Mutations in Escherichia coli altering an apurinic endonuclease, endonuclease II, and exonuclease III and their effect on in vivo sensitivity to methylmethanesulfonate.

The levels of endonuclease II, an apurinic endonuclease, and exonuclease III in the parent strains (AB 1157) of Escherichia coli and in various mutants were determined by chromatography on DEAE-cellulose. AB 3027 and NH 5016 lacked endonuclease II and exonuclease III. BW 2001 lacked the apurinic endonuclease and exonuclease III while BW 2007, BW 9093, and BW 9059 lacked only exonuclease III. Deletion mutants BW 9101 and BW 9109 lacked all three enzymes. The latter mutants locate the genes for the two endonucleases in the region of exonuclease III (chith) of 38.2 min (White et al., 1976). All of the mutants which were sensitive to methylmethanesulfonate in vivo lacked exonuclease III, but not all mutants lacking exonuclease III were MMS sensitive. The deletion mutants and NH 5016 were the exceptions.

Endonuclease II, apurinic acid endonuclease, and exonuclease III.

An endonuclease of Escherichia coli active on a DNA treated with methylmethane sulfonate has been separated from an endonuclease active on depurinated sites. The former enzyme is disignated here as endonuclease II, while the latter enzyme is designated as apurinic acid endonuclease. Endonuclease II is also active on DNA treated with methylnitrosourea, 7-bromomethyl-12-methylbenz[a]anthracene, and gamma-irradiation. A third fraction which contains activities for both depurinated and alkylated sites needs further study. Endonuclease II, molecular weight 33,000, has been purified 12,500-fold and does not have exonuclease III activity. Apurinic acid endonuclease, molecular weight 31,500, has been purified 11,000-fold and does not have exonuclease III activity. Exonuclease III, molecular weight 26,000, has been purified 2300-fold and does not have endonucleolytic activity at depurinated reduced sites or at alkylated sites in DNA. Therefore, these are three separate proteins. Exonuclease III can produce, presumably by its exonucleolytic activity, double-strand breaks in heavily alkylated DNA under conditions where it does not make single-strand endonucleolytic breaks at either depurinated-reduced or alkylated sites.