ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair–deficient escherichia coli K‐12

Abstract

We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt- bacteria to those in their isogenic ogt+ parental counterparts. The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt- and ogt+ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L-arabinose resistance. Induced mutations occurred only in excision repair-defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt- bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt+ and ogt- bacteria were not dependent on the ogt- null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging agent. The mechanism of this effect has yet to be established.

Reactions of Glyceraldehyde 3-Phosphate Dehydrogenase Sulfhydryl Groups with Bis-Electrophiles Produce DNA–Protein Cross-Links but Not Mutations

“…Subsequently this phenomenon was also identified in systems overexpressing AGTs (11). bis-Electrophiles induced mutations in these systems through induction of DNA-GSH and DNA-AGT crosslinks by reaction between nucleophilic sulfhydryls, bis-electrophiles, and DNA (15,19).…”

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

“…At present, we cannot definitively identify the adduct or adducts that give rise to the majority of the mutants which were G:C to A:T transitions and this is the subject of ongoing investigation. These could arise from an AGT protein-DNA cross-link at either the O 6 or N 2 positions. The extent to which by-pass DNA polymerases (37,48) are able to extend past such protein-DNA cross-links is unclear and there may be an initial "repair" event that removes part of this bulky adduct and leaves a lesion that is more readily by-passed.…”

“…Future studies are also needed to test the role that other DNA repair pathways play in protecting from DBE (4,6).…”

“…AGT repairs O 6 -alkylguanine adducts that are of major importance in initiating mutations and cytotoxicity. This repair process is mediated by transfer of the alkyl group from the O 6 atom of Gua to the active site Cys residue in the AGT protein. AGT is inactivated by the alkyl group transfer, and therefore additional repair requires new protein synthesis.…”

“…AGT is inactivated by the alkyl group transfer, and therefore additional repair requires new protein synthesis. 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).…”