The dnaG Gene Product of Escherichia Coli: Characterization of the Catalytic Activity

Abstract

The initiation of oX174 replicative form synthesis from viral single-stranded DNA in vitro, occurs in at least two distinct stages (1–3). The first step requires the products of the dnaB and dnaC gene, the DNA-binding protein, and additional protein factors1 to “activate” oX174 single-stranded DNA in an ATP-dependent reaction. This activated DNA may be isolated by gel filtration after incubation of DNA with ATP and a partially purified cell extract (1) or resolved protein fractions (2). Furthermore “activated” DNA promoted immediate complementary strand synthesis, requiring the dnaG gene product in addition to DNA polymerase and factors as opposed to the kinetic lag observed for unreacted DNA. The absolute requirement for the dnaG protein in this latter stage suggests that it plays an essential role in DNA chain initiation. It has been reported that the dnaG protein functions, (phage G4 replicative form synthesis), by synthesizing an oligoribonucleotide primer in a rifampicin-resistant reaction (4). It was suggested that the template for this reaction: binding protein complexed with single-stranded DNA, is the functional equivalent of “activated” oX174 DNA; and that dnaG protein catalyzes the same reaction on both DNAs.