Bacterial DNA replication is carefully controlled at the initiation stage, possible by the regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence that indicates membranes have a profound influence on DnaA protein. The long term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses both biochemical approaches with defined components and genetic and physiological studies to directly test the hypothesis that acidic phospholipids of the cellular membrane participate in the regulation of DnaA protein activity.
The Specific Aims are to; 1. Identify the membrane binding site on DnaA protein by chemical crosslinking. Disrupt the membrane binding function by site-directed mutagenesis and isolate the mutant DnaA proteins. Confirm with defined components that the other replication activities of DnaA protein have not been altered. 2. Examine in vivo the replication activity of the mutant Dna A proteins are viable, and if so, if they initiate replication at aberrant times during the cell cycle. Localize mutant forms of DnaA protein within the cell by subcellular fractionation and immuno-gold microscopy to examine the importance of DnaA protein-membrane association for regulated initiations. 3. Generate and screen randomly mutagenized dnaA genes for the ability to suppress the growth arrest of acidic phospholipid-deficient cells. Map the suppressor mutations in dnaA and examine the replication activities of the mutant proteins in vitro. Determine the cellular location of the mutant proteins and analyze their effect on the cell cycle control of chromosomal replication in vivo. This work should provide insight into the regulation of the initiation of DNA replication , which is a key control point in the prokaryotic cell cycle and in the determination of eukaryotic cellular quiescence or proliferation. Furthermore, knowledge gained from the proposed studies of DnaA protein may guide future investigations of how phospholipids may act as regulators of enzymatic activities.
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