The genetic material is constantly subjected to solar ultraviolet radiation (UV), and living organisms place a high priority on the correction of potentially lethal or mutagenic UV-induced DNA damage. It has long been known that individuals afflicted with relatively rare heritable DNA repair disorders such as xeroderma pigmentosum exhibit UV hypersensitivity and a high incidence of skin cancer. More recently, the observed correlation between increased terrestrial UV exposure due to atmospheric ozone destruction, and an increasing incidence of human skin cancer, highlight the health-related significance of studying basic mechanisms by which cells repair UV damage to their DNA. Bacteria of the genera Bacillus and Clostridium produce spores which are 10-100 fold more resistant to killing by UV than vegetatively growing cells. UV resistance of bacterial spores is due to (i) the unique photochemistry of spore DNA and (ii) the ability of germinating spores to accurately repair this unique DNA damage, known as """"""""spore photoproduct"""""""", or SP, by using two major repair systems, one of which is apparently specific for SP. The available evidence strongly implicates shifting of the DNA helix between the B-form and an A-like form during sporulation and germination in the production and subsequent repair of SP. The long-range objective of this project is to elucidate the structure, regulation, and mechanism of the SP-specific DNA repair system in Bacillus subtilis called spl. Since submission of the first application, DNA correcting a mutation in the spl system has been cloned, mapped on the B. subtilis chromosome, and sequenced; the spl gene sequence encodes a 40 kilodalton protein with no homolog in the current sequence databases. The objectives of this revised version of the application are: identifying both the cis-acting regulatory sites of the spl gene(s) and corresponding trans-acting factors; investigating how these regulatory components interact to direct the proper temporal and compartmental expression of the gene(s) during sporulation; identifying and purifying the enzyme(s) involved in SP repair, and cloning possible additional spl genes; and studying the mechanism and requirements of the SP repair reaction in vitro, using purified components.
