Unique properties of Staphylococcus aureus primase

Abstract

Bacterial DnaG primase catalyzes the de novo synthesis of a short RNA polymer on the leading strand and repeatedly on the lagging strand template, providing a free 3′-hydroxyl group for elongation by DNA polymerase; a process that is essential for DNA replication. Because DnaG from Escherichia coli and Staphylococcus aureus share only 39.8% amino acid sequence similarity, we hypothesized that bacterial primases initiation specificities and replicative helicase interactions would differ at the genus level. The ability of S. aureus primase to utilize different ssDNA templates was examined using denaturing high-pressure liquid chromatography for analysis of primer composition and quantity. Primer synthesis was demonstrated from templates containing either 5′-dCTA-3′ or 5′-dTTA-3′, in contrast with E. coli DnaG that utilizes templates containing 5′-dCTG-3′. In addition, primer initiation was shown to begin complementary to the second T in both of these trinucleotides. Experiments assessing the ability of replicative helicases from both S. aureus and E. coli to stimulate either S. aureus or E. coli primase showed that helicase stimulation of primase was not cross-reactive. Enzyme and ssDNA concentration dependence studies demonstrated that S. aureus DnaG functions as a dimer, as has been suggested from structural studies. Given the essentiality, species-specificity, and divergence of eubacterial primases from their eukaryotic counterpart, primase is a prospective target for antibiotic development.