Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine

Abstract

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, τ, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimuiated ATPase activity comparable with E. coli τ. S. aureus τ also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III·τ. We demonstrate that the extreme C-terminal residues of PolC bind to and function with β clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus δ wrench of the clamp loader binds to E. coli β. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes β clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous β clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them. © 2005 by The American Society for Biochemistry and Molecular Biology. Inc.