The objective of this proposed study is to build upon a successful phase I study to establish a complete DNA replication system for a prototypical gram-positive pathogen, Streptococcus pyogenes. As in our phase I studies, we plan to continue to exploit available genomic information to permit expression of the S. pyogenes homologs of known replication proteins. We will also exploit templates containing the Streptococcal replication origin to provide a functional assay for Streptococcal replication and initiation factors. Factors not accessible through genomic searches will be isolated by biochemical procedures using their requirement to reconstitute replicative reactions to monitor their purification. The corresponding structural genes for isolated factors will be identified and expressed. The resulting multicomponent replication system will be developed during phase III into a high-throughput assay and compound collections screened for novel lead compounds that can be developed into antibacterials that target the gram-positive replication apparatus.
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