The central theme of the proposal is to analyze how initiator proteins activate replication origins by """"""""action at a distance"""""""". Specifically, long range activation of distant origins by pi mediated enhancer-origin interaction, shutting down of the potential origin at the enhancer of plasmid R6K will be analyzed by a multifaceted approach, structure-function analysis of normal pi and mutants by both molecular genetic and structural analysis by X-ray crystallography and NMR spectroscopy. The shorter range looping of pSC101 origin promoted by DnaA protein and the role of RepA and the DNA-binding protein IHF will also be investigated. Finally, the """"""""handcuffing"""""""", i.e. protein mediated intermolecular pairing of origin interon as a viable model for copy control will be critically examined in vivo and in vitro by isolation and analysis of mutants defective in the process. The investigators will also analyze whether other origin-binding proteins modulate and perhaps enhance or reverse handcuffing process.
