Our major objective is to understand the molecular mechanism underlying regulated initiation of DNA replication. DNA replication, like other high precision DNA transactions such as recombination and transcription is largely influenced by protein- DNA and protein-protein interactions. Bacterial plasmids provide one of the most advanced and convenient systems to study this fundamental biological process. We have developed genetic and biochemical tools which are and excellent point of entry to use plasmid R6K as a model system in the analysis of components, and the events that assure the controlled duplication of a prokaryotic genome. The experiments proposed in the first section are directed toward the identification of key domains in the R6K replication region which are critical in forming higher order structures involving the multifunctional replication protein, PI. The experiments in the second section will take advantage of the availability of PI protein variants purified from mutants altered in a negative control of replication initiation to probe the role of PI in negative control loop. In the third section, we propose studies on the role of the E. coli Integration Host Factor (IHF) in the plasmid R6K replication. Our preliminary data convince us that the IHF protein could play a critical role in the architecture of the plasmid origin. Since a large group of prokaryotic and eukaryotic chromosomes exhibit remarkable structural similarities in the organization of a basic replicon, we believe that by studying plasmid R6K, we will ultimately learn about fundamental mechanisms that are underlying the duplication of genetic material in general.
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