Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12

Abstract

The fpg+ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg+ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1::Knr mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Knr mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Knr mutation; (iii) phage P1 mediated transduction of the fpg-1::Knr mutation in the AB1157 background. The resulting fpg- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N'-nitro-N-nitroso-guanidine, (iii) ultraviolet light, (iv) gamma-radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.