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 alkyltr ansferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endoge nous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoetha ne (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permea ble to exogenous drugs, with the AGT inhibitor O-6-benzylguanine (BG) aboli shed the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the obse rvation that E. coli expressing either the C145A AGT mutant, which is inact ive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, whi ch are inactive due to alteration of the DNA binding domain, did not enhanc e the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, whi ch is active in repairing methylated DNA but is totally resistant to inacti vation by BG due to alterations in the active site pocket, was unable to en hance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K 165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moder ately resistant to BG, did increase mutations in response to DBE but was le ss active than wild type. These results suggest that human AGT is able to i nteract with a DNA lesion produced by DBE but, instead of repairing it, con verts it to a more genotoxic adduct. This interaction is prevented by mutat ions that modify the active site of AGT to exclude BG. (C) 2000 Elsevier Sc ience B.V. All rights reserved.