Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region. A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We show that DnaA protein actively mediates the entry of DnaB at oriC. One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance. A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the Nterminal 62 residues. Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.The initiation of chromosomal DNA replication in all freeliving organisms is a two-stage process that first involves assembly of the initiation machinery at specific sequences called the origin of DNA replication (see Ref. 1 for a recent review). Once formed, the initiation complex then assembles proteins that act at the replication fork for semiconservative DNA synthesis. In the yeast Saccharomyces cerevisiae, origin sequences are targeted by the six-member origin recognition complex (ORC).1 ORC in a prereplication complex is then recognized by Cdc6 protein, Cdc45 protein, and minichromosome maintenance proteins (reviewed in Ref.2). The initiation complex is somehow activated by cyclin-dependent kinases and Cdc7-Dbf4 protein kinase, leading to the import of required proteins at the replication forks and DNA synthesis.In Escherichia coli, DnaA protein is the functional counterpart to ORC in that it recognizes specific sequences in the replication origin, oriC. Once bound, it directs formation of the initiation complex through a series of discrete steps. First, it induces a local distortion of an AT-rich region near the left boundary of oriC to form an intermediate, the open complex, in an ATP-dependent process that is assisted by either HU or IHF (3-5). The prepriming complex is then formed by the binding of DnaB helicase from the DnaB-DnaC complex (6, 7). Replication fork assembly and DNA replication follow by the coordinated activities of primase and the subunit of DNA polymerase III holoenzyme, each forming contacts with DnaB for concerted primer formation, primer extension, and replication fork movement (8, 9). Processive DNA synthesis also requires singlestranded DNA-binding protein (SSB) and DNA gyrase to relieve positive superhelicity ahead of the replication fork.In the above sequence of events, the entry of DnaB protein at oriC is an important step in the initiation process because this protein is required for bidirectional replication fork movement. How this occurs is poorly understood and limited to the following...

SummaryDuring exponential growth, the level of Dps transiently increases in response to oxidative stress to sequester and oxidize Fe 2+ , which would otherwise lead to hydroxyl radicals that damage the bacterial chromosome. We report that Dps specifically interacts with DnaA protein by affinity chromatography and a solid phase binding assay, requiring the N-terminal region of DnaA to interact. In vitro, Dps inhibits DnaA function in initiation by interfering with strand opening of the replication origin. Comparing isogenic dps + and dps::kan strains by flow cytometry and by quantitative polymerase chain reaction assays at either the chromosomally encoded level, or at an elevated level encoded by an inducible plasmid, we show that Dps causes less frequent initiations. Results from genetic experiments support this conclusion. We suggest that Dps acts as a checkpoint during oxidative stress to reduce initiations, providing an opportunity for mechanisms to repair oxidative DNA damage. Because Dps does not block initiations absolutely, duplication of the damaged DNA is expected to increase the genetic variation of a population, and the probability that genetic adaptation leads to survival under conditions of oxidative stress.