Alexia Chandor-Proust
Study of Spore Photoproduct Lyase, a “Radical-SAM” enzyme involved in DNA repair
Published on 28 November 2008
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Thesis presented November 28, 2008
Abstract:
The major photoproduct of UV-irradiated DNA from bacterial spores is 5-(a-thyminyl)-5,6-dihydrothymine, a unique thymine dimer called Spore Photoproduct (SP). At the beginning of germination, this photoproduct is specifically repaired by the Spore Photoproduct Lyase (SPL), regenerating the two thymine monomers. SPL belongs to a family of iron-sulfur enzymes named « Radical-SAM ». In this familyy, proteins have a [4Fe-4S] cluster, chelated by three cysteines highly conserved constituting the “Radical-SAM” motif CxxxCxxC, and use S-Adenosylmethionine as a cofactor. They all use a radical based mechanism, initiated by 5’-deoxyadenosyl radical formation, derived from the homolytic cleavage of S-Adenosylmethionine by the reduced [4Fe-4S] cluster. Here, we have characterized, by biochemical and spectroscopic methods, the recombinant SPL from
Clostridium acetobutylicum or
Bacillus subtilis. Furthermore, we have synthesized a novel substrate called SPTpT under the form of a dinucleoside monophosphate. The structural characterization of this compound, using NMR spectroscopy, has been carried out. Moreover, this substrate is recognized and efficiently repaired by this enzyme, providing a convenient way to investigate the repair mechanism. Finally, the sequence of all SPL contains a fourth well-conserved cysteine. Site-directed mutagenesis shows that this residue is strongly involved in the repair mechanism but not in the cluster formation. The role of this cysteine 141 in the catalytic mechanism has been studied.
Keywords:
Spore, DNA, DNA lesion, photoproduct, DNA repair, SPL, Radical-SAM, iron-sulfur cluster
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