Stoichiometry of DnaA and DnaB Protein in Initiation at the Escherichia coli Chromosomal Origin

Abstract

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4 -5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.Initiation of Escherichia coli chromosomal DNA replication involves the stepwise assembly of proteins at the chromosomal origin, oriC 1 (reviewed in Refs. 1 and 2). This DNA locus is first recognized by the binding of DnaA protein to individual DnaA boxes in oriC in an ordered manner (3). Unwinding of an AT-rich region near the left border of oriC then occurs and requires the ATP-bound form of DnaA protein (4, 5). HU or IHF assist at this step to form an intermediate termed the open complex (6). The binding of ATP to DnaC, a prerequisite, forms the DnaB-DnaC complex (7,8), which is then recruited to oriC to form the prepriming complex (9). At this step, DnaB interacts with two functional domains of DnaA protein; the stabilization of the prepriming complex also may involve a cryptic single-stranded DNA binding activity of . Upon the release of DnaC from the prepriming complex, DnaB is then free to act as a helicase to unwind further the parental DNA template. Its distributive interaction with primase leads to the priming of both leading and lagging strands that are then concurrently extended by each catalytic unit of the dimeric DNA polymerase III holoenzyme in the enzymatic machinery at each replication fork (Ref . 13 and references therein).The E. coli replication origin is a locus at which DNA replication is controlled. This site also serves as the assembly site for the enzymatic machinery destined to function at each of the two replication forks that move in opposite directions on the bacterial chromosome. The work described below addresses two questions. First, what is the molecular composition of complexes formed at oriC in assembling the replication fork machinery? Second, because DnaB is the replicative helicase that drives replication fork movement, is the entry of DnaB at oriC somehow modulated so that one and only one helicase molecule is bound per replication fork, or can multiple DnaB molecules bind? The stoichiometries of DnaA and DnaB protein in the prepriming complex assembled...

Helicase Loading at Chromosomal Origins of Replication

“…On the bottom strand the area protected is to the left of DnaA box R1 that overlaps the right 13-mer. Quantitative analysis of this protein complex assembled at oriC supports the conclusion from footprinting studies that two DnaB -DnaC complexes are bound (Fang et al 1999;Carr and Kaguni 2001).…”

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

“…In bacteria, origin DNA is initially unwound by DnaA, which promotes the DnaA-and DnaC-assisted loading of 2 single hexameric DnaB helicase complexes onto ssDNA (34). Large T-antigen, the replicative helicase from SV40, forms a hexamer in solution.…”

An Essential Tryptophan of Escherichia coli DnaA Protein Functions in Oligomerization at the E. coli Replication Origin

“…Complexed to ATP, DnaA next unwinds a region within oriC and then recruits the replicative helicase, DnaB, to form the prepriming complex (3,4). After DnaB translocates to the apex of each replication fork, the helicase both unwinds the parental DNA and interacts transiently with primase (5,6). Primers synthesized by primase are then extended by DNA polymerase III holoenzyme under a bidirectional mode of DNA replication.…”