This proposal is focused on DNA-protein interactions that precisely time new rounds of chromosome replication, with the long-term objective of dissecting molecular mechanisms controlling bacterial growth. Studies are proposed to investigate interactions of oriC, the unique E. coli chromosomal replication origin with initiator protein DnaA and DNA bending proteins Fis and IHF. The intracellular nucleoprotein complex formed with these proteins and oriC is not static, but changes its composition as cells progress through the cell cycle. Two major goals of this proposal are to enhance our understanding of the dynamics of conversion from one complex to the next, and to begin evaluating the role each component plays in ensuring that DNA replication initiates synchronously from all copies of oriC at the appropriate time.
The Specific Aims are as follows: 1. To use DNA footprinting and unwinding assays to assess the role of Fis in the assembly and functionality of DnaA and Dna/IHF complexes on supercoiled oriC templates in vitro; 2. To use insitu DNA footprinting and unwinding assays on permeabilized, synchronized cells to determine if a correlation exists between the timing of plasmid oriC unwinding in the cell cycle and duration of IHF binding; 3. To test the hypothesis that growth rate regulation of Fis affects the dynamics of Dna binding site accessibility of oriC by examining assembly of Dna-oriC complexes at various concentration ratios of Fis and IHF; 4. To evaluate the role of non-R box DnaA binding sites on nucleoprotein complex formation and oriC function using site-specific mutagenesis; 5. To begin development of a PCR-based footprinting method suitable to produce genomic oriC footprints in situ. Our methodologies and the results obtained should provide new insight into the function of growth regulatory machinery in all living cells. This information is immensely important for understanding the control of bacterial growth, as well as cell growth defects, and can help to identify new targets used to guide the design of novel cell growth inhibitors.
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