Domains of DnaA Protein Involved in Interaction with DnaB Protein, and in Unwinding the Escherichia coli Chromosomal Origin

Marszałek, Jarosław; Zhang, Wenge; Hupp, Ted R.; Margulies, Carla; Carr, Kevin M.; Cherry, Scott; Kaguni, Jon M.

doi:10.1074/jbc.271.31.18535

Cited by 83 publications

(139 citation statements)

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“…Prepriming Complex Formation-Plasmid DNAs and purified proteins have been described previously (10,14). To separate the steps of assembly of the prepriming complex from downstream events of priming and DNA replication, staged incubations were performed (15).…”

Section: Methodsmentioning

confidence: 99%

“…The highly negatively supercoiled DNA termed F1* is the product of this assay. Finally, prepriming complex formation can be shown by the specific retention of DnaA and DnaB (and also DnaC if the prepriming complex is formed with ATP␥S instead of ATP) at oriC by immunoblot analysis of isolated complexes (10) or by electron microscopy (9,19).…”

Section: Conditions Optimal For Preprimingmentioning

confidence: 99%

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

Carr

Kaguni

2001

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Conditions Optimal For Preprimingmentioning

confidence: 99%

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

Carr

Kaguni

2001

Journal of Biological Chemistry

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“…Specific interaction between DnaA and the DnaB helicase (9,10) recruits the helicase and contributes to its loading by a helicase loader, the DnaC protein (11). Interactions between DnaB and the τ-subunit of polymerase (12), as well as DnaB and primase (13), contribute to replisome assembly at Escherichia coli oriC.…”

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confidence: 99%

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

Wawrzycka

Gross

Wasaznik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β-and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.DNA replication initiation | polymerase III | β-clamp | Rep | plasmid RK2

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“…The DnaA complex unwinds and opens several AT-rich segments of doublestranded DNA within the origin and stabilizes the resulting single-strands. DnaA then recruits the DnaB helicase with help from DnaC to form the "prepriming complex" [1]. After priming of the singlestranded DNA, replication occurs by the action of DNA Polymerase III.…”

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confidence: 99%

“…In addition to oligomerization, cross-linking studies have shown that the N-terminal domain of DnaA interacts directly with both DnaB [1] and the regulatory protein DiaA [21]. For DnaB, the regions of interaction were further defined by solid phase binding assays, which showed that residues 24-86 from the N-terminal domain, as well as residues in domain III, were important [20].…”

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NMR structure of the N-terminal domain of the replication initiator protein DnaA

Lowery

Chandonia

Kim

et al. 2007

J Struct Funct Genomics

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DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1 -100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/-0.2 Å based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.Keywords: replication, DnaB, DiaA, ATPase, initiation Page 2 12/12/06The bacterial protein DnaA plays a central role in the initiation of chromosomal replication and is involved in the activation and repression of several genes, including its own gene (dnaA) in E.coli. Replication begins with the formation of oligomers of DnaA on recognition sites at the origin of replication (OriC). The DnaA complex unwinds and opens several AT-rich segments of doublestranded DNA within the origin and stabilizes the resulting single-strands. DnaA then recruits the DnaB helicase with help from DnaC to form the "prepriming complex" [1]. After priming of the singlestranded DNA, replication occurs by the action of DNA Polymerase III. ATP hydrolysis acts as the switch between active (ATP-bound) and inactive (ADP-bound) forms of DnaA. Initiation begins only with the ATP-bound form (for reviews see [2][3][4] , as well as domain IV bound to DNA [8] showed that domain III resembles other members of the AAA+ family of ATPases, and that domain IV adopts a helix-turn-helix DNA-binding fold. In contrast, the secondary structure of domain I has been predicted based on multiple sequence alignments [5,9,10], however, little is known about its tertiary structure.DnaA recognizes five high affinity 9 base pair sites as well as several lower affinity 6 base pair sites within OriC via the C-terminal helix-turn-helix DNA-binding domain (domain IV) [11,12]. In addition to direct binding, the formation of oligomers containing 20 to 30 molecules of DnaA at the origin has been demonstrated by electron microscopy [13,14]. The mechanism of oligomer formation has been studied extensively [3,15], but the nature of the complex remains poorly understood.Replacement of the dimerization domain of the lambda cI repressor with the N-terminal domain of DnaA resulted in a functional chimeric protein, suggesting direct interactions between the N-terminal ...

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Domains of DnaA Protein Involved in Interaction with DnaB Protein, and in Unwinding the Escherichia coli Chromosomal Origin

Cited by 83 publications

References 44 publications

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

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

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

NMR structure of the N-terminal domain of the replication initiator protein DnaA

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