Replication of phage G4. A novel and simple system for the initiation of deoxyribonucleic acid synthesis (original) (raw)

1975, Journal of Biological Chemistry

Conversion in uitro of single-stranded circular DNA of phage G4 (related to phage 4X174) to the double-stranded replicative form (RF-II) depends on a novel and relatively simple group of three proteins: a priming protein of approximately 65,000 daltons, the DNA unwinding protein, and the DNA polymerase III holoenzyme. Stimulation by ATP and GTP suggests an RNA synthetic step in the priming of DNA synthesis. The synthetic strand in the RF-II contains a small gap at a unique position relative to the template strand; the 5' end of the gap is about 250 nucleotide residues (5% of the genome length) away from the single site of cleavage by a restriction endonuclease (Eco RI). Studies of the conversion of single-stranded circular DNA to the duplex form in vitro have disclosed two distinctive systems for initiation of DNA synthesis in extracts of Escherichia coli (1). One, exemplified by filamentous phage M13, is inhibited by rifampicin because RNA polymerase action is required for priming of DNA replication (2). The other, illustrated by the polyhedral phage @X174, is resistant to rifampicin (3). The @X174 system depends upon seven or more proteins, including those inferred from genetic studies to be needed for initiation of the E. coli chromosome at its origin (4). Upon examining the replication of the DNA of phage G4 (5), a particle resembling @X174, we have now come upon a third system. As with 4X174, the enzymes required for initiation of DNA synthesis on the G4 template are resistant to rifampicin. Unlike $X174, the G4 system is far simpler and depends on only three of the replication proteins. In this report, we (a) compare the G4 replication system with the others, (6) describe a partially purified protein which primes G4 DNA synthesis, and provide some evidence for its RNA polymerase action, and (c) show that initiation is at a unique location on the viral template. A subsequent report' will present further information using purified enzymes for G4 replication and details of the novel RNA synthetic action by the priming protein, tentatively identified as the dnaG protein. MATERIALS AND METHODS Materials were from sources described previously (2, 6). Bacterial and &age Strains-Escherichia cob H560 (F+, pol A-, *This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. This is the ninth paper of the series dealing with the initiation of DNA synthesis. The preceding paper is Ref. 16.