O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O6-alkylguanine adducts. The interaction of dibromomethane (CH2Br2) and bromomethyl acetate (BrCH 2OAc) with AGT was studied in vitro, and the effect of AGT on their toxicity and mutagenicity was investigated using Escherichia coli strain TRG8 (lacking endogenous AGT) that expressed human AGT or its inactive C145A mutant. Both CH2Br2 and BrCH2OAc reacted with AGT at its cysteine acceptor site, abolishing its DNA repair activity with the latter agent being much more potent. The formation of AGT-Cys145S-CH 2OAc by BrCH2OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys145S-CH2Br adduct from CH 2Br2 was too unstable for such characterization. In the presence of CH2Br2, AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5′-d(AG)8-3′, but no cross-link was formed by BrCH2-OAc. Survival of cells exposed to CH 2Br2 was reduced, and the number of mutants was greatly increased when wild-type AGT was present. The cytotoxicity of CH 2Br2 was similar to that of BrCH2CH 2Br2, but the mutagenicity was about four times less. Virtually all of the AGT-mediated mutants induced by CH2Br 2 in the rpoB gene were at G:C sites with equal numbers of transitions to A:T and transversions to T:A. In contrast, BrCH2OAc was more than 10-fold less genotoxic than CH2Br2 and the survival of cells exposed to BrCH2OAc was not affected by AGT. The number of mutations (almost all G:C to A:T transitions) induced by BrCH 2OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. These results with CH 2Br2 are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys145S- CH2Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity. The results with BrCH2OAc suggest that it reacts directly with DNA to form O6-(CH2OAc) guanine, which, if unrepaired, causes G:C to A:T transitions. Our experiments reveal two novel pathways (direct inactivation of AGT and formation of AGT-Cys145S-CH2-DNA adducts) by which CH 2Br2 may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.
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