Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.Initiation of Escherichia coli chromosomal replication occurs at the chromosomal origin, oriC, a unique sequence that contains all necessary cis-acting elements (42, 47). To initiate a round of replication, approximately 20 molecules of DnaA protein bind to the DnaA boxes in oriC (10,13,14,38,41,62), promote strand opening of the AT-rich 13-mers (2,3,17), and facilitate the loading of DnaB helicase (14,40). The remaining components of the replisome are subsequently recruited to the sites of the newly formed replication forks (33,39).Given the central role of DnaA protein in the initiation process, its regulation is most likely crucial for normal, cellcycle-controlled chromosomal replication. While dnaA mRNA levels fluctuate throughout the cell cycle, DnaA protein levels remain relatively constant (51). Therefore, other mechanisms must regulate the protein's activity. Prominent among these in vitro is the influence on the replicative action of DnaA protein by the tight binding of ATP and ADP (52,65). Both the ADP and ATP forms of DnaA protein are capable of binding oriC (52). However, only the binding of ATP-DnaA leads to duplex melting and the ensuing initiation events (52,53,65). The ATP bound to DnaA protein is slowly hydrolyzed in a DNA-dependent manner, with the resulting ADP remaining tightly bound, thus producing inactive ADP-DnaA protein. A partially purified factor, termed IdaB, and ␤-subunit, the processivity factor of DNA polymerase III, stimulate the conversion of ATPDnaA to 29).Treatment of ADP-DnaA with anionic phospholipids causes the release of the bound nucleotide (8, 54). When exposed to anionic phospholipids in the presence of oriC DNA and excess ATP, replicatively inactive ADP-DnaA is rejuvenated into the active ATP form (9, 54). An examination of DnaA proteolytic fragments demonstrated that a distinct segment of DnaA is necessary for productive interaction with lipid bilayers (15). Amino acid sequence analysis of this region suggests the existence of an amphipathic helix (57), a motif commonly found in peripherally associated membrane proteins. Chemical cro...

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