An Enzyme System for Replicating the Duplex Replicative Form of ϕX174 DNA

Abstract

Because the small coliphages M13, G4, and ϕX174 rely almost completely on host enzymes for development, their viral DNAs are model templates for investigating the role of these enzymes in the replication of the host chromosome.

The life cycle of these viruses includes three stages of DNA replication: (I) conversion of the viral single-stranded DNA circle to the duplex replicative form (SS → RF), (II) multiplication of the replicative form (RF → RF), and (III) synthesis of viral single strands using the complementary strand of the replicative form as template (RF → SS) (Denhardt 1975; Baas and Jansz; Ray; Dressler et al.; all this volume).

The enzymatic events in the SS → RF conversion, resolved and reconstituted, include three distinct mechanisms for initiating DNA chains (McMacken et al.; Wickner; both this volume). In this article we summarize the progress that has been made in resolving and reconstituting the second stage in the life cycle, the replication of RF DNA.

A Soluble Enzyme System for RF DNA Replication
A soluble enzyme fraction (fraction II, Table 1) prepared from uninfected E. coli cells was shown to support the synthesis of complementary-strand DNA on (ϕX SS DNA templates. The result was the production of duplex RF DNA. Fraction II also supported DNA synthesis on RF DNA templates provided the original fraction II was supplemented with a fraction II prepared from ϕX-infected cells. The products of this reaction, RFI (superhelical, covalently closed, circular, duplex DNA) and RFII (circular, duplex DNA in which at least...