The DNA Replication Machine of a Gram-positive Organism

This report outlines the protein requirements and subunit organization of the DNA replication apparatus of Streptococcus pyogenes , a Gram-positive organism. Five proteins coordinate their actions to achieve rapid and processive DNA synthesis. These proteins are: the PolC DNA polymerase, τ, δ, δ′, and β. S. pyogenes dnaX encodes only the full-length τ, unlike the Escherichia coli system in which dnaX encodes two proteins, τ and γ. The S. pyogenes τ binds PolC, but the interaction is not as firm as the corresponding interaction in E. coli , underlying the inability to purify a PolC holoenzyme from Gram-positive cells. The τ also binds the δ and δ′ subunits to form a τδδ′ “clamp loader.” PolC can assemble with τδδ′ to form a PolC·τδδ′ complex. After PolC·τδδ′ clamps β to a primed site, it extends DNA 700 nucleotides/second in a highly processive fashion. Gram-positive cells contain a second DNA polymerase, encoded by dnaE , that has homology to the E. coli α subunit of E. coli DNA polymerase III. We show here that the S. pyogenes DnaE polymerase also functions with the β clamp.