The DNA replication machine of a Gram-positive organism

Abstract

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 tothe 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.