Studies on the In Vitro Synthesis of ϕ X174 RFI DNA and Circular Single-stranded DNA

Abstract

The replication of ϕ X174 DNA in vivo has been studied extensively. Physically, the cycle can be divided into three steps: (1) the conversion of the entering viral circular single-stranded DNA to a circular duplex replicative form (SS → RF); (2) the duplication of the RF DNA to produce progeny RF molecules (RF → RF); and (3) the asymmetric synthesis of progeny viral SS DNA (RF → SS) (Sinsheimer 1968).

During the first stage, SS → RF, no new proteins other than those encoded in the host are required. This system has been reconstructed with purified proteins and has been shown to depend on a number of proteins known to be involved in Escherichia coli DNA replication (Wickner and Hurwitz 1975a; Schekman et al. 1975). These include the E. coli dna gene products dnaB, dnaC, dnaG, dnaE (DNA polymerase III), and dnaZ as well as a number of other proteins (E. coli DNA-binding protein, replication factors X, Y, and Z, and the DNA elongation factors I and III [EFI and EFIII]) that have not yet been defined genetically. It has been established that the proteins dnaB, dnaC(D), DNA-binding protein, and replication factors X, Y, and Z generate a protein complex with ϕ X SS DNA in the presence of ATP (Wickner and Hurwitz 1975b). The dnaG protein recognizes this complex and synthesizes an oligonucleotide which is used for priming of replication. The elongation of the primed DNA template requires the action of the DNA elongation system, which is comprised of...