Al-Ani G et al. (2014), ISWI remodels nucleosomes through a random walk.

XB-ART-49764

Biochemistry 2014 Jul 15;5327:4346-57. doi: 10.1021/bi500226b.

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ISWI remodels nucleosomes through a random walk.

Al-Ani G , Malik SS , Eastlund A , Briggs K , Fischer CJ .

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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.

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Species referenced: Xenopus laevis
Genes referenced: smarca5

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References [+] :

Al-Ani, Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides. 2014, Pubmed, Xenbase

	Figure 1. Native gel-based repositioning of various nucleosome substrates by ISWI. (A) Repositioning of 51N51, 71N71, and 91N91 nucleosomes (50 nM) by ISWI (25 nM). ISWI and nucleosomes were incubated together at 25 Â°C, and repositioning reactions were initiated by addition of 1 mM ATP. Reactions were stopped at the indicated time points by the addition of stopping buffer and resolved using a 5% TBEâacrylamide native gel. The first lane in each gel (C) shows a control reaction without ISWI that was allowed to proceed for 120 min before being stopped. Gels were stained for DNA and imaged as indicated in Experimental Procedures. (B) Analysis of changes in translational positions over time for the 91N91 nucleosome substrate.
	Figure 2. Fluorescence anisotropy-based repositioning of F18N18F by ISWI. Measurements of changes in anisotropy (Îr) of 10 nM fluorophore-labeled F18N18F nucleosome incubated with 10 nM ISWI and 1 mM ATP (â), without ISWI (â ), or without ATP (â²).
	Figure 3. Fluorescence anisotropy-based repositioning of F18N18F and F24N24F. Measurements of changes in anisotropy (Îr) of 10 nM fluorophore-labeled F18N18F (â) or 24N24 (â ) nucleosomes incubated with 10 nM ISWI and 1 mM ATP. The solid lines represent single-exponential fits of the data.
	Figure 4. Fluorescence anisotropy-based repositioning of F18N18F and F24N24F in the presence of various ISWI concentrations. Measurements of changes in anisotropy (Îr) of 10 nM F18N18F (â) or F24N24F (â ) incubated with (A) 5, (B) 10, (C) 15, or (D) 20 nM ISWI. The reaction was started by the addition of 1 mM ATP. Isotherms were analyzed as described in Experimental Procedures. The solid lines represent fits of the data.
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