Initiation and termination of the bacteriophage øX174 rolling circle DNA replicationin vivo: packaging of plasmid single–stranded DNA into bacteriophage øX174 coats

Abstract

The bacteriophage øX174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting øX174 phages. A consequence of this interaction is packaging of single–stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with tow øX (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (> 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (> 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.