A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H202, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.Endonuclease IV (23) of Escherichia coli is an apurinicapyrimidinic (AP) DNA endonuclease, i.e., a DNase specific for apurinic and apyrimidinic sites in DNA. It catalyzes the cleavage of a phosphodiester bond 5' to the AP site and is an example of the most prevalent form of AP endonuclease found throughout nature. Within E. coli, however, the enzyme is a minor one; it accounts for no more than 10% of the AP endonucleolytic activity measured in crude extracts (24). The major AP endonucleolytic activity of E.

The xth gene of Escherichia coli K-12, which encodes exonuclease III, has been sequenced. Exonuclease III from a cloned copy of the E. coli K-12 gene has been purified and characterized. The molecular weight (30,921), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme. The xth promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence reveals that a region of dyad symmetry which could form a hairpin stem-loop structure in RNA characteristic of a rho-dependent terminator lies immediately downstream from the xth gene.

The nfo gene of Escherichia coli K-12 which encodes endonuclease IV has been sequenced. The predicted gene product has a molecular weight of 31,562, in good agreement with the size of the gene product estimated by maxicell analysis. The nfo promoter was mapped by primer extension of in vivo transcripts. Inspection of the nucleotide sequence revealed no regions of potential secondary structure corresponding to a transcriptional terminator downstream from the structural gene; however, there was a potential open reading frame immediately downstream from the nfo structural gene.