DNA degradation in an amber mutant of Escherichia coli K12 affecting DNA ligase and viability

Abstract

Isolation of an amber mutant lig-321 (or dnaL321) if Escherichia coli K12 with a defect in DNA ligase activity was previously reported (Nagata & Horiuchi, 1974). This was the first demonstration that, in E. coli, conditionally lethal nonsense mutants can be isolated selectively. Unlike the hitherto available E. coli K12 DNA ligase-deficient ( lig) mutants, the DNA of this mutant is degraded under lethal conditions. This paper describes its further characterization. The DNA degradation was found to be an energy-requiring process, in which endonuclease I did not seem to participate. Kinetic analyses of prelabeled DNA indicated that the parental strands were degraded. The sedimentation profile of prelabeled DNA in an alkaline sucrose gradient showed that the extensive degradation was preceded by a step in which the parental strands were broken into relatively large pieces. At least in the early phase of degradation, which we examined by alkaline sucrose gradient centrifugation of pulse-labeled DNA, synthesis of discontinuous daughter chains (Okazaki fragments, Okazaki et al., 1968) was confirmed. Joining of the nascent chains, however, was completely inhibited. Genetic analyses revealed that the mutant allele is recessive to the wild type. This agrees with in vitro studies in which the mutant crude extract was found not to inhibit DNA ligase activity of the wild type extract. These and other properties of the lig-321 mutant were compared with the other DNA ligase-deficient mutants of E. coli. The role of this enzyme in DNA replication, repair and recombination is discussed.