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 coupled with the efficient repair of accumulated damage. Exposure of bacterial spores to UV radiation results in the formation of a methylene-bridged thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), as the primary photoproduct. 1-3 SP accumulates in UV-irradiated spores, however it is rapidly repaired upon germination, thus giving rise to the extraordinary UV resistance of bacterial spores. 4,5 The repair of SP is catalyzed by the enzyme spore photoproduct lyase (SPL), and involves the direct reversal of SP to two thymines without base excision (Scheme 1).SPL is a member of the radical AdoMet superfamily, and utilizes a [4Fe-4S] cluster and Sadenosylmethionine (AdoMet) as essential cofactors in SP repair. 6-9 We have previously shown that SP repair is initiated by abstraction of H• from C6 of SP by an AdoMet-derived 5′-deoxyadenosyl radical; 10,11 this H-atom abstraction is thought to initiate a radical-mediated β-scission of the C5-C bridge bond in the photoproduct, as originally proposed by Mehl and Begley. 12 While two distinct diastereomers of SP (5R or 5S, Fig 1) 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. 13 The 5S configuration, therefore, is possible only in less well-defined DNA structures or as an interstrand crosslink. It was thus quite surprising when two recent reports concluded that SPL repairs only the 5S, and not the 5R, isomer of a synthetic SP substrate. 14,15 We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemicallydefined synthetic SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. This stereospecific repair of 5R-SP by SPL is consistent with the longstanding hypothesis that SP is a result of UV-induced dimerization of adjacent thymines in double-helical DNA.SPL was cloned from Clostridium acetobutylicum, overexpressed in Escherichia coli, and purified using a method similar to published procedures. 11 The enzyme contained 2.9 (± 0.2) Fe per SPL, and had UV-visible and EPR spectroscopic properties characteristic of an ironsulfur enzyme. The 5R and 5S diastereomers of protected (N-SEM, O-TES, and O-TBDMS) SP were synthesized using modifications of published procedures, 13,15 and were subsequently deprotected (Supporting Information). The structures of the fully protected, the di-SEM protected, and fully deprotected dinucleoside spore products were confirmed by 1 H and 13 C NMR techniques, and NOESY and ROESY were used to assign the stereochemistry at C-5 (S.I.). In order to remove any potential ambiguity associated with the assignment of stereochemistry at C-5 in the open dinucleoside forms of SP, the...