Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase

Liping Liu, Anthony E. Pegg, Kevin M. Williams, F. Peter Guengerich

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Abstract

The presence of the DNA repair protein O6-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 Cys145. A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the Gly136-Arg147 peptide from tryptic digests of AGT reacted with DBE. Incubation of AGT with DBE and oligodeoxyribonucleotides led to the formation of covalent AGT-oligonucleotide complexes. These results indicate that DBE reacts at the active site of AGT to generate an S-(2-bromoethyl) intermediate, which forms a highly reactive half-mustard at Cys145. In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys145-SCH2CH2OH.

Original languageEnglish (US)
Pages (from-to)37920-37928
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number40
DOIs
Publication statusPublished - Oct 4 2002

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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