Proofreading dynamics of a processive DNA polymerase

Ibarra, Borja; Chemla, Yann R.; Plyasunov, Sergey; Smith, Steven B.; Lázaro, José Portolés; Salas, Margarita; Bustamante, Carlos

doi:10.1038/emboj.2009.219

Cited by 99 publications

(160 citation statements)

References 44 publications

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“…Other alternative models, such as the model that describes φ29 pol and exo activity,28 are not consistent with our data. For φ29, the total velocity was presented as a sum of exo and pol activity, such that fluctuations between the states determined the total velocity.…”

Section: Resultscontrasting

confidence: 87%

“…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%

“…Specifically, in contrast to pol III core, the dwell time of φ29 is much longer than its time of switching between pol and exo activities. The average velocity of φ29 catalysis appears to be a continuous function of the force with several fine features suggesting intermediate steps in the pol to exo switching process 28. In contrast, ε by itself has been shown to be an ssDNA exonuclease, and the catalytic activity of ε is similar on ssDNA and mispaired primer termini.…”

Section: Discussionmentioning

confidence: 98%

“…The 8.1 kbp construct was created by first linearizing the pBacgus11 (a gift from Borja Ibarra) dsDNA vector (8041 bp) with BamHI and SacI (NEB). A digoxigenin (DIG) labeled dsDNA handle with a complementary sticky end to the BamHI sequence was generated as described 28. A biotinylated oligonucleotide (5′‐bbCTCbTCTCbTCTCTTCTCTCTT CTCTTGGCCAGCT‐3′, IDT) with a 3′ end complementary to SacI sequence, and the dsDNA DIG‐handles were then ligated to their complementary positions at the linearized pBacgus11 DNA using T4 DNA ligase.…”

Section: Methodsmentioning

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. The key difference between pol III core and these polymerases is that the editing and polymerization activities of pol III core are carried out by distinct subunits, ε and α, respectively.…”

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: Resultscontrasting

confidence: 87%

Section: Introductionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

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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Mechanical stability of low‐humidity single DNA molecules

et al. 2011

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DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone, which together with base sequence, further confer its higher order structure. The authors overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. The authors found that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70 and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules, which interact with DNA in vivo.

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A toolbox for generating single‐stranded DNA in optical tweezers experiments

et al. 2013

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Essential genomic transactions such as DNA-damage repair and DNA replication take place on single-stranded DNA (ssDNA) or require specific single-stranded/double-stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single-molecule studies of DNA-protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single-molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force-induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force-induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA.

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Proofreading dynamics of a processive DNA polymerase

Cited by 99 publications

References 44 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

Mechanical stability of low‐humidity single DNA molecules

A toolbox for generating single‐stranded DNA in optical tweezers experiments

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