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Priming of DNA Synthesis on Viral Single-stranded DNA In Vitro

Roger McMacken, Lee Rowen, Kunihiro Ueda, Arthur Kornberg


Among the many viral, bacterial, and animal DNA polymerases isolated thus far none can start a chain in vitro (Kornberg 1974). In addition to a template strand, each requires a 3′-OH-terminated chain (primer terminus). Because they lack such termini, the coliphage circular, single-stranded (SS) DNA chromosomes have proved to be ideal templates for studying the mechanisms used by Escherichia coli to initiate new DNA chains (Schekman et al. 1974; Wickner and Hurwitz 1975a).

Because RNA polymerases are known to initiate new RNA chains, it appeared that an RNA transcript might prime DNA synthesis (Brutlag et al. 1971), and rifampicin, a specific inhibitor of E. coli RNA polymerase, should therefore inhibit DNA synthesis. RNA polymerase was shown to be essential in the conversion of phage M13 SS DNA to the duplex replicative form (RF). Later it was demonstrated that soluble extracts from gently lysed E. coli cells support the conversion of various bacteriophage SS DNAs to their replicative forms (Wickner et al. 1972; Schekman et al. 1974). Fractionation and identification of the proteins involved in this apparently simple replication showed not only that synthesis of the complementary DNA chain is surprisingly complex, but also that there are at least three different DNA-strand initiation systems in E. coli (Schekman et al. 1974; Wickner and Hurwitz 1974; Schekman et al. 1975; Kornberg 1977). The chromosomes of the filamentous phage M13 and the isometric phages G4 and ϕX174 are each replicated in vitro by a different set of bacterial replication proteins, which differ primarily...

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