic studies. In contrast to bacterial DNA, UV irradiation of bacterial spores results in the formation of the spore product as the major lesion. We will develop a simple model system for the conversion of bis-thymines to spore product. We will develop a synthetic route to the dinucleotide spore product and incorporate this into short oligonucleotides to determine the minimum substrate required for the repair enzyme. we will explore the mechanism of spore product formation and repair using a combination of stereochemical, isotope effect and radical or carbanion trapping studies.