O⁶-alkylguanine-DNA alkyltransferase has opposing effects in modulating the genotoxicity of dibromomethane and bromomethyl acetate

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.