Abstract
DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3'-O-(N-methylantraniloyl)-5'-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.
Highlights
EFFECT OF SURFACE MOLECULAR CROWDING*In Escherichia coli DnaA binds to the five DnaA-boxes on oriC and promotes the replication complex assembly [5]
Initiation of DNA replication is the central event in the bacterial cell cycle [1]
There is an indication that clustering, rather than the concentration of acidic phospholipids, in such a membrane is essential for nucleotide dissociation [26]
Summary
In Escherichia coli DnaA binds to the five DnaA-boxes on oriC and promotes the replication complex assembly [5] This first event in the multistep initiation process is the target for regulation: the initiating activity of DnaA depends on the type of the bound nucleotide, and only the ATP-DnaA form is functional [6]. Our working hypothesis is that such domains may provide the binding sites for integral and peripheral (amphitropic) membrane proteins and serve as spatially and temporally localized signal [32] The dynamics of such domains during the cell cycle may trigger the accessibility of the activating acidic phospholipids to DnaA. The nucleotide binding kinetics, followed by the fluorescent derivative, 3Ј-O-(N-methylanthraniloyl)-adenosine 5Ј-triphosphate (MANT-ATP), appeared to be strongly sensitive to the protein density on the membrane We will discuss this finding in relation to the timing of the initiation of DNA replication in the bacterial cell cycle
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