The role of human O(6)-alkylguanine-DNA alkyltransferase in promoting 1,2-dibromoethane-induced genotoxicity in Escherichia coli.
The expression of the DNA repair protein human O(6)-alkylguanine-DNA alkyltransferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endogenous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoethane (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permeable to exogenous drugs, with the AGT inhibitor O(6)-benzylguanine (BG) abolished the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the observation that E. coli expressing either the C145A AGT mutant, which is inactive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, which are inactive due to alteration of the DNA binding domain, did not enhance the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, which is active in repairing methylated DNA but is totally resistant to inactivation by BG due to alterations in the active site pocket, was unable to enhance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moderately resistant to BG, did increase mutations in response to DBE but was less active than wild type. These results suggest that human AGT is able to interact with a DNA lesion produced by DBE but, instead of repairing it, converts it to a more genotoxic adduct. This interaction is prevented by mutations that modify the active site of AGT to exclude BG.
Liu
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Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, 17033-0850, USA.
Xu-Welliver
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Pegg
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D004274:DNA, Recombinant / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D015946:Ethylene Dibromide / Q000633:toxicity*; D006801:Humans; D009152:Mutagenicity Tests; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D015946:Ethylene Dibromide; D019853:O(6)-Methylguanine-DNA Methyltransferase