Abstract
Spore photoproduct lyase (SPL) repairs a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct, or SP, in germinating endospores. SP is the exclusive DNA photo-damaging product found in endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. Early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair SP in the absence of light. Recently, it has been established that SPL belongs to the radical S-adenosylmethionine (SAM) superfamily. The enzymes in this superfamily utilize a tri-cysteine CXXXCXXC motif to bind a [4Fe-4S] cluster. The cluster provides an electron to the S-adenosylmethionine (SAM) to reductively cleave its C5′-S bond, generating a reactive 5′-deoxyadenosyl (5′-dA) radical. This 5′-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. The H atom donor is suggested to be a conserved cysteine141 in B. subtilis SPL; the resulting thiyl radical likely interacts with a neighboring tyrosine99 before oxidizing the 5′-dA to 5′-dA radical and, subsequently, regenerating SAM. These findings suggest SPL to be the first enzyme in the large radical SAM superfamily (>44,000 members) to utilize a radical transfer pathway for catalysis; its study should shed light on the mechanistic understanding of the SAM regeneration process in other members of the superfamily.
Highlights
The germinating spores utilize two major pathways to repair SP: the general nucleotide excision repair pathway (NER) mediated by the uvr genes [17] and a spore-specific DNA repair system, which involves in situ monomerization of SP into two thymine residues mediated by an enzyme, named spore photoproduct lyase (SPL, Figure 1), which is encoded by the spl gene [18,19,20,21]
Spore photoproduct lyase (SPL) is a member of the so-called radical S-adenosylmethionine (SAM) superfamily, which is defined by the characteristic CXXXCXXC motif [33], recent evidence suggests that other three-cysteine motifs facilitate this radical chemistry [34,35,36]
Only SPL, lysine-2,3-aminomutase and 7-carboxy-7-deazaguanine synthase have been shown to use SAM catalytically [34,39,80], though more are expected, considering the large number of radical SAM enzymes discovered to date [38]
Summary
Bacterial endospores are the longest-lived cells known on earth. They are so resistant to harsh environments, such as vacuum, heat, desiccation and irradiation, that they may survive in outer space as hitchhikers clinging to the outside of spacecraft [1]. The genomic DNA adopts a B-conformation, and thymine photo-dimerization leads to the formation of cyclobutane pyrimidine dimer (CPD) and pyrimidine (6-4) pyrimidone photoproduct ((6-4) PD) [4]. Of the total photoproducts in spore’s genomic DNA, and the cis-syn CPDs comprise ~5% of the photo-lesions [12]. SPs formed under UV irradiation could account for as many as 8% of the total thymine residues in spore genomic DNA [11,14]. They must be repaired, as SPs prove lethal to the germinated bacteria [9,16]
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