O6-alkylguanine-DNA alkyltransferase - mediated toxicity of bis-electrophiles: Biochemical mechanisms

The studies described in this thesis have examined human alkyltransferase (hAGT)-mediated toxicity with other bis-electrophiles, specifically butadiene diepoxide (BDO) and epibromohydrin (EBH), which are epoxide compounds. In vitro studies were performed to investigate the reaction of hAGT with BDO and EBH. Covalent binding to DNA was observed when purified recombinant wild-type hAGT (wt-hAGT) was incubated with either BDO or EBH in the presence of an [35S]-labelled oligonucleotide. Unlike similar experiments with DBE, hAGT-DNA cross-link formation was also observed with both compounds when a mutant hAGT protein was used with its Cys145 active site residue altered to an alanine (C145A). This verified the ability of these epoxide compounds to cross-link at another residue on hAGT aside from its active site.;Studies in hAGT-expressing E. coli and CHO cells have confirmed the potentiation of toxicity by BDO, EBH and the dibromoalkanes in both model systems. With EBH and BDO, the enhancement in genotoxicity was also observed in E. coli expressing the inactive C145A-hAGT mutant, albeit not to the same degree as seen with wt-hAGT. This AGT-mediated toxicity was eliminated following expression of a C145A/C150S-hAGT double mutant indicating that covalent DNA adduct formation at the Cys150 residue were likely responsible for the increased toxicity observed in E. coli expressing the C145A mutant.;Investigation into the types of genomic alterations induced at the bacterial rpoB gene locus and the mammalian CHO-HPRT gene locus reveal at least two distinct mechanisms for mutagenesis mediated by bis-electrophiles upon reaction with AGT. Previous mass-spectrometry studies revealed an N7-guanine adduct formed by an in vitro reaction between AGT and DBE, which can depurinate resulting in toxic abasic sites. Subsequent rounds of replication result in preferential mis-incorporation of adenine across from the adducted base resulting in G:C to T:A transversions, which we have detected in our mutation spectrum analyses. The other pathway(s) responsible for the additional mutations observed with our model systems has yet to be defined. However, our work in E. coli with and without a functional nucleotide excision repair (NER) pathway have yielded some interesting insight into alternative processing of bis-electrophile induced covalent AGT-DNA cross-links.;Data from our laboratory utilizing E. coli with a proficient and deficient NER system indicate that this DNA repair pathway may augment hAGT-mediated dibromoalkane toxicity. Furthermore, we have extended these findings to include both BDO and EBH. Bacteria lacking a functional NER system are less susceptible to the mutagenic potential of the hAGT-DNA adducts formed during bis-electrophile exposure suggesting that the UvrABC complex of the bacterial NER pathway potentially binds to the site of the lesion. In this way, the UvrABC complex effectively shields the adduct from proteolysis and/or the action of other, more efficient repair proteins, while enhancing its susceptibility to error-prone trans-lesion synthesis, which manifests as an increase in mutation frequency of NER-proficient bacteria upon exposure to these compounds.;Overall, these studies have extended the hAGT-repertoire of genotoxic activation to include epoxides, another class of bis-electrophiles. These compounds, unlike the dibromoalkanes, can react at both the Cys 145 and Cys150 residues of hAGT. Furthermore, both BDO and EBH can potentiate their toxicity in the presence of AGT by two different mechanisms -- via a compound-AGT intermediate or a compound-DNA intermediate. The ability to cross-link at two residues on hAGT as well as the two modes of mediating toxicity probably contribute to the increased toxicity seen in vivo. Our NER findings suggest that this pathway amplifies the genotoxicity observed with hAGT and bis-electrophiles and demonstrates the interaction of two DNA repair pathways to promote DNA damage induced by a group of environmental carcinogens. (Abstract shortened by UMI.)...