In vivo analysis of the initiation of bacteriophage T7 DNA replication.

We have examined the initiation of bacteriophage T7 DNA replication in vivo using a pulse-labeling technique. The pulse-labeling technique permits the rapid identification of initiation sites on the T7 chromosome and a determination of the rate of movement of the replication fork. This technique has been used to analyze a number of phage mutants having alterations in the nucleotide sequence of the primary origin. The experiments confirm the results obtained by electron microscope analysis on the mapping of the primary origin region and demonstrate the requirement for a T7 promoter in the primary origin. The secondary origins were found to be located near the center and at the right end of the genome. Analysis of T7 phage harboring mutations in the essential replication genes of T7 shows that they fell into three classes. The first, including those mutated in genes 4 and 5, do not initiate DNA synthesis. The second, in genes 3, 6, and 1.2, initiate and elongate as wild-type phage, albeit some with lower rates of synthesis, during the first round of replication and then cease DNA synthesis. Mutations in gene 2 have no apparent effect on initiation or elongation.