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...

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe-4S] + cluster accounting for 3-4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe-4S] + cluster is observed that accounts for 34-45% of total iron. Addition of S-adenosyl-methionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-Lmethionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min −1 mg −1 , whereas no repair was observed for the 5S diastereomer.

The first step in purine biosynthesis converts PP1‐ribose‐P (PPRP) and glutamine into β‐P‐ribosylamine (PRA) and glutamate. The enzyme that catalyzes this reaction is called phosphoribosylpyrophosphate amidotransferase (GPATase or PurF). This enzyme has been extensively characterized in both prokaryotes (Bacillus subtilis) and in eukaryotes (human) but has never been characterized in an archaeon. In Sulfolobus solfataricus, a well characterized archaeon, the gene most likely to code for the PurF protein is SSO0632. The neighboring gene, SSO0633, while similar to other PurF‐like genes, does not code for the necessary active site cysteine. Both genes have been successfully cloned in pMAL‐c5E vector. Protein expression and experimental characterization of these proteins will be discussed. Funding for this project was received from a Cottrell College Science Award.

In this experiment methyltransferases RumA and RumB were studied. These proteins are part of a group of enzymes that conduct post transcriptional modifications on RNA. RumB specifically methylates uracil 747 on the 23S subunit of ribosomal RNA. RumA methylates uracil 1939 on the 23S subunit of ribosomal RNA. Both RumA and RumB transfer a methyl group from a donor molecule (S‐Adenosylmethionine) to a receptor molecule (uracil on RNA). These two proteins share significant similarity in sequence as well as the incorporation of an Fe‐S cluster. Due to the similarity it is proposed that RumB should follow RumA characteristics. The RumA and RumB genes were successfully amplified using PCR, ligated into a plasmid, transformed, sequenced, and induced. The proteins were expressed using the pET‐45b expression vector. Cell lysis was not successful as the protein of interest remained in the cell pellet as opposed to the supernatant. Future research will include protein purification and analysis.