The long term goal of our research is to understand the mechanisms regulating the initiation of DNA replication. In both prokaryotic and eukaryotic cells, the rate of chromosomal duplication is controlled by the rate of initiation at the origin of replication, not by the rate of chain elongation. Consequently, the determinants of the replication rate and its control are necessarily involved in the initiation step. In the bacterial cell, initiation of replication at the bacterial origin, oriC, requires an RNA polymerase-mediated transcription event and the presence of the DnaA protein. This project seeks to define these events in molecular terms. We are using genetic and biochemical approaches to elucidate both the involvement of RNA polymerase in initiation, whether it be priming or transcriptional activation, and the interaction of DnaA protein with oriC in forming the initial complex. Only with a detailed knowledge of these two events can regulation of DNA replication be understood. In addition, we plan to study the regulation of expression of the dnaA gene, because expression of this gene may be critical in linking the initiation event to the cell cycle. Specifically, we plan to: . Characterize the involvement of sequences adjacent to oriC in initiation of DNA replication. Deletions of regions next to oriC will be introduced into the chromosome and characterized. These deletions will be replaced with transcription termination sequences and G+C blocks. . Establish if the original Fraction II in vitro system requires RNA polymerase. . Define the structural features and other parameters that contribute to forming the oriC initial complex. Does DnaA protein induce bending at one DnaA binding site and what contributions to bending occur when more binding sites are added, in parallel positions or in alternating positions, as is seen in oriC? . Investigate regulation of DnaA protein expression. Use our single round in vitro transcription assay and site-directed mutagenesis to assess sequence specifications for growth rate regulation and other controlling molecules. Search for other factors that regulate dnaA gene expression with gel shift assays and Southwesterns.
