Evidence that both growing DNA chains at a replication fork are synthesized discontinuously.

Escherichia coli, Bacillus subtilis, and T7-infected E. coli have been labeled with short pulses of [3H] thymidine, and the labeled DNA has been examined by sedimentation in alkaline sucrose. In all three systems, the great majority of the DNA labeled by a short pulse is found in the form of small DNA chains of 10S, the so-called Okazaki pieces. The B. subtilis and T7 nascent DNA fragments hybridize with equal efficiency to the separated strands of B. subtilis and T7 DNA, respectively. The results suggest that both growing DNA chains at a given replication fork are synthesized discontinuously in the case of E. coli, B. subtilis, and T7. We have found that the method used to terminate the pulse affects the size distribution of the labeled DNA; some methods allow joining of nascent DNA fragments after termination of the pulse. Previous reports of discontinuous DNA synthesis on only one growing DNA chain and continuous synthesis on the other DNA chain are probably due to preferential joining of Okazaki pieces on the DNA chain growing in the overall 5' leads to 3' direction.