Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Abstract

The overwhelming majority of DNA photoproducts in UVirradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3-5)-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3-5)-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a singleor double-stranded DNA for recognition and repaired by the SP lyase enzyme.The DNA of all organisms is subject to modifications upon exposure to a wide variety of chemical and physical agents. Among them, solar ultraviolet radiation is known to induce dimerization reactions between adjacent pyrimidines (1). In the vast majority of living systems, the resulting photoproducts are cyclobutane pyrimidine dimers (CPDs) 5 and pyrimidine (6-4) pyrimidone photoproducts (Scheme 1A) that can be generated at any of the four bipyrimidine doublets (TT, CT, TC and CC), although the yields of the lesions depend on the bases involved (2). These lesions induce mutations and can be lethal because of blocking of the replication machinery. The photochemistry in bacterial spores is quite different. Indeed, in this dormant form produced by some bacteria such as Bacillus subtilis, the only photoproduct produced upon exposure to UV light corresponds to two thymines linked by the methyl group of one of the bases (3, 4). The formation of this specific lesion, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP) (Scheme 1A), is explained by specific features of the spores, including DNA conformation (A form), dehydration, the presence of dipicolinic acid in the core, and binding of small acid-soluble proteins to DNA (5-8). The formation of SP as the unique DNA lesion in irradiated spores is proposed to account for their extreme resistance to UV radiation. Indeed, spores express a specific repair enzyme, the spore photoproduct lyase (SP lyase) that directly reverts SP to two unmodified thymines upon germination (9, 10), much more efficiently than dimeric photoproducts are removed from other cell types by the classical nucleotide excision repair pathway. The specific photochemistry of DNA in spores combined with the action of SP lyase appears to be a major evolutionary advantage for spore-forming bacteria in resistance to UV radiation.In their N-terminal half, all SP lyase enzymes contain a strictly conserved a...

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

“…The insertion of a cysteine residue inside the mutated enzyme active-site thus completely restored the SP lyase repair activity. Furthermore, kinetic experiments revealed that, while the wild-type SP lyase had a DNA repair rate of 1.66 nmol min À1 mg À1 (in the range of the reported values for SP lyases 5,12 ), the double mutant proved to be more than two-times faster (3.48 nmol min À1 mg À1 ) (Fig. 2).…”

“…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.…”

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

“…At each time point (0, 15 30, 60, 120, 215, and 280 min) 20 l and 10 l of the solution were transferred into Eppendorf tubes, and the reaction was stopped by flash-freezing in liquid nitrogen. Conversion of the spore photoproduct (SPTpT) into the unmodified dinucleoside monophosphate (TpT) and other products in SPL-treated samples was quantified by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS), using the same conditions as for samples treated by wt SPL (7).…”

“…Iron and Sulfide Binding to WT and C141A B. subtilis SPL-[Fe-S] cluster reconstitutions of wt and C141A B. subtilis SPL were carried out under strictly anaerobic conditions in a glove box containing Ͻ2 ppm O 2 as previously described (7).…”

Chemical investigation of light induced DNA bipyrimidine damage and repair

“…Removal For enzymatic assays the SP-containing DNA and the SP dinucleotide are normally produced via irradiation of dried oligonucleotides together with dipicolinic acid (DPA). 5,14,16,[46][47][48][49] However, in order to obtain defined nucleotide sequences, necessary for many applications such as crystallization, this method is limited to short oligonucleotides and the yields are very unsatisfactory.…”