Properties of a temperature-sensitive radiation-sensitive mutant of Escherichia coli.

We have described a temperature-sensitive, radiation-sensitive mutant of Escherichia coli. In vitro assays have demonstrated that DNA ligase activity is markedly reduced in this strain relative to that of the parent strain. Okazaki fragments of newly replicated DNA accumulate in this mutant strain upon growth at the restrictive temperature. These results imply that the same enzyme, DNA ligase, functions both in normal semiconservative replication and in nonconservative repair replication of DNA. In a previous communication' we reported the isolation and characterization of a temperature-sensitive, radiation-sensitive mutant of Escherichia coli. The evidence presented therein demonstrated that this mutant is able to excise py- rimidine dimers induced by ultraviolet irradiation from DNA, and is also able to perform nonconservative repair replication of DNA at the restrictive tempera- ture. We concluded that the defect lay in a late step of dark repair, possibly in DNA ligase.2. 3 We present in this communication results of in vitro assays of DNA ligase activity, which allow the conclusion that the defect in this mutant strain is in this function. Recent evidence4-8 suggests that DNA ligase may play a role in normal, semiconservative replication of DNA, in addition to its postulated role in non- conservative repair replication. Newly replicated DNA is recovered as single- stranded fragments after denaturation, suggesting that DNA replication occurs in a discontinuous manner. It has been proposed that the fragments are joined together subsequent to their synthesis in a reaction catalyzed by DNA ligase to form continuous strands of DNA. Such fragments of newly replicated DNA would be expected to accumulate in a DNA ligase-deficient mutant. We pre- sent below evidence that such fragments do accumulate in our temperature- sensitive, radiation-sensitive mutant of E. coli upon growth at the restrictive temperature. Materials and Mlethods.-E. coli TAU-bar9 and the mutant derivative thereof, ts-7, were used as previously described. DNA ligase assays: Two-liter cultures of TAU-bar and ts-7 were grown at 25?C with aeration in tryptone broth containing, per liter: Bacto-Tryptone, 10 gm, NaC1, 5 gm. All growth media were supplemented with 2.0 ug/ml thymine (Calbiochem). The cul- tures were grown to a density of 4 to 5 X 108 cells/ml, harvested by centrifugation, and resuspended in 4-ml sonication buffer (0.05 M glycylglycine, 0.001 1M EDTA, 0.001 M glutathione-SH, pH 7.0) per gram packed cells. The cells were disrupted by sonication, and cell debris was removed by centrifugation. The crude extracts were partially puri- fied by streptomycin precipitation and ammoniumn sulfate precipitation, as described by Olivera and Lehman.3 Protein concentrations were determined by the biuret method. 1195