Interaction of O6-alkylguanine-DNA alkyltransferase with nitric oxide and dihaloalkanes: Biochemical mechanisms and implication in carcinogenesis

The studies described in this thesis revealed the biochemical mechanisms underlining the interaction of DNA repair protein O 6-alkylguanine-DNA alkyltransferase (AGT) with nitric oxide (NO) and environmental toxicants dihaloalkanes; the effects of these interactions on the toxicity of these agents, the function of AGT and carcinogenesis.; NO reversibly inhibits human AGT (hAGT) activity by forming S-nitrosocysteine at the alkyl acceptor site Cys145. This led to an enhanced protein loss in vitro and in vivo. The reduction in hAGT stability was due to an increase in hAGT ubiquitination and its subsequent degradation by the ubiquitin/proteasomal system. NO-induced ubiquitination did not occur with the C145S or C145A mutants. These results suggest a protein conformational change following the nitrosylation of Cys145 that leads to degradation of the nitrosylated hAGT, an effect analogous to alkylation of Cys145 having on the stability of hAGT.; Several models were initially proposed for the mechanism of AGT-promoted dibromoethane (DBE) genotoxicity. However, in vivo studies using hAGT-expressing E. coli cells pointed to bioactivation of DBE by AGT. Furthermore, in vitro studies detected a covalent binding between purified recombinant hAGT and DBE. This results in the loss of hAGT repair activity and the formation of S-(2-bromoethyl)-Cys 145-hAGT. In the presence of DNA, the DNA-binding function of hAGT facilitates the reaction of the half mustard with DNA and the formation of hAGT-DNA adducts, most notably on guanines. The hAGT protein was observed cross-linked to the N7-guanine, a site where adduct formation leads to increased incidence of abasic sites. This suggests that hAGT-mediated dihaloalkane toxicity may involve cytotoxic and mutagenic abasic sites. Furthermore, the DNA-hAGT adducts that do not dissociate from DNA may induce mutations by activating error-prone DNA synthesis.; hAGT is involved in dual mechanisms that induce genotoxicity. Exposure to NO caused an irreversible loss of capacity for DNA repair of alkyl adducts. Reduction in hAGT stability may contribute to the development of tumors in cells upon chronic exposure to NO as such exposure may also lead to the formation of N-nitroso-compounds that can act as alkylating agents. Bromoalkanes, in addition to depleting active alkyltransferases, induce toxic DNA-hAGT adducts. Moreover, the hAGT-promoted dihaloalkane cytotoxicity indicates a potential for development of a new class of chemotherapeutical agents.