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

Abstract

The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(␣-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed.

Post-translational Modification of Ribosomal Proteins

“…Further experiments are needed to substantiate this mechanism. Most of the radical AdoMet enzymes that have been functionally studied display a decoupled AdoMet cleavage, which refers to a reductive cleavage of AdoMet in the absence of substrate, or a reductive cleavage of AdoMet that exceeds the stoichiometry required for catalysis (37,38). In the case of RimO this decoupling is observed only in the presence of the substrate (AdoH/product around 5).…”

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

“…Instead, an alanine residue (A138) occupies this position, a substitution known to preclude DNA repair in Gt SP lyase. 6,7 Interestingly, we identified in the clostridial structural model, a cysteine residue (C74) in close proximity to the methylene bridge of the SP lesion suggesting mechanistic adaptation among SP lyases. Exploiting the differences between clostridial and bacilli SP lyases, we aimed to reorient the H-atom transfer pathway in Gt SP lyase and investigate functional and structural diversities in this enzyme family.…”

“…a sulfinic acid adduct). 6 We solved the crystal structure of SP lyase from Geobacillus thermodenitrificans (Gt) and discovered that this crucial cysteine residue (C140 in Gt) is located in close proximity to the SP lesion. 7 The position and the distance of this cysteine residue, relative to the a-methylene carbon atom of the 3 0 -thymine moiety (4.5 Å), led us to propose a function as ultimate H-atom donor to the DNA lesion and a key role in the ill-defined migration and control of radicals inside the enzyme active site.…”

“…S6 and Table S1, ESI †). The formation of the 6-mer* and the 6-mer products in the absence of C140 is explained by the quenching of the allylic radical intermediate by a SO 2 À radical (originating from sodium dithionite) 6 or its reaction with DTT used as a reductant, respectively. 11 Since in the wild-type enzyme C140 fulfills the function of radical quenching, we hypothesized that positioning another cysteine residue in the vicinity of the DNA lesion should rescue the repair activity of the C140A mutant.…”

Chemical investigation of light induced DNA bipyrimidine damage and repair

“…[51][52][53] Indeed, a mechanism has been proposed and is supported by experiments such as mutation studies on SP lyase, but structural information such as crystal structures is still lacking. 54,55 …”