Al-Ani G et al. (2014), Quantitative determination of binding of ISWI t...

XB-ART-49763

Biochemistry 2014 Jul 15;5327:4334-45. doi: 10.1021/bi500224t.

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Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides.

Al-Ani G , Briggs K , Malik SS , Conner M , Azuma Y , Fischer CJ .

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The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.

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Species referenced: Xenopus laevis
Genes referenced: smarca5

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	Scheme 1. ISWI (P) Binding to DNA (D) and the Nucleotide Analogue (A)Î²1, Î²A, Î²1,A, and Î²A,1 represent the stoichiometric macroscopic equilibrium constants.
	Scheme 2. ISWI (P) Binding to Nucleosomes (N)Î²1 and Î²2 represent the stoichiometric macroscopic equilibrium constants.
	Figure 1. Fluorescence anisotropy measurements (Îr) of binding of ISWI to DNA and nucleosome substrates. (A) A 20 bp FITC-labeled DNA substrate [(â) 10 and (â) 25 nM] was titrated with ISWI concentrations ranging from 6 to 183 nM, and changes in fluorescence anisotropy were monitored. Isotherms were analyzed using Scheme 1 as described in Experimental Procedures. The solid line represents the fit of the data to this scheme, which returned a 1/Î²1 value of 18 Â± 2 nM. (B) Electrophoretic mobility shift assay performed by titrating a nonlabeled 10N5 nucleosome substrate (50 nM) with increasing ISWI concentrations ranging from 12 to 200 nM. Samples were analyzed using a 5% TBEâacrylamide native gel. Gels were stained using a DNA staining dye and imaged using a Typhoon imager. Independent experiments showed that high-molecular weight smearing is caused by interaction of ISWI with free DNA present (<2%) in the reconstituted nucleosome sample. (C) Fluorescence anisotropy measurements of binding of ISWI to doubly labeled Alexa488 (F10N5F) and singly labeled Alexa488 (F10N5) nucleosomal substrates. Nucleosomes at 2.5 nM (â and â²) and 10 nM (â and â ) were titrated with increasing concentrations of ISWI ranging from 3 to115 nM. Equilibrium binding isotherms were analyzed using Scheme 2 as described in Experimental Procedures. The solid line represents the fit of the data to this scheme, which returned a 1/Î²1 value of 1.3 Â± 0.6 nM and a 1/Î²2 value of 13 Â± 7 nM2. (D) Computer simulations according to Scheme 2 of the fraction of free nucleosome (N), singly bound nucleosome (PN), and doubly bound nucleosome (P2N) species present as a function of the concentration of ISWI. In these simulations, the total nucleosome concentration was 10 nM, and a 1/Î²1 value of 1.3 Â± 0.6 nM and a 1/Î²2 value of 13 Â± 7 nM2 were taken from the analysis of the data in panel C.
	Figure 2. Fluorescence anisotropy measurements (Îr) of equilibrium binding of ISWI to DNA and nucleosomes in the presence of nucleotides. (A) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ATP-Î³-S. These data were analyzed using Scheme 1 as described in Experimental Procedures. The solid lines in the figure represent the fits of the data to this scheme, which returned the following values: 1/Î²A = 140 Â± 30 Î¼M, 1/Î²A,1 = 390 Â± 70 Î¼M, and 1/Î²1,A = 42 Â± 8 nM. (B) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ADP. The solid line in this figure represents the fit of equilibrium DNA binding data collected in the absence of nucleotide (Figure 1A). (C) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ATP-Î³-S. (D) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ADP. The solid lines in panels C and D are the fits of the equilibrium nucleosome binding data collected in the absence of nucleotides (Figure 1C).
	Figure 3. ISWI binding to nucleosome substrate with long flanking DNA. (A) EMSA performed by titrating a 10N18 nucleosome substrate (50 nM) with increasing concentrations of ISWI ranging from 12 to 300 nM. Samples were analyzed using a 5% TBEâacrylamide native gel. Gels were stained using a DNA staining dye and imaged using a Typhoon imager. (B) Fluorescence anisotropy measurements (Îr) of equilibrium binding of ISWI to Alexa488-labeled 10N18 nucleosomes in the presence of 2 mM nucleotides. To more readily determine the effect of ADP on ISWI binding, two different concentrations [2.5 nM (â, â², and â) and 10 nM (â and â¼)] of the 10N18 substrates were used in the associated binding experiments. The solid line in this panel represents the fit of the equilibrium nucleosome binding data collected in the absence of nucleotides. (C) Electrophoretic mobility shift assay performed by titrating a nonlabeled 5N71 nucleosome substrate (50 nM) with increasing concentrations of ISWI ranging from 12 to 300 nM. Samples were analyzed using a 5% TBEâacrylamide native gel. Gels were stained using a DNA staining dye and imaged using a Typhoon imager.

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