Mechanical Measurement of Single-molecule Binding Rates: Kinetics of DNA Helix-destabilization by T4 Gene 32 Protein

Pant, Kiran; Karpel, Richard L.; Rouzina, Ioulia; Williams, Mark C.

doi:10.1016/j.jmb.2003.12.025

Cited by 83 publications

(165 citation statements)

References 53 publications

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“…(8), (9) 49. We find that at least two base pairs ( n exo,init =1.84 ± 0.20 bp) are required to be destabilized through thermodynamic fluctuations to enter the exo‐active state of pol III core.…”

Section: Discussionmentioning

confidence: 92%

“…It has been shown that destabilization of the primer‐template junction increases the susceptibility of DNA to the exonuclease activity of pol III core and its exo subunit ε 30, 32, 33. Because template tension uniformly destabilizes all base pairs of stretched DNA,49 the force dependence of k init is likely due to the increased probability of the duplex fraying, which can be described as49

k_{init} = k_{F} e^{n_{exo, init} false(Δ G^{†} false(F false) - Δ G_{0}^{†} false) / k_{normals} T}

…”

Section: Resultsmentioning

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: Discussionmentioning

confidence: 92%

k_{init} = k_{F} e^{n_{exo, init} false(Δ G^{†} false(F false) - Δ G_{0}^{†} false) / k_{normals} T}

…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…To determine the equilibrium melting force, F eq , a series of time-dependent experiments have been performed. [100][101][102][103] The DNA-protein complex may be stretched into the overstretching plateau, specifically to a length of 0.42 nm per base pair. Alternatively, the DNA may be stretched, and then relaxed back to a length of 0.42 nm per base pair.…”

Section: Figurementioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

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The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 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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“…In contrast to temperatureinduced melting, these force studies can be performed at physiological temperatures, thus avoiding protein denaturation. Pant et al 34,66 pioneered this method to study the thermodynamic and kinetic DNA-binding properties of the T4 bacteriophage SSB, the gene 32 protein (gp32). A characterization of the dependence of the gp32 equilibrium binding constant on the ionic strength revealed that protein binding is regulated by intramolecular conformational changes (For more information, see the review by McCauley and Williams in this issue).…”

Section: Dna Denaturation By Single-stranded Dna Binding Proteinsmentioning

confidence: 99%

“…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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Mechanical Measurement of Single-molecule Binding Rates: Kinetics of DNA Helix-destabilization by T4 Gene 32 Protein

Cited by 83 publications

References 53 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

Mechanisms of DNA binding determined in optical tweezers experiments

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

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