Understanding the mechanism of controlled initiation of DNA replication remains a fundamental and largely unsolved problem in cell biology. Our long-term goal is to contribute to the solution of this problem by investigating Escherichia coli plasmid R6K (pR6K). The major focus of our research during the current granting period will be the biochemical analysis of the activator and replication inhibitor activities of pR6K- encoded initiator protein pi and the determination of the role of so- called enhancer region in replication. Because the gamma ori to which pi proteins binds shares a number of structural features with origins of many other plasmids, the proposed studies are likely to have broad biological significance. However, it is important to note that the gamma ori also differs from the other origins in many respects. Thus, our studies may elucidate novel mechanisms of control of DNA replication.
Our specific aims i n this proposal are: SA#1. To determine minimal sequence requirements for the activity of gamma ori replication in vivo. SA#2. To characterize in vitro the replication properties of gamma ori templates with and without the enhancer sequences. SA#3. To determine how the replication enhancer works. SA#4. To elucidate the role of pi protein in setting initiation frequency by using wt and mutant pi proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040314-07
Application #
2392056
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1989-03-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1999-03-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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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