The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at

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... C restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure. The Escherichia coli chromosomal origin (oriC) has two major roles (reviewed in Refs. 1 and 2). One is to act as a site where DNA replication is controlled so that it occurs only once per cell cycle. The second is to serve as a locus where the replication fork machinery is assembled, involving a series of orchestrated steps. An important event at oriC is the binding of DnaA protein to specific sequences named DnaA boxes (3). A second essential step in the assembly process is the DnaA-dependent recruitment of DnaB (4, 5). Studies on the native structure of DnaB have firmly established that it is a hexamer of identical subunits arranged as a toroidal structure with a central opening (6 -9). Its stability requires the presence of Mg 2ϩ ion; removal of the metal ion by dialysis or chelation is needed to dissociate the DnaB hexamer into its subassemblies (10). However, the form of DnaB that is required at the stage of initiation at oriC is as a complex with its partner, DnaC (11, 12). Assembly of this complex requires the binding of ATP to DnaC, with the nucleotide serving to alter the conformation of an N-terminal domain of DnaC so that it can bind to DnaB (13). Each DnaC monomer is present at a 1:1 ratio with each DnaB protomer (14 -16). Whereas it is the DnaB-DnaC complex that is active at the stage of initiation, DnaB liberated from DnaC is active during DNA synthesis. The association of DnaC with DnaB inhibits the enzymatic activities of this essential helicase, and the hydrolysis of ATP bound to DnaC is required to release DnaC from DnaB (5, 12, 17). Once DnaB is situated at the apex of the replication fork, the helicase acts to unwind the parental DNA as each DNA strand is copied by DNA polymerase III holoenzyme. These events are facilitated by a functional coupling, involving an interaction between DnaB as it moves in the 5Ј-to-3Ј direction on the lagging strand template and the tau subunit of DNA polymerase III holoenzyme (18). When this physical interaction is maintained, DnaB moves at a 20-fold faster rate than the speed of DnaB translocation alone. At oriC, it has been proposed that an AT-rich region unwound by DnaA protein serves as the entry site for DnaB (19) . Footprinting studies map DnaB to this region, in support of the model (20). Other results indicate that only two DnaB hexamers are bound at oriC (20, 21). Because DnaA induces a limited degree of unwinding, one model is that only two DnaB hexamers can bind because the available single-stranded DNA (ssDNA) 1 is only sufficient for one DnaB hexamer for each DNA strand. We have relied on a simple replication system to study the process of recruitment of DnaB onto DNA and to address the question of whether the availability of ssDNA influences the number of helicase molecules that can bind. With a singlestranded DNA carrying a DnaA box in a hairpin structure (M13 A-site), DnaA bound to this site forms a structure that in turn is recognized by the DnaB-DnaC complex to form an intermediate termed the prepriming complex (5, 22). Following the release of DnaC, DnaB is then free to move on the ssDNA. The transient binding of primase to DnaB results in primers that are formed at apparently random locations (23, 24). These primers are then extended by DNA polymerase holoenzyme in conversion of the ssDNA to duplex form. In this system, DNA replication is dependent on a single DnaA box-containing sequence, and only one DNA strand is synthesized on the ssDNA template. By comparison, DNA replication from oriC is more complicated because a duplex DNA is involved, and each parental DNA strand is bound by DnaB to support bidirectional replication fork movement. Priming and DNA synthesis occur on both strands of the parental duplex. In this report, we sought to characterize further the molecular composition of the complex formed by the binding of DnaA, DnaB, and DnaC protein to the ssDNA carrying the DnaA box hairpin. The major objective was to test the model that the amount of ssDNA available influences the number of DnaB molecules that can bind. Several independent methods were used. In the first, a 379-nucleotide-long ssDNA bearing the DnaA box hairpin and covered by SSB was used to demonstrate