The only carefully studied bacterial replication system, Escherichia. coli, requires 23 proteins for in vitro reconstitution of an authentic replication system. The overall objective of these proposed studies is to establish a complete DNA replication system from an important gram- positive pathogen, Streptococcus pyogenes. We will exploit available genomic information to express the Streptococcus homologs of known replication proteins. we will construct templates containing the Streptococcal replication origin to provide a functional assay for Streptococcal replication factors. Factors not accessible through genomic searches will be isolated by biochemical procedures using their requirement to reconstitute Streptococcus-specific replication. The corresponding structural genes for isolated factors will be identified and expressed. The resulting multicomponent replication system will be developed into a high throughput assay format and, together with a collaborative pharmaceutical partner, existing compound collections will be screened for novel lead compounds that can be developed into anti- bacterials that target the replication apparatus. The goal of this phase I application is to clone four centrally important components: the origin, the origin binding initiator protein, the replicative polymerase and the processivity factor. The protein products will be purified and characterized. The origin will be characterized in terms of its interaction with the origin specific initiator protein.
The resulting Streptococcus multiprotein replication system will be used to attract a pharmaceutical company partner to exploit it for high throughput screens for lead compounds for the development of novel anti- bacterials. Given the expanding drug resistance of a number of important gram-positive pathogens, a new antibiotic should achieve commercial viability in the $23 billion antibacterial market.
