Replication of ϕX174 RF DNA In Vivo

Abstract

The first clue toward understanding the mode of ϕX174 DNA replication was the discovery by Sinsheimer et al. (1962) of an intracellular double-stranded form (replicative form [RF]) of bacteriophage ϕX DNA. Until that time it was a puzzle how a single-stranded DNA molecule could replicate. The mediation of a double-stranded form seemed to solve this problem, and a few years later Denhardt and Sinsheimer (1965a) and Stone (1967) were able to show that ϕX RF DNA replicates in a semiconservative manner.

Three stages of DNA synthesis can be distinguished during the ϕX life cycle (for reviews see Sinsheimer 1968; Denhardt 1975, 1977; Dressler et al., this volume). The first RF in the ϕX-infected Escherichia coli C host derives from the de novo synthesis of a complementary strand on the infecting viral single-stranded (SS) DNA (SS→RF), which is accomplished by preexisting host enzymes. This parental duplex RF DNA then replicates to form a pool of some 20 RF molecules (RF→RF) at 15 minutes after infection; this requires several host enzymes as well as one phage-encoded enzyme, the gene-A protein. In the final stage of the ϕX life cycle, SS DNA of the progeny virus is derived from RF DNA by asymmetric synthesis (RF→SS). This paper reviews the second stage of ϕX DNA replication (RF→RF) in vivo and is for the most part limited to labeling studies and to analyses of replicating intermediates by biochemical and electron microscopic methods. Other approaches, namely, the effect of blocking host-cell functions by mutation and studies...