Replication initiation at the Escherichia coli chromosomal origin

Abstract

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.

Figure 1

Escherichia coli oriC carries recognition sites for DnaA, including DnaA boxes, I-sites and τ-sites. Also shown are binding sites for IHF and Fis, and the 13-mer sequences that become unwound by DnaA complexed to ATP. Following the binding of DnaA to the sites described above and unwinding, DnaA loads one DnaB₆-DnaC₃ complex each on the top and bottom strands of the unwound region. The first primers synthesized by primase on the top and bottom strands both induce the dissociation of DnaC from DnaB so that DnaB can act as a DNA helicase to unwind the parental duplex DNA, and prime the synthesis of each leading strand for the rightward and leftward progressing replication forks. The subsequent occasional interaction of primase with DnaB leads to primers (not shown) for Okazaki fragment synthesis.

Figure 2

Functional domains of DnaA, DnaB, and DnaC protein. (A) In the domain organization of DnaA, a region near the N-terminus is involved in the interaction between DnaA and DnaB, HU, Dps, DiaA, or ribosomal protein L2 (see DnaA-dependent unwinding of oriC). A region within this domain is also required for DnaA oligomerization. Domain II functions as a flexible linker joining domain I and IIIa and IIIb, but this domain is not entirely dispensable. Domain III is involved in ATP binding and carries the amino acid sequence motifs shared among AAA+ family members. Domain IV is involved in DNA binding. (B) Domains of DnaB identified by trypsin digestion correlate with the X-ray crystallographic structure of Bacillus stearothermophilus DnaB [42,90,91]. Regions of DnaB that interact with primase, DnaC and ATP, which include the Walker A and Walker B box and the arginine finger (arginine 409 of E. coli DnaB) are shown. (C) The interacting domains of DnaC with DnaB and with ATP, including the AAA+ motifs and the conserved arginines in box VII are shown. The numbers in the respective lines refer to the coordinates for E. coli DnaA, DnaB and DnaC protein.