Initiation is the key step in control of DNA replication in both procaryotic and eucaryotic cells. Our long term goal is to understand how this process is regulated in E. coli and how the regulatory system responds to the growth rate of the cell. The dnaA gene product plays a key role in regulating initiation and is itself subject to autoregulation. The gene has two promoters separated by a region containing a binding site for the DnaA protein; it also has an untranslated leader sequence of unknown function. We propose to dissect this regulatory system to gain insight into the way in which the gene is regulated. The DnaA protein is known to bind to a specific DNA sequence repeated four times within the origin of replication and present at numerous other chromosomal sites including the dnaA regulatory region; the protein may also bind to RNA polymerase. We will use a biochemical and genetic approach to study DNA binding and interactions with other proteins that might be important in initiation. Certain dnaA mutants show an overinitiation phenotype which leads to cessation of cell growth and an overall increase in DNA/mass ratio. We will compare the properties of the DnaA protein produced by one of these mutants, dnaAcos, with those of the wild type protein since we feel that understanding of the unregulated phenotype will throw light on the normal process. Proposed studies include analysis of properties such as DNA binding, interactions with other proteins and function in an in vitro initiation assay. Additional approaches to understanding dnaA function include studying the effects of overproducing the protein on initiation frequency, looking at the effects of total shut-off of dnaA expression on initiation, and cloning and DNA sequence analysis of the dnaA gene from Klebsiella pneumoniae, a comparative study to see which regions of the dnaA gene have been conserved.
