Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

Wuite, Gijs J. L.; Smith, Steven B.; Young, Mark C.; Keller, David L.; Bustamante, Carlos

doi:10.1038/35003614

Cited by 456 publications

(469 citation statements)

References 20 publications

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“…We observe that the mechanical tension applied to the substrate DNA promotes the switching between exonucleolysis and polymerization functions, which agrees with previous single molecule studies on DNA polymerases Klenow Fragment, T7 gp5, and φ29 DNA polymerase 27, 28, 29. The force dependence of T7 polymerization velocity is modeled as a function of the free energy change involved in ssDNA‐dsDNA conversion 27.…”

Section: Introductionsupporting

confidence: 88%

“…The instantaneous velocities of T7 and Klenow Fragment DNA polymerases were previously modeled,27, 29 primarily attributing the force dependence to the activation enthalpy of converting n bases from the ss to the ds geometry. In that model, the projections of DNA segments along the direction of applied external force were determined “globally” by the change in the extension of ds and ss DNA extension as a function of force, averaged over thousands of bases.…”

Section: Resultsmentioning

confidence: 99%

“…The force dependence of T7 polymerization velocity is modeled as a function of the free energy change involved in ssDNA‐dsDNA conversion 27. The kinetic scheme proposed for φ29 DNA polymerase describes the intramolecular primer transfer as a consequence of a conformational change in the φ29 pol ‐ DNA assembly induced by the applied tension on the DNA template 28.…”

Section: Introductionmentioning

confidence: 99%

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Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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Section: Introductionsupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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“…An intrinsic structural property of DNA is that ssDNA is substantially shorter than dsDNA at forces lower than 6 pN (Fig. 2c) [26][27][28] . As a consequence, conversion from dsDNA to ssDNA can be monitored through a decrease in total DNA length.…”

Section: Resultsmentioning

confidence: 99%

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

Tanner

Hamdan

Jergic

et al. 2008

Nat Struct Mol Biol

137

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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“…The results of these studies have contributed to new insights in the areas of transcription, [18][19][20][21][22][23] recombination, [24][25][26][27][28][29][30] repair, 31 and replication. [32][33][34][35][36][37] The physical properties of DNA itself have been thoroughly characterized by stretching individual DNA molecules and monitoring their length as a function of parameters such as force and torque. 38 In this review, I will discuss how changes in the physical properties of DNA can be exploited to study the dynamics of nucleic-acid enzymes at the single-molecule level.…”

Section: Introductionmentioning

confidence: 99%

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

Oijen

2006

Biopolymers

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The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single‐ and double‐stranded DNA can be used as a probe to study the dynamics of these enzymes at the single‐molecule level. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 144–153, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

Cited by 456 publications

References 20 publications

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

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

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

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