Conversion of Phage Single-stranded DNA to Duplex DNA In Vitro

Abstract

In recent years there has been much progress toward understanding the molecular mechanisms of DNA replication of small Escherichia coli phages. Three enzymatic pathways have been identified by which phage circular single-stranded (SS) DNA (fd[M13], St-1 [G4], and ϕ X174) is converted to duplex DNA. Each of these pathways has been fractionated into many protein components, all of which are derived from the host. Some of the proteins required for the replication of phage SS DNA are also required for the replication of E. coli DNA. Some of the genes whose functions are required for E. coli DNA replication have been identified by the isolation of mutants conditionally lethal for DNA synthesis. Functions required for the initiation of E. coli DNA replication include dnaA, dnaB, dnaC(D), dnaI, and dnaP gene products and RNA polymerase (Wechsler 1978). Those required for continuing a round of DNA replication include dnaB, dnaC(D), dnaG, dnaZ, polC, nalA, and cou gene products (Wechsler 1978). By means of in vitro complementation assays it has been possible to isolate the proteins required for phage SS DNA replication that are also required for E. coli DNA replication. Extracts prepared from some E. coli mutants temperature-sensitive for DNA replication were found to be active for phage DNA synthesis at low temperature but not at high temperature; these included extracts of dnaB, dnaC(D), polC, dnaG, and dnaZ mutants (Wickner et al. 1972; Schekman et al. 1972; Wickner and Hurwitz 1976). The corresponding wild-type proteins have been purified from wild-type cells by...