Characterization of a radical DNA repair enzyme: the spore photoproduct lyase – SPOREPAIR

Bacterial spores are dormant forms adopted by some bacteria under stress conditions. Specific features of spores include a high degree of dehydration, the compaction of the genome in a core protected by walls and membranes, and the absence of metabolic activity. As the result of these specificities, spores are more resistant than vegetative bacteria against a series of physical and chemical agents such as ionizing radiation, desiccation, chemicals and ultraviolet radiation. In the latter case, the presence of spores drastically reduces the efficiency of sterilization procedures based on the use of UVC that targets DNA in bacteria.
Resistance of spores to UV radiation is accounted for by two specific aspects. First, absorption of UV photons by DNA in spores does not lead to the formation of the classical pyrimidine dimers (cyclobutane dimer and (6-4) photoproducts) like in all other cell types. Rather, and because of the specific environment of DNA in spores, a unique lesion is produced that involves the formation of a methylene bridge between two adjacent thymines. This lesion, 5-(?-thyminyl)-5,6-dihydrothymine) is referred to as the spore photoproduct (SP).
Upon germination of the spore, the accumulated photoproducts have to be repaired quickly. The second specificity of spore photobiology occurs at this stage. Instead of using the classical nucleotide excision repair pathway, which is slow and ATP-consuming, SP are repaired by the enzyme "spore photoproduct lyase" (SPL), which reverts SP to the two initial thymines. SPL belong to the "radical SAM" family of enzymes. It possesses a [4Fe-4S] cluster that, in combination with the biological sulfonium S-adenosyl-methionine, initiates the repair reaction by production of 5’-deoxyadenosyl radical (Ado°). This radical abstracts a hydrogen atom from the saturated thymine of SP. This leads to the cleavage of the methylene bridge with regeneration of a first thymine. In addition, this rearrangement leads to the production of a methyl-centred radical on the second thymine. A hydrogen atom transfer completes the mechanism.
Although SPL is now well known, a numbers of unexplained points remain which will be addressed in the present project. We will study several aspects:
- 1) influence of the nature of the substrate (nucleoside, DNA duplex) on the efficiency of the reaction.
- 2) the chemical nature of the H atom donor in the end of the enzymatic process (AdoH or a cysteine of the protein).
- 3) the energetic and thermodynamic aspects of the reaction and iv) the tridimensional structure of SPL bound to its substrate.