Endonuclease III (nth) mutants of Escherichia coli

Abstract

Two strains that overproduce endonuclease III were found in a colony bank containing hybrid ColE1-Escherichia coli plasmids. The enzyme was identified in crude extracts by the degradation of partially depyrimidinated DNA in the presence of EDTA, by its sedimentation velocity, and by its associated thymine glycol-DNA glycosylase activity. An insertion mutation was produced by cloning the kanamycin-resistance gene of Tn5 into the plasmid copy of the nth gene. The mutation was then transferred to the chromosome in the following steps: (i) selection for chromosomal integration of the plasmid at 42 degrees C in a temperature-sensitive polA strain, (ii) curing via temperature shifts, and (iii) phage P1-mediated transduction of a new host. The insertion mutant, as well as a separately isolated deletion mutant, had no measurable glycosylase activity for DNA containing thymine glycol. Although such residues are common lesions in oxidized or irradiated DNA, the mutants were not unusually sensitive to H2O2 or gamma-rays. The insertion mutation had a mutator effect (4- to 22-fold enhancement) on one tested allele.