Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase

Quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method

Roberto Galletto, Maria J. Jezewska, Wlodzimierz Bujalowski

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63 Citations (Scopus)

Abstract

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining ∼11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(±0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25°C unwinds ∼291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P=0.89(±0.03) , little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

Original languageEnglish (US)
Pages (from-to)83-99
Number of pages17
JournalJournal of Molecular Biology
Volume343
Issue number1
DOIs
StatePublished - Oct 8 2004

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DnaB Helicases
Base Pairing
Escherichia coli
DNA
Enzymes
Adenosine Triphosphate
Temperature
Single-Stranded DNA
Proteins

Keywords

  • E. coli DnaB helicase
  • helicase mechanism
  • processivity
  • rapid quench-flow
  • single turnover kinetics

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: Quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method",
abstract = "Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining ∼11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(±0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25°C unwinds ∼291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P=0.89(±0.03) , little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.",
keywords = "E. coli DnaB helicase, helicase mechanism, processivity, rapid quench-flow, single turnover kinetics",
author = "Roberto Galletto and Jezewska, {Maria J.} and Wlodzimierz Bujalowski",
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TY - JOUR

T1 - Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase

T2 - Quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method

AU - Galletto, Roberto

AU - Jezewska, Maria J.

AU - Bujalowski, Wlodzimierz

PY - 2004/10/8

Y1 - 2004/10/8

N2 - Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining ∼11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(±0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25°C unwinds ∼291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P=0.89(±0.03) , little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

AB - Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining ∼11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(±0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25°C unwinds ∼291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P=0.89(±0.03) , little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

KW - E. coli DnaB helicase

KW - helicase mechanism

KW - processivity

KW - rapid quench-flow

KW - single turnover kinetics

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DO - 10.1016/j.jmb.2004.07.055

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JF - Journal of Molecular Biology

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