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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Bowers, Lisa M; Filutowicz, Marcin (2008) Cooperative binding mode of the inhibitors of R6K replication, pi dimers. J Mol Biol 377:609-15
Bowers, Lisa M; Kruger, Ricardo; Filutowicz, Marcin (2007) Mechanism of origin activation by monomers of R6K-encoded pi protein. J Mol Biol 368:928-38
Kunnimalaiyaan, Selvi; Rakowski, Sheryl A; Filutowicz, Marcin (2007) Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface. J Bacteriol 189:4953-6
Peng, Yanyu; Rakowski, Sheryl A; Filutowicz, Marcin (2006) Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity. FEMS Microbiol Lett 261:245-52
Kunnimalaiyaan, Selvi; Inman, Ross B; Rakowski, Sheryl A et al. (2005) Role of pi dimers in coupling (""handcuffing"") of plasmid R6K's gamma ori iterons. J Bacteriol 187:3779-85
Kunnimalaiyaan, Selvi; Kruger, Ricardo; Ross, Wilma et al. (2004) Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K. J Biol Chem 279:41058-66
Kruger, Ricardo; Rakowski, Sheryl A; Filutowicz, Marcin (2004) Isomerization and apparent DNA bending by pi, the replication protein of plasmid R6K. Biochem Biophys Res Commun 313:834-40
Bowers, Lisa M; Lapoint, Kathleen; Anthony, Larry et al. (2004) Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340:11-8
Kruger, Ricardo; Filutowicz, Marcin (2003) pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K. Nucleic Acids Res 31:5993-6003
Kruger, Ricardo; Filutowicz, Marcin (2003) Characterization of His-tagged, R6K-encoded pi protein variants. Plasmid 50:80-5

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