The long term objectives of this proposal are to understand how DNA-protein interactions trigger properly timed, physiologically important events, with the goal of dissecting molecular mechanisms that control cellular growth and replication. Studies will be focused on how nucleoprotein complexes that trigger new rounds of DNA replication are assembled during the cell cycle. The key questions addressed are: Do regulatory proteins which bind to replication origins do so in an ordered fashion during the cell cycle? Does this order change as a function of growth rate? How does loss of individual origin binding proteins or disruption of an individual protein binding site affect assembly of the initiation complex? To answer these questions, interaction of the regulatory proteins, DnaA, FIS, and IHF, with their specific binding sites within the Escherichia coli chromosomal replication origin, oriC, will be measured.
The specific aims are: 1) generate detailed footprints of DnaA, IHF, and FIS interactions with oriC using three different reagents: dimethylsulfate, UV light, and potassium permanganate to modify nonmutant and mutant minichromosomal oriC DNA in vitro and in wild-type and fis, him, and dnaAts mutant strains in vivo; 2) generate oriC in vivo footprints at regular intervals throughout the entire cell cycle in synchronously-growing cultures and determine when DnaA, IHF, and FIS bind to their sites; 3) examine the effect of decreasing growth rate on cell cycle footprints and test the hypothesis that the duration of FIS and IHF binding to oriC varies with growth rate; 4) measure the effect of site-specific mutations in DnaA, IHF, or FIS binding sites on nucleoprotein complex assembly during the cell cycle by comparing to nonmutant oriC in vivo and in vitro; and 5) measure chromosome and minichromosome replication timing and cell cycle footprints in growth synchronized fis, him, and seqA mutant strains to determine the degree to which these mutations perturb normal initiation control and nucleoprotein complex formation. Protein interaction with minichromosomal oriC, in vivo and in vitro will be assayed using alkaline primer extension analysis of modified DNA. The baby machine will be used to produce synchronously- growing E. coli cultures, and DNA replication will be measured by incorporation of radiolabeled precursor. Our methodology and the results obtained should provide new insight into the workings of cell growth regulatory machinery as it functions in living cells. This perspective is crucial for understanding the control of bacterial growth, as well as cell growth defects, and for the design of novel cell growth inhibitors.
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