INITIATION OF DNA REPLICATION--DNAA, RNA POLYMERASE, & COUPLING TO CELL CYCLE

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The long term goal of this project is to understand how initiation of DNA replication is coupled to growth rate. Our objective is to understand how DNA replication, cell growth, and cell division are so precisely and coordinately regulated. In both prokaryotic and eukaryotic cells, the rate of chromosomal duplication is controlled by the rate of initiation at the origin(s) of replication, not by the rate of chain elongation. The rate-limiting steps of the initiation event remain unidentified. To understand how the frequency of the initiation event is determined, the mechanisms that regulate the expression of the DnaA protein must be understood. Both genetics and biochemistry are used to elucidate the regulatory circuits controlling expression of the dnaA operon promoters. The major dnaA promoter is growth rate regulated and under stringent control. Guanosine 3',5'-bispyrophosphate(ppGpp) may provide the molecular basis for growth rate regulation of DnaA protein expression. Mutations in this promoter and in the dnaA box nearby that eliminate promoter activity and binding of DnaA protein to the dnaA box will be introduced into the chromosome and assayed for growth rate regulation and stringent control in order to identify which regulatory sequences affect growth rate regulation and stringent control. A single round in vitro transcription assay is being used to ascertain the contribution of ppGpp and DnaA protein to regulation of the activity of each of the dnaA promoters. Mutants defective in the inhibitory response to ppGpp will be examined for growth rate regulation and precise timing of initiation of DNA replication in the cell cycle. We will determine whether the dnaA gene is still under growth rate control in cells that lack ppGpp -- is there a growth rate regulation mechanism other than ppGpp that controls the expression of DnaA protein? As in the past, MBRS students will be involved in both the technical and scientific aspects of the research.