A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX4 forms DnaX3deltadelta'

Pritchard, A. E.; Dallmann, H. Garry; Glover, Bradley P.; McHenry, Charles S.

doi:10.1093/emboj/19.23.6536

Cited by 109 publications

(111 citation statements)

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“…6. The clamp loader assemblies τ 1 γ 2 δδ', τ 2 γ 1 δδ', and τ 2 γ 1 δδ'χψ were reconstituted by sequential mixing of purified subunits and separated by chromatography on a MonoS column (GE Healthcare), using modifications of the methods of Pritchard et al 50 , as described in Supplementary Fig. 7.…”

Section: Replication Proteinsmentioning

confidence: 99%

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Tanner

Hamdan

Jergic

et al. 2008

Nat Struct Mol Biol

136

145

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We present single-molecule studies of the replication machinery of Escherichia coli and describe the visualization of individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kb, 8-fold higher than that of primer extension by Pol III alone. Addition of the primase DnaG to the replisome causes a 3-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG proteinprotein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of 2-3 DnaG monomers to the propagating DnaB destabilizes the replisome. The modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.Complete and accurate replication of DNA involves the coordinated activity of a large number of proteins. The replisome, the molecular machinery of DNA replication, unwinds the doublestranded DNA (dsDNA), synthesizes primers to initiate synthesis, and polymerizes nucleotides onto each of the two growing strands 1 . The replication system of Escherichia coli is ideal for studying the dynamic interplay among the various components at the replication fork. The enzymes of the E. coli replisome duplicate DNA with remarkable efficiency: the replication fork moves at a rate approaching 1000 nucleotides per second while maintaining coordination between continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand 1,2 . A fully functional replisome that displays all the fundamental enzymatic reactions characterizing DNA replication can be reconstituted in vitro with a limited number of purified key protein components: the DnaB helicase unwinds dsDNA; the DnaG primase synthesizes short oligoribonucleotides for priming of synthesis of the lagging strand; and the DNA polymerase III (Pol III) holoenzyme polymerizes nucleotides onto each nascent strand ( Fig. 1 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe Pol III holoenzyme is composed of three subassemblies: a core polymerase, sliding clamp, and clamp loader complex. The core polymerase is a heterotrimer of three subunits: α, the DNA polymerase; ε, proofreading exonuclease; and θ, which stabilizes ε 4 . The αεθ core is a poorly processive polymerase that only incorporates <20 nucleotides before dissociating from the primer-template 5 . However, when tethered to the sliding clamp, a ring-shaped homodimer of β subunits that encircles dsDNA, the processivity of the core increases dramatically to several kilobases (kb) at ~750 bp/s 5 . The loading of the β 2 clamp onto the primer/template strand requires opening of the ring by the γ multiprotein clamp-loading complex ...

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Section: Replication Proteinsmentioning

confidence: 99%

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Tanner

Hamdan

Jergic

et al. 2008

Nat Struct Mol Biol

136

145

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“…The clamp loader, ␥ complex, consists of five different subunits, ␥, ␦, ␦Ј, , and . The ␦, ␦Ј, , and subunits are present in single copy in the complex (15,16), and ␥ is present as a homotrimer (17). The ␤ subunit interacts most strongly with the ␦ subunit (18), although it also shows a week interaction with the ␥ and subunits (A. Yuzhakov and M.O.D., unpublished results).…”

Section: Pcna and Gp45 Participate In Many Dna Metabolic Processesmentioning

confidence: 99%

“…ATP hydrolysis is promoted by primed DNA and results in dissociation of ␥ complex from ␤, leading to the closed ring on DNA (ref. 17, and reviewed in ref. 1).…”

Section: Pcna and Gp45 Participate In Many Dna Metabolic Processesmentioning

confidence: 99%

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

Saro

O'Donnell

2001

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

212

203

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The ␤ and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli ␤ clamp is known to function with DNA polymerases II and V, indicating that ␤ also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the ␤ clamp. Thus, ␤ interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of ␤ interactive proteins suggests that the prokaryotic ␤ ring participates in a wide variety of reactions beyond its role in chromosomal replication. PCNA ͉ mismatch repairThe Sliding Clamp in Replication

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“…The E. coli holoenzyme is held together by multiple proteinprotein interactions among subunits with the stoichiometry (␣⑀ ) 2 2 ␥␦␦Ј (␤ 2 ) 2 2 (27). Pol III (␣⑀ ) forms a stable, isolable complex held together by ␣-⑀ and ⑀-interactions (22, 28 -30).…”

mentioning

confidence: 99%

“…The ␣ and ⑀ subunits enhance the activity of one another (30 -32). Within the DnaX complex, three DnaX subunits form a pentameric core with structurally related proteins, ␦ and ␦Ј (19,27,33). ␥ binds -and appears to reside adjacent to ␦Ј within the pentameric ring that makes up the core of the DnaX complex (16,19,33,34).…”

mentioning

confidence: 99%

DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

Bullard¹,

Williams²,

Acker³

et al. 2002

Journal of Biological Chemistry

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DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the ␤ sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of ␣ (pol III catalytic subunit), ␤ (sliding clamp processivity factor), and the essential DnaX ( /␥), ␦ and ␦ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including ␦-␦ interaction, ␦␦ -/␥ complex formation, and ␣-interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50°C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72°C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.DNA replication in all biological systems is performed by specialized multiprotein replicases (1, 2). Cellular replicases consist of three major subassemblies: a sliding clamp processivity factor, a clamp loader, and a specialized polymerase. Replicases, especially bacterial replicases, are rapid and processive consistent with the requirement for them to synthesize a several megabase genome from a single origin in less than one hour.In the prototypic Escherichia coli replication system, a key determinant of processive DNA synthesis is the interaction between the ␤ processivity factor and pol III 1 (3, 4). The dimeric ␤ subunit is a bracelet-shaped molecule that clamps around DNA permitting it to rapidly slide along duplex DNA without dissociating (5). ␤ binds to the pol III ␣ subunit through protein-protein contacts preventing the polymerase from dissociating from the template, ensuring high processivity. Efficient loading of the ␤ subunit onto DNA requires ATP-dependent opening and closing of the clamp by the DnaX complex. The DnaX complex contains the essential DnaX, ␦ and ␦Ј subunits plus two ancillary proteins, and (6 -9). The dnaX gene encodes two proteins, ␥ and , by programmed ribosomal frameshifting (10 -15). Both and the shorter ␥ product share ATPbinding domain I, domain II, and domain III that is responsible for DnaX oligomerization, -binding, and binding of ␦-␦Ј (16 -19). contains two unique domains. domain IV forms a link with the DnaB helicase and domain V binds pol III (17,20,21). Pol III consists of ␣, the catalyti...

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A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX4 forms DnaX3deltadelta'

Cited by 109 publications

References 36 publications

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase

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