Mechanism of DNA chain growth, II. Accumulation of newly synthesized short chains in E. coli infected with ligase-defective T4 phages.

The most recently replicated portion of the bacterial and T4 phage chromosome, selectively labeled by an extremely short radioactive pulse, can be isolated, after denaturation, as short DNA chains with an average sedimentation coefficient of 7-11S.1-3 This supports an hypothesis of discontinuous DNA chain growth, by which short stretches of DNA are synthesized at the replicating point and subsequently connected to the older portion of the growing strands by formation of phosphodiester linkages. The joining of these short chains is assumed to be carried out by polynucleotide ligase, an enzyme discovered recently in normal and T4 phage-infected E. coli.7-12 If DNA replicates in vivo by such a mechanism, the newly synthesized short DNA chains would accumulate in the cell under conditions where the function of ligase is temporarily impaired. In the present work, this prediction was tested and verified by experiments with T4 phage mutants, which produce thermosensitive polynucleotide ligase. 13 Materials and Methods.-Bacteria and bacteriophages: Escherichia coli B/5, bacteriophage T4 ts A80, and T4 ts B20 were generous gifts of Dr. R. S. Edgar. T4 ts A80 and T4 ts B20 are gene 30 mutants, which induce temperature-sensitive polynucleotide ligase.13 T4D (wild-type) was supplied by Dr. J. Tomizawa. Infection of bacteriophages: E. coli B/5 was grown in a glucose salt medium (Medium A described previously3) at 370 to 5 X 108 cells/ml, and incubated at 200 for 20 min. DL-tryptophan (40 4g/ml) and bacteriophages (m.o.i. = 10) were then added to the culture and the incubation was continued at 200 with shaking. Temperature shift: When the volume of a culture was 10 ml or less, temperature shift from 200 to 30 44° was attained simply by pouring the culture into a flask being shaken in a water bath of the new temperature. For larger volumes, the culture was warmed to a new temperature by being shaken in a 500 water bath before being placed in the water bath of the new temperature. By these methods temperature shift-up was completed in 1 min. For temperature shift-down, the culture was chilled in ice water to the desired temperature. It took about 10 see to change the temperature of a 30-ml culture from 43 to 30°. Other methods: Pulse-labeling with H3-thymidine, DNA extraction, and alkaline sucrose gradient sedimentation were carried out as described previously.3 Results.-Pulse-labeling at 43-440: Our previous study (ref. 3, Fig. 6) showed that H3-thymidine that had been incorporated into T4 wild-type infected cells during the period of active phage DNA synthesis (70 min after infection at 200) first appeared in short DNA chains having an average sedimentation rate of 8-9S (in alkali) before transition to large chains with sedimentation coefficients of 30-60S. In order to see if temporal inhibition of polynucleotide ligase causes accumulation of the nascent short DNA chains, E. coli B/5 infected with T4 ts A80 or ts B20 (gene 30) was pulse-labeled with H3-thymidine 65-70 minutes