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. 2004 Oct 1;23(19):3836-43.
doi: 10.1038/sj.emboj.7600364. Epub 2004 Sep 9.

Reaction cycle of the yeast Isw2 chromatin remodeling complex

Daniel J Fitzgerald  1 , Carl DeLuca, Imre Berger, Hélène Gaillard, Raphael Sigrist, Kyoko Schimmele, Timothy J Richmond
Affiliations

Reaction cycle of the yeast Isw2 chromatin remodeling complex

Daniel J Fitzgerald et al. EMBO J. .

Abstract

Members of the ISWI family of chromatin remodeling factors hydrolyze ATP to reposition nucleosomes along DNA. Here we show that the yeast Isw2 complex interacts with DNA in a nucleotide-dependent manner at physiological ionic strength. Isw2 efficiently binds DNA in the absence of nucleotides and in the presence of a nonhydrolyzable ATP analog. Conversely, ADP promotes the dissociation of Isw2 from DNA. In contrast, Isw2 remains bound to mononucleosomes through multiple cycles of ATP hydrolysis. Solution studies show that Isw2 undergoes nucleotide-dependent alterations in conformation not requiring ATP hydrolysis. Our results indicate that during an Isw2 remodeling reaction, hydrolysis of successive ATP molecules coincides with cycles of DNA binding, release, and rebinding involving elements of Isw2 distinct from those interacting with nucleosomes. We propose that progression of the DNA-binding site occurs while nucleosome core contacts are maintained and generates a force dissipated by disruption of histone-DNA interactions.

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Figures

Figure 1
Figure 1
Recombinant Isw2 interacts with a mononucleosome model substrate. (A) Purity of the Isw2 preparation. Coomassie brilliant blue stained section of a 10% SDS gel. Isw2 comprises the Itc1p (145 kDa) and Isw2p (130 kDa) proteins. (B) Nucleosome-stimulated ATPase activity of recombinant Isw2. ATP hydrolysis rates (ATP/min/complex) are measured for Isw2 alone (−) and in the presence of histone octamer (Oct), 601-50 DNA (DNA), 601-50 nucleosome (Nuc), and 601-50 nucleosome including a 10:1 molar ratio of ATPγS to ATP (γS). ATP hydrolysis by Isw2(K215R) was measured in the presence of 601-50 nucleosomes (K215R). Error bars correspond to 1 standard deviation. (C) Isw2/601-50 nucleosome complexes visualized by native gel electrophoresis. Components of each reaction are listed above the gel in order of addition (top to bottom) for lanes 2–7. Lanes M and 1 are 100 bp marker and 601-50 DNA, respectively. Band identities are indicated on the right.
Figure 2
Figure 2
Isw2 binding to DNA is nucleotide dependent. (A) Native gel analysis of Isw2 binding to DNA. Isw2 was incubated with 2 M equivalents of 601-50 DNA and 1 mM of the indicated nucleotide (top) for lanes 2–5. Lanes M and 1 are 100 bp marker and 601-50 DNA, respectively. Band identities are indicated on the right. (B) Real-time Isw2/DNA binding studies. Representative SPR sensograms of four separate experiments are shown. Isw2 was passed over immobilized 601-50 DNA and followed with a wash solution. Isw2 dissociation was measured in the presence of the indicated nucleotides. (C) Native gel analysis of Isw2(K215R) binding to 601-50 DNA. Isw2(K215R) was incubated with 2 M equivalents of 601-50 DNA and 1 mM of the indicated nucleotide (top) for lanes 2–5. Lane 1 is 601-50 DNA. Band identities are indicated on the right.
Figure 3
Figure 3
Sedimentation velocity analysis of Isw2 in the presence of nucleotides. Isw2 was sedimented alone (none) and in the presence of 1 mM ADP or ATPγS. Representative s-value distributions are shown for each of the three conditions. The sedimentation boundaries shown for Isw2 correspond to centrifugation for 3 h in the presence of either 1 mM ADP or 1 mM ATPγS. The predicted boundary shapes from the s-value distribution model are also shown and superimpose on the measured boundaries for both conditions (inset).
Figure 4
Figure 4
Nucleotide dependency of Isw2 binding to 601-50 nucleosomes. (A) Native gel analysis of Isw2 binding to mononucleosomes. Isw2 was incubated with 1.3 M equivalents of 601-50 nucleosomes and 1 mM of the indicated nucleotides (top) for lanes 2–6. Lanes M and 1 are 100 bp marker and 601-50 DNA, respectively. Band identities are indicated on the right. (B) Real-time Isw2/mononucleosome binding studies. Isw2 was passed over immobilized 601-50 nucleosome and followed with a wash solution. Isw2 dissociation was measured in the presence of the indicated nucleotides. (C) Native agarose gel analysis of Isw2 binding to tetranucleosomes. Tetranucleosomes were incubated with 3.0 M equivalents of Isw2 and 1 mM of the indicated nucleotides (top) for lanes 2–6. Lanes M and 1 are 2-log DNA marker and tetranucleosome, respectively. Band identities are indicated on the right.
Figure 5
Figure 5
Exo III analysis of the Isw2/601-50 nucleosome complex. Isw2 was incubated with 1.0 M equivalent 601-50 nucleosomes and 1 mM of the indicated nucleotides (top). Exo III digestion was allowed to proceed for the indicated times in minutes (top) at 32°C, and the samples were analyzed on a 6% denaturing gel. The 601-50 nucleosome is depicted schematically by a dark oval (right). The distance in base pairs along the DNA flanking the 601 nucleosome core is also indicated. Isw2-specific pauses are marked by arrows. Lane numbers are indicated (bottom).
Figure 6
Figure 6
Model for coupling the Isw2 nucleosome remodeling reaction with an ATP hydrolysis cycle. The Isw2 complex (two black ovals) bound to a nucleosome (core: circle; flanking DNA: rod) is depicted in four states (I–IV), which represent differences in (1) Isw2 nucleotide binding, (2) Isw2 DNA binding, (3) Isw2 conformation, and (4) DNA position in the nucleosome core. The reaction cycle is described in the text. The depiction is not intended to suggest relative Isw2p and Itc1p subunit locations.
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