Replication of the Double‐Stranded Replicative‐Form DNA of Bacteriophage M‐13 in Plasmolysed Escherichia coli Cells

Escherichia coli cells, infected with bacteriophage M‐13 am‐5, when plasmolysed by treatment with 2 M sucrose incorporate radioactivity from deoxyribonucleoside triphosphates into phage‐specific DNA. After suppressing host DNA synthesis by treatment with mitomycin C prior to infection, more than 80% of the incorporated label is found in superhelical replicative form I molecules. If the incorporation is carried out in the presence of bromodeoxyuridine triphosphate, the radioactive label is found in equal amounts in two classes of hybrid replicative form molecules in which either the viral or the complementary strand is labeled. DNA synthesis depends on a functional gene‐2 product and shows a specific requirement for ATP and magnesium as divalent cation. Incorporation is inhibited by mitomycin C, nalidixic acid, phenyl ethanol, and sulfhydryl‐blocking agents. It is concluded that the observed DNA synthesis occurs by a semi‐conservative replication process resembling replicative form replication in vivo.