title: Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.
abstract: In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Abril
authors.forename: T
authors.initials: T
authors.lastname: Roldán-Arjona
authors.forename: M J
authors.initials: MJ
authors.lastname: Prieto-Alamo
authors.forename: A A
authors.initials: AA
authors.lastname: van Zeeland
authors.forename: C
authors.initials: C
authors.lastname: Pueyo
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D001426:Bacterial Proteins / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D004260:DNA Repair; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014157:Transcription Factors
chemical_list: D000477:Alkylating Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D005020:Ethyl Methanesulfonate; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; C067511:Ogt protein, E coli