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

Abstract

Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class 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.

Understanding of the activity of a protein involved in DNA repair by biochemical, structural and in silico approaches

“…We used the Sulfolobus solfataricus thermostable ortholog (SsOGT) as a model for AGTs [1], by performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNAbound and alkylated version of the protein. With this protein, we were able to highlight conformational changes and perturbations of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N-and C-terminal domains of SsOGT is important for protein activity and stability [2]. All the data allowed us to propose a general model of structural rearrangements occurring during the reaction cycle of AGTs [3], and proposing it as a starting point to design strategies to modulate AGT activity in therapeutic settings.…”

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

“…The adduction of such chemicals or their reactive metabolites to DNA, alters DNA structure and can impede DNA/DNA and DNA/protein interactions. 4,5 DNA adducts that escape repair can disrupt cell division, or induce mutations which can lead to downstream cellular dysfunctions. 6–8 DNA adducts of genotoxicants have been employed as biomarkers in molecular epidemiology studies designed to assess the role of chemical exposures in the etiology of cancer.…”

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

“…Despite the different primary structures (Figure 2a), thermophilic enzymes show a typical AGT protein architecture, consisting of two domains [37]: a highly conserved C-terminal domain (CTD), surprisingly superimposable for all available AGT structures (Figure 2b), and a N-terminal domain (NTD), which is very different among AGTs and whose function is not well understood (likely involved in regulation, cooperative binding, and stability [6,38,39]). The CTD contains the DNA binding helix-turn-helix motif (HTH); the Asn hinge, which precedes the -V/IPCHRVV/I-amino acid sequence containing the conserved catalytic cysteine (except the Caenorhabditis elegans AGT-2 that has the -PCHPsequence [40,41]); and the active site loop, responsible for the substrate specificity.…”

“…Because AGT covalently binds a benzyl-fluorescein moiety of its substrate after reaction, it is possible to immediately load the protein product on a SDS-PAGE-the gel-imaging analysis of the fluorescence intensity gives a direct measure of the protein activity because of the 1:1 stoichiometry of protein/substrate (Figure 3). Signals of fluorescent protein (corrected by the amount of loaded protein by Coomassie staining analysis) obtained at different times are plotted, and a second order reaction rate is determined [38,39,46,52,59,60]. This method can be applied to all AGTs that bind O 6 -BG, with the exception of the E. coli Ada-C [61,62].…”