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Biochemistry. 2014 Jul 15;53(27):4346-57. doi: 10.1021/bi500226b. Epub 2014 Jun 30.

ISWI remodels nucleosomes through a random walk.

Author information

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

Abstract

The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.

PMID:
24898619
PMCID:
PMC4100782
DOI:
10.1021/bi500226b
[Indexed for MEDLINE]
Free PMC Article

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