DNA Replication, Bacteria Cell Cycle, and Cell Growth

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The goal of this project is to understand how initiation of DNA replication is coupled to growth rate. 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 being used to elucidate the regulatory circuits controlling expression of the dnaA operon promoters. The growth rate regulator and alarmone, guanosine tetraphosphate (ppGpp), inhibits transcription from the dnaA P2 promoter. The molecular basis of this inhibition is being characterized in vitro. 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? We will investigate DnaA protein-DNA interactions by isolation and characterization of DnaA mutant proteins with altered DNA-binding specificities. We will construct a chromosomal mutation with genes on either side of oriC deleted, and the resulting mutant will be examined for loss of precise timing in the cell cycle. %%% This research studies the control of a key step in cell divison and should provide significant insights into a basic question in biology, as well as abberant conditions suchs as uncontrolled cell mutiplication.