A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors

Bacterial DNA primase DnaG is an attractive target for antibiotic discovery since it plays an essential role in DNA replication. Over the last 10 years, we have developed and optimized a robust colorimetric assay that enabled us to identify and validate inhibitors of bacterial primases. Here, we provide a detailed protocol for this colorimetric assay for DnaG from three different pathogenic bacteria (Mycobacterium tuberculosis, Bacillus anthracis, and Staphylococcus aureus), which can be performed in high throughput. We also describe secondary assays to characterize hits from this high-throughput screening assay. These assays are designed to identify inhibitors of the coupled enzyme inorganic pyrophosphatase, DNA binding agents, and elucidate the mode of inhibition of primase inhibitors.