Replication of M-13 DNA in plamolysed Escherichia coli cells. Formation of fully synthetic duplex DNA

Abstract

The replication of the double-stranded replicative-form DNA of bacteriophage M-13 was studied in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Newly synthesized DNA was density-labelled with bromodeoxyuridine triphosphate and analysed by equilibrium centrifugation in neutral CSCL. After a 60-min incubation at 30 degrees C 15-20% of the radioactive label in corporated from (32P)DGTP was found in fully synthetic duplex DNA, corresponding to 7-9 replicative form molecules/cell. The plasmolysed cell system is therefore capable of re-initiating new rounds of replicative form replication in vitro. The kinetics of labelling indicate that molecules are selected for replication at random from an intracellular pool of approximately 150 replicative form molecules. Rifampicin and nalidixic acid, which interfere with the semiconservative replication of replicative form DNA, completely prevent the formation of fully synthetic duplex DNA.