Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein

The alkylated DNA-protein alkyltransferases (AGTs, OGTs or MGMTs; EC 2.1.1.63), evolutionary conserved from prokaryotes to higher eukaryotes, directly repair highly mutagenic and carcinogenic O6-guanine-alkylation lesions on DNA. Because of their irreversible reaction mechanism, the alkylated form of this class of proteins is inactive, leading to degradation via ubiquitination in eukaryotes. Great interest raised for the human ortholog, hAGT, because it is involved in resistance to alkylating chemotherapy drugs acquired by many tumor cells. Understanding the mechanism of hAGT inactivation upon the alkylation reaction might be helpful to prepare appropriate mechanism-based inhibitors, in order to restore the efficacy of the alkylating agents treatment. Biochemical and structural analyses suggested that conformational changes occur, but the intrinsical instability of alkylated-hAGT, which rapidly leads to structure destabilization, prevented the possibility of detailed description of these changes. We report here studies on the alkylated DNA-protein alkyltransferase, SsOGT, from the hyperthermophilic archaeon Sulfolobus solfataricus. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of this protein in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins.