The ongoing emergence of drug-resistant bacteria is rapidly reducing the efficacy of existing antibiotics leading to increased mortality, morbidity and public health burden due to bacterial infections. Since most drugs in late-stage clinical development are analogs of existing drugs to which resistance is expected to develop rapidly, there is an acute need for novel, mechanistically distinct antibacterials. To date, none of the antibacterials target any of the central components of the DNA replication system of bacteria. The overall aim of this project is to assemble a minimal DNA replication system from Pseudomonas aeruginosa, a clinically important pathogen in which the emergence of multidrug resistance is beginning to limit treatment options. We anticipate that 5 subunits will be minimally required to reconstitute the replicative polymerase capable of rapid and processive DNA synthesis: DnaE (DNA polymerase III subunit), DnaN (sliding clamp processivity factor), DnaX (clamp loader ATPase), HoIA and HoIB (accessory subunits of the clamp loader complex). All of these genes are apparent in the recently completed Pseudomonas aeruginosa genome. We plan to clone, express and purity these five proteins and use them to reconstitute a functional DNA replicase. This multiprotein assembly will then be used in high-throughput screens to identify new drug candidates that inhibit this and other bacterial replication systems.
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| Jarvis, Thale C; Beaudry, Amber A; Bullard, James M et al. (2005) Discovery and characterization of the cryptic psi subunit of the pseudomonad DNA replicase. J Biol Chem 280:40465-73 |
| Jarvis, Thale C; Beaudry, Amber A; Bullard, James M et al. (2005) Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors. J Biol Chem 280:7890-900 |
