Inactivation and degradation of O(6)-alkylguanine-DNA alkyltransferase after reaction with nitric oxide.

Abstract

O(6)-Alkylguanine-DNA alkyltransferase (AGT) plays a critical role in protection from the carcinogenic effects of simple alkylating agents by repairing O(6)-alkylguanine adducts via a direct transfer reaction. Nitric oxide (NO) or species derived from it are known to be able to initiate neoplastic growth and cannot only damage DNA, either directly or via the formation of intermediates such as nitrosamines, but can also inhibit some DNA repair processes. We have studied the inactivation of AGT by NO in detail in vitro and in vivo using wild-type human AGT (hAGT) and mutants at key residues. Our results show that hAGT is readily but reversibly inactivated by the formation of S-nitrosylcysteine at Cys-145, which is the alkyl acceptor site. The facile reaction of this cysteine residue with NO is attributable to its interaction with other residues in hAGT including His-146 and Glu-172 that activate the sulfhydryl group of Cys-145 to allow its nucleophilic attack on DNA adducts. Although the S-nitrosylcysteine adduct in hAGT is readily reversible by reaction with other cellular thiols, the formation of S-nitrosocysteine at Cys-145 was found to lead to the rapid degradation of the hAGT protein in vivo. This degradation is brought about by the ubiquitin/proteasomal system. The formation of an S-nitrosylcysteine at Cys-145 in hAGT in response to NO led to a large increase in the ubiquitination of the protein. This NO-mediated increase did not occur with the C145S or C145A mutants. A conformational change in hAGT, which involves opening of an asparagine hinge, normally occurs after alkylation of the protein in its role in DNA repair and causes degradation of the alkylated hAGT. Our results indicate that a similar effect occurs after reaction of the protein with NO. Thus, exposure to NO causes an irreversible loss of DNA repair capacity for alkylation adducts. This may contribute toward the potential development of tumors in cells upon chronic exposure to NO because of inflammation or infection. This may be of particular importance because such exposure may also lead to the formation of N-nitroso compounds that can act as alkylating agents.