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Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
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ISWI remodels nucleosomes through a random walk.

Gada Al-Ani1, Shuja Shafi Malik, Allen Eastlund

  • 1Department of Molecular Biosciences, University of Kansas , 2034 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, Kansas 66045, United States.

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The ISWI chromatin remodeler moves histone octamers on DNA via a random walk mechanism. A single ISWI monomer is sufficient for nucleosome repositioning, with ATP hydrolysis not being rate-limiting.

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Area of Science:

  • Molecular Biology
  • Chromatin Dynamics
  • Biochemistry

Background:

  • The ISWI chromatin remodeler plays a crucial role in organizing nucleosomes on DNA.
  • Understanding the precise mechanism of ISWI-mediated nucleosome repositioning is vital for comprehending chromatin structure and function.

Purpose of the Study:

  • To investigate the mechanism by which the ISWI chromatin remodeler repositions nucleosomes on DNA.
  • To quantify the rate of nucleosome repositioning and determine the role of ISWI concentration and ATP hydrolysis in the process.

Main Methods:

  • Standard electrophoresis assays to monitor ISWI-catalyzed nucleosome repositioning.
  • Spectrophotometric assays to characterize ISWI activity on short nucleosome substrates.
  • Quantitative analysis of repositioning experiments at varying ISWI concentrations.

Main Results:

  • ISWI repositions histone octamers between distinct, stable positions on DNA through a random walk mechanism.
  • The macroscopic rate of nucleosome repositioning was determined.
  • A single ISWI monomer is sufficient for repositioning; additional ISWI binding does not increase the rate.
  • ATP hydrolysis is poorly coupled to nucleosome repositioning, indicating it is not energetically rate-limiting.

Conclusions:

  • ISWI utilizes a random walk mechanism for nucleosome repositioning.
  • Nucleosome repositioning by ISWI is primarily mediated by a single ISWI monomer.
  • The energy from ATP hydrolysis is not the sole determinant of the rate-limiting step in ISWI-driven DNA translocation.