Persistence of Deoxyribonucleic Acid Polymerase I and Its 5′ → 3′ Exonuclease Activity in PolA Mutants of Escherichia coli K12

Abstract Partially purified enzyme preparations derived from a strain of Escherichia coli K12 with an amber mutation (polA1) in the DNA polymerase I gene contain approximately 1% as much polymerase I activity as the preparations from wild type strain purified by the same procedure. Two other polymerase I mutants, one temperature-sensitive (polA12) and the other nonsuppressible (polA5), also contain significant levels of polymerase I (12% and 35% of wild type, respectively). In the case of polA12, the polymerase I activity is abnormally thermolabile. The level of polymerase I observed in a given mutant is dependent, in part, on the pH and the template-primer used. Despite their low DNA polymerase I levels, these mutants possess nearly normal amounts of the 5' → 3' exonuclease activity that is known to be part of DNA polymerase I and is separable as a 2.8 S fragment by proteolytic cleavage. In the temperature-sensitive and nonsuppressible mutants, the 5' → 3' exonuclease and polymerase I activities cosediment at approximately 5.4 S just as in the wild type. In contrast, the polymerase I activity in the amber mutant sediments at 5.4 S, but the 5' → 3' exonuclease activity sediments at 2.8 S. These findings suggest that the residual DNA polymerase I activity sedimenting like the wild type enzyme in the polA1 mutants is the result of a low level read-through of the amber mutation, and that the 5' → 3' exonuclease represents the amber peptide, or a proteolytic cleavage product of it.