The DNA-alkyltransferase of Sulfolobus solfataricus: in vivo and in vitro studies on a conserved protein involved in repair of DNA alkylation damage

DNA-alkyltransferases are ubiquitous proteins, which act by a unique reaction mechanism, leading to irreversible protein alkylation and direct repair of highly mutagenic and carcinogenic DNA alkylation damages. The alkylated form of DNA-alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation (Daniels et al., 2000). We report here in vivo and in vitro studies on the DNA-alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). Transcriptional and post-translational regulation of SsOGT by DNA damage was studied, showing that although alkylating agent treatment induces increase of the transcript level, the protein is degraded by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability. The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. The data obtained on the wild type and a mutant protein impaired in DNA binding activity, clearly showed their higher stability to common chemical and physical denaturing agents as well as protease attack, in comparison with the human AGT (Mollwitz et al., 2012). The utilization of these archaeal catalysts as alternative protein tags to the commercially available SNAP-tag(TM) (Keppler et al., 2003) in extremophilic model organisms is evaluated. References oG. Perugino, A. Vettone, G. Illiano, A. Valenti, M.C. Ferrara, M. Rossi, and M. Ciaramella (2012). Activity and regulation of archaeal DNA-alkyltransferase: conserved protein involved in repair of DNA alkylation damage. J Biol Chem 287, 4222-4231. oD.S. Daniels, C.D. Mol, A.S. Arvai, S. Kanugula, A.E. Pegg, and J.A. Tainer (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor, and extrahelical base binding. EMBO J 19, 1719-1730. oA. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson (2003). A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86-89. oB. Mollwitz, E. Brunk, S. Schmitt, F. Pojer, M. Bannwarth, M. Schiltz, U. Rothlisberger, and K. Johnsson (2012). Directed evolution of the suicide protein O6-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability. Biochemistry 51, 986-994.