Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics

Wlodzimierz Bujalowski, Maria J. Jezewska

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Abstract

Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, dεA(pεA)19, that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer dεA(pεA)9, which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. The value of the bimolecular rate constant, k1, is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)1. These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)831-852
Number of pages22
JournalJournal of Molecular Biology
Volume295
Issue number4
DOIs
StatePublished - Jan 28 2000

Fingerprint

DnaB Helicases
Single-Stranded DNA
Escherichia coli
Fluorescence
Proteins
Binding Sites
Adenylyl Imidodiphosphate
Protein Binding
Nucleic Acids
Adenosine Triphosphate
Enzymes

Keywords

  • DNA replication
  • Helicase
  • Protein-ssDNA interactions
  • Stopped-flow kinetics

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics",
abstract = "Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, dεA(pεA)19, that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer dεA(pεA)9, which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. The value of the bimolecular rate constant, k1, is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)1. These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed. (C) 2000 Academic Press.",
keywords = "DNA replication, Helicase, Protein-ssDNA interactions, Stopped-flow kinetics",
author = "Wlodzimierz Bujalowski and Jezewska, {Maria J.}",
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T1 - Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics

AU - Bujalowski, Wlodzimierz

AU - Jezewska, Maria J.

PY - 2000/1/28

Y1 - 2000/1/28

N2 - Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, dεA(pεA)19, that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer dεA(pεA)9, which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. The value of the bimolecular rate constant, k1, is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)1. These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed. (C) 2000 Academic Press.

AB - Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, dεA(pεA)19, that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer dεA(pεA)9, which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. The value of the bimolecular rate constant, k1, is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)1. These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed. (C) 2000 Academic Press.

KW - DNA replication

KW - Helicase

KW - Protein-ssDNA interactions

KW - Stopped-flow kinetics

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U2 - 10.1006/jmbi.1999.3378

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EP - 852

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

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