Characterization of a New Thermophilic Spore Photoproduct Lyase from Geobacillus stearothermophilus (SplG) with Defined Lesion Containing DNA Substrates

Abstract

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S] ؉ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.Spores of various Bacillus and Clostridium species are extremely resistant to harsh physical, chemical, and biological conditions allowing them to survive even under extreme conditions (1, 2). The oldest known viable spore was discovered from a Bacillus species, designated 2-9-3, in a 250 million-yearold salt crystal from the Permian Salado Formation (3). The resistance of spores from Geobacillus stearothermophilus toward heat is even so high that the survival of the organism during heat sterilization is used as a bioindicator for insufficient heat treatment (4).Particularly noteworthy is the unusually high stability of spores in the presence of UV light. For example under typical UV sterilization conditions, only about 70% of thermophilic G. stearothermophilus spores are inactivated. Under the same conditions, typical pathogens such as herpes simplex or polio viruses are fully destroyed (5, 6). In addition, UV irradiation of spores gives rise to different DNA lesions (7). Although in normal cells mostly cyclobutane pyrimidine dimers and (6-4) lesions (8) are formed, in spores the unusual photoproduct 5-thyminyl-5,6-dihydrothymine (SP), 2 depicted in Scheme 1 (7, 9), is exclusively generated (10). These differences in the photoreactivity may be because of an unusual packing of the DNA in spores (1, 11, 12) and the high amounts of dipicolinic acid (DPA) present in spores (13).During germination, the SP lesion is repaired either by the general nucleotide excision repair pathway (14, 15) or by a single enzyme, called spore photoproduct lyase, which is able to split the SP lesions directly back into two thymidines. Recent studies by Nicholsen et al. (16) and Broderick and co-workers (17) performed with the SP-lyase from Bacillus subtilis showed that the enzyme requires S-adenosylmethionine (AdoMet) as a cofactor for repair. A detailed sequence comparison, spectroscopic studies (18,19), and a recent labeling experiment (17) all provide evidence that the SplG is a member of the radical AdoMet enz...

DNA Repair and Free Radicals, New Insights into the Mechanism of Spore Photoproduct Lyase Revealed by Single Amino Acid Substitution

“…Further production of AdoH still took place after 60-min incubation reflecting the ability of the wt SPL enzyme to reductively cleave AdoMet in the absence of substrate, a property also reported by others (see Fig. 1B) (21,22). These data thus suggest that, at least with SPTpT as a substrate, the SPL enzyme reaction is not catalytic with regard to AdoMet and that AdoH does not serve as a hydrogen atom donor during the final step of the reaction, leading to TpT (see Scheme 1B).…”

“…This is consistent with the fact that the SPL-C141A enzyme chelates a [4Fe-4S] cluster with high yield, as shown by Mössbauer spectroscopy, and catalyzes the reductive cleavage of AdoMet in the absence of substrate, as does wt SPL (our results and Refs. 21,22). Thus, in both cases the reaction starts with the reductive cleavage of AdoMet by the reduced cluster generating the 5Ј-deoxyadenosyl radical, which then abstracts one of the H atoms at the methylene C-6 position.…”

Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase

“…Several SP lyases from Bacilli 7,12,15,16 and one from Clostridia 9 have been biochemically characterized. However, it was not clear so far how the repair activity takes place in Clostridia as the crucial cysteine residue found in Bacilli (C140 in Gt) is not conserved among clostridial SP lyases.…”

Synthesis and Stereochemical Assignment of DNA Spore Photoproduct Analogues

“…[42] The reconstitution of the {Fe-S} cluster and the repair assays were performed under anaerobic conditions in a glove box. For enzyme activity, the amount of 5'-deoxyadenosine and repaired SP was determined and analyzed by using rp-HPLC.…”