Steve Hinrichs, MD, & Marilynn Larson, PhD

Staphylococcal research in our laboratory entails studying the regulation, function, and interaction of proteins required for DNA replication in bacterial pathogens. In particular, our research focuses on the mechanism of action for S. aureus DnaG primase and replicative helicase. Our investigations to date have elucidated the structural and molecular basis for primase initiation specificity and the requirements for a productive primase-helicase interaction. In addition to gaining a greater understanding of these biological processes, our ongoing work targets these essential S. aureus DNA replication enzymes for antibiotic discovery.

During the initial stages of DNA replication, bacterial DnaG primase is recruited to the replication fork and stimulated by replicative helicase.* This interaction catalyzes the de novo synthesis of RNA primers on the leading and lagging strand templates by primase, and is required for subsequent DNA elongation. Amazingly, this priming process occurs through the specific interaction of primase with only three nucleotides in the DNA template. Our group was the first to demonstrate that S. aureus primase uses a different recognition trinucleotide sequence than the well-characterized Escherichia coli primase for the initiation of primer synthesis. We have also shown that the interaction of primase with the DNA template and replicative helicase exhibits both general and specific features. More specifically, primase trinucleotide specificity correlates with bacterial class and a functional primase-helicase interaction only occurs between closely related bacteria.

Both DnaG and replicative helicase are highly conserved, genome-persistent enzymes that are present in low copy number and are essential for bacterial survival. The modular structure of bacterial primase provides many possible targets for small molecular weight inhibitory drugs and the required functional interaction of DnaG with helicase provides additional regions for targeted inhibition. Further, these bacterial proteins are structurally unrelated to eukaryotic primases and helicases, making them excellent targets for inhibitory compounds without any expected impact on the infected mammalian host. For antibiotic discovery, we have developed two unique, non-radioactive methods for screening drug-like compounds that inhibit bacterial primase activity, namely a fluorescent assay suitable for high-throughput screening and a denaturing HPLC assay for confirmation and further characterization of those inhibitors.

In summary, our studies have determined class-specific functions of primase and the primase-helicase interaction, providing insight on how these essential bacterial enzymes could be exploited for drug targeting. Our goal is to discover small molecular weight inhibitors of primase activity and/or the primase-helicase interaction in S. aureus, leading to the identification of novel antibiotics. Since bacterial primases share many common features, we ultimately hope to identify broad-spectrum drug-like inhibitors of bacterial primer synthesis, as well as provide more insight on the mechanisms that regulate this process to prevent pathogen proliferation.

*For more information on the molecular process of bacterial DNA replication, see website of our collaborator Dr. Mark Griep, who is at the University of Nebraska Lincoln campus.