The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids.
DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication. DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive. Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer. We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process. Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC. Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids. A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.
Crooke
E
E
Department of Biochemistry, Stanford University School of Medicine, California 94305.
Castuma
C E
CE
Kornberg
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000502:physiology*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism / Q000494:pharmacology*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D010743:Phospholipids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate