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Mutagenesis and DNA repair for alkylation damages in Escherichia coli k‐12
Author(s) -
Abril Nieves,
RoldánArjona Teresa,
PrietoAlamo MariaJosé,
Van Zeeland Albert A.,
Pueyo Carmen
Publication year - 1992
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850190405
Subject(s) - mutagenesis , mutant , mutation , dna repair , biology , nucleotide excision repair , dna , ethylnitrosourea , dna damage , escherichia coli , biochemistry , methyl methanesulfonate , in vivo , mutation frequency , mutagen , microbiology and biotechnology , gene , genetics
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 (O 6 ‐alkG, N 7 ‐alkG, N 3 ‐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 (Δ uvrB ) and unable to induce the adaptive response ( ada ::Tn10). The very low initial levels for O 6 ‐meG and O 6 ‐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, O 6 ‐alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N 3 ‐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). This is in accordance with the relative inefficiency of these agents as inducers of the adaptive response, as well as the relative inefficiency of the ada ATase in dealing with alkylation products longer than methyl adducts. The ogt protein provided additional resistance to mutation by low doses of methylating agents, as it was observed in ada deficient bacteria. Thus the broad substrate specificity of ogt ATase would appear to be also of biological relevance.