The effectiveness of the O6-alkylguanine-DNA alkyltransferase encoded by the ogtST gene from S. typhimurium in protection against alkylating drugs, resistance to O6-benzylguanine and sensitisation to dibromoalkane genotoxicity

Williams

Guengerich

et al. 2004

Chem. Res. Toxicol.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes O(6)-alkylguanine adducts. The interaction of dibromomethane (CH(2)Br(2)) and bromomethyl acetate (BrCH(2)OAc) 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 CH(2)Br(2) and BrCH(2)OAc 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-Cys(145)S-CH(2)OAc by BrCH(2)OAc was confirmed by mass spectral analysis, but the presumed AGT-Cys(145)S-CH(2)Br adduct from CH(2)Br(2) was too unstable for such characterization. In the presence of CH(2)Br(2), AGT was covalently cross-linked to an oligodeoxyribonucleotide, 5'-d(AG)(8)-3', but no cross-link was formed by BrCH(2)OAc. Survival of cells exposed to CH(2)Br(2) was reduced, and the number of mutants was greatly increased when wild-type AGT was present. The cytotoxicity of CH(2)Br(2) was similar to that of BrCH(2)CH(2)Br(2), but the mutagenicity was about four times less. Virtually all of the AGT-mediated mutants induced by CH(2)Br(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, BrCH(2)OAc was more than 10-fold less genotoxic than CH(2)Br(2) and the survival of cells exposed to BrCH(2)OAc was not affected by AGT. The number of mutations (almost all G:C to A:T transitions) induced by BrCH(2)OAc was slightly reduced by the presence of wild-type AGT and substantially increased by the inactive C145A mutant. These results with CH(2)Br(2) are consistent with a mechanism in which reaction at the active site Cys145 residue followed by attack of AGT-Cys(145)S-CH(2)Br at guanine in DNA forms a covalent adduct, which leads to cytotoxicity and to mutagenicity. The results with BrCH(2)OAc suggest that it reacts directly with DNA to form O(6)-(CH(2)OAc)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-Cys(145)S-CH(2)-DNA adducts) by which CH(2)Br(2) may cause damage to the genome in addition to the well-recognized pathway involving activation by GSTs.

Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

O ⁶-Alkylguanine-DNA Alkyltransferase Has Opposing Effects in Modulating the Genotoxicity of Dibromomethane and Bromomethyl Acetate

Williams

Guengerich

et al. 2004

Chem. Res. Toxicol.

“…It was therefore quite unexpected when it was found that the overexpression of Ogt (one of the two Escherichia coli AGTs) actually increased the toxicity of dibromoethane (DBE) and dibromomethane (DBM) toward E. coli (7). The enhancement of the mutagenicity and toxicity of DBE in E. coli has been confirmed and extended to AGTs from other species, including human AGT (hAGT) and other mammalian AGTs (8 -10), the E. coli Ada (9) and the Salmonella typhimurium Ogt, although the latter was only weakly active (11). The expression of the E. coli Ada was also reported to increase the killing of mammalian cells by DBE (12).…”

mentioning

confidence: 93%

Paradoxical Enhancement of the Toxicity of 1,2-Dibromoethane byO 6-Alkylguanine-DNA Alkyltransferase

Journal of Biological Chemistry

Pegg

Williams

et al. 2002

148

The presence of the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-haloethyl)glutathiones that mimic the reactive product of the reaction of DBE with glutathione, which is catalyzed by glutathione S-transferase. These experiments support a mechanism by which hAGT activates DBE. Studies in vitro showed a direct reaction between purified recombinant hAGT and DBE resulting in a loss of AGT repair activity and a formation of an hAGT-DBE conjugate at Cys 145 . A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the

“…Although an early study reported that the mutagenicity of 1,2-dibromoethane (DBE) was not affected by enzymes that repair alkylation lesions (4), there is now convincing data that AGT paradoxically promotes the toxicity of DBE (5,6). Overexpression of human AGT (hAGT) or AGTs from other species enhances the mutagenicity and lethality of DBE in Escherichia coli (5)(6)(7)(8)(9) and induces growth retardation in mammalian cells (10).…”

mentioning

confidence: 99%

Characterization of a Mutagenic DNA Adduct Formed from 1,2-Dibromoethane by O6-Alkylguanine-DNA Alkyltransferase

Journal of Biological Chemistry

Hachey

Valadez

et al. 2004

137

It has been proposed that the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive halfmustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N 7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N 7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N 7 -alkylation by alkyltransferase-S-CH 2 CH 2 Br and depurination, plus another as yet uncharacterized system(s).