Abstract
Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA, which results in accumulation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), coupled with the efficient repair of accumulated damage by the enzyme spore photoproduct lyase (SPL). SPL is a member of the radical AdoMet superfamily of enzymes, and utilizes an iron-sulfur cluster and S-adenosylmethionine to repair SP by a direct reversal mechanism initiated by H atom abstraction from C-6 of the thymine dimer. While two distinct diastereomers of SP (5R or 5S) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure; the 5S configuration is possible in less well-defined DNA structures or as an interstrand cross-link. We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. The observation that 5R-SP, but not 5S-SP, is a substrate for SPL is consistent with the expectation that 5R is the SP isomer produced in vivo upon UV irradiation of bacterial spore DNA.