Anisotropy-Based Nucleosome Repositioning Assay

Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes....

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 1805; p. 333
Main Authors Briggs, Koan, Al-Ani, Gada, Eastlund, Allen, Fischer, Christopher J
Format Journal Article
LanguageEnglish
Published United States 2018
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Abstract Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.
AbstractList Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.
Author Briggs, Koan
Fischer, Christopher J
Al-Ani, Gada
Eastlund, Allen
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  surname: Al-Ani
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  givenname: Allen
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  organization: Department of Physics and Astronomy, College of Liberal Arts and Sciences, The University of Kansas, Lawrence, KS, USA
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  givenname: Christopher J
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  fullname: Fischer, Christopher J
  email: shark@ku.edu
  organization: Department of Physics and Astronomy, College of Liberal Arts and Sciences, The University of Kansas, Lawrence, KS, USA. shark@ku.edu
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Keywords Sequential n-step mechanism
Chromatin remodeler
Fluorescence anisotropy
Motor protein
Nucleosome repositioning
ATPase
Language English
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PublicationTitle Methods in molecular biology (Clifton, N.J.)
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Snippet Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called...
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StartPage 333
SubjectTerms Adenosine Triphosphate - metabolism
Animals
Anisotropy
Binding Sites
Biological Assay - methods
Chromatin Assembly and Disassembly
DNA - metabolism
Hydrolysis
Kinetics
Nucleosomes - metabolism
Substrate Specificity
Time Factors
Xenopus laevis
Title Anisotropy-Based Nucleosome Repositioning Assay
URI https://www.ncbi.nlm.nih.gov/pubmed/29971726
Volume 1805
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