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Nucleosome sliding by Chd1 does not require rigid coupling between DNA-binding and ATPase domains - PubMed

Nucleosome sliding by Chd1 does not require rigid coupling between DNA-binding and ATPase domains

Ilana M Nodelman et al. EMBO Rep. 2013 Dec.

Abstract

Chromatin remodellers are ATP-dependent motor proteins that physically reposition and reorganize nucleosomes. Chd1 and Iswi-type remodellers possess a DNA-binding domain (DBD) needed for efficient nucleosome mobilization; however, it has not been clear how this domain physically contributes to remodelling. Here we show that the Chd1 DBD promotes nucleosome sliding simply by tethering the remodeller to nucleosome substrates. Nucleosome sliding activity was largely resistant to increasing length and flexibility of the linker connecting the DBD and ATPase motor, arguing that the ATPase motor does not shift DNA onto the nucleosome by pulling on the DBD.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Overview of S. cerevisiae Chd1 constructs used in this study. (A) Chd1 variants possessing the native Chd1 DBD. All constructs were on the basis of an N- and C-terminally truncated variant spanning residues 118–1,274, which includes the double chromodomains, ATPase motor and DBD, and are indicated with the subscript ΔNC. All changes to the DBD linker are denoted with superscripts, with the locations of the deletions, replacement and insertions labelled on the schematics. GGSx9 stands for nine Gly-Gly-Ser repeats, which replaced Chd1 residues 971–1,000. The +RAM(121) construct contains a 121-residue insertion of the RAM segment (residues 1,763 to 1,883) from the Drosophila melanogaster Notch receptor. (B) Chd1 variants possessing the foreign AraC DBD. All constructs contained the DBD of the AraC transcriptional regulator (residues 175–281) in place of the native Chd1 DBD, denoted with the subscript ΔDBD/+AraC. As in (A), superscripts refer to changes in the DBD linker, with Δ1,015, Δ978 and Δ961 referring to the beginning of the deletion replaced by the AraC DBD. The +ANK(233) construct contained the ankyrin domain (residues 1,907–2,139) of D. melanogaster Notch. Note that domains in A and B are not drawn to scale. (C) and (D) Purified proteins containing the native Chd1 DBD (C) or with the foreign AraC DBD (D) on SDS–PAGE gels stained with GelCode Blue. ANK, Ankyrin; DBD, DNA-binding domain; SDS–PAGE, SDS–polyacrylamide gel electrophoresis

Figure 2
Figure 2

Chd1 tolerates increased length and flexibility in the DBD linker, but requires a minimal length linker for coordinated action of DBD and ATPase domains. (A) Nucleosome sliding as assessed by native PAGE. Data shown are representative experiments (n≥4). (B) Quantification of nucleosome sliding reactions shown in (A). Error bars represent standard deviations (s.d.) (n≥4). (C) Relative rate constants for nucleosome sliding. Rate constants were obtained from single exponential fits to the data shown in (B), with the relative rates shown as krel=kobs, Chd1 variant/kobs, Chd1ΔNC. For Chd1ΔNC, kobs was 0.33±0.02 min−1. Error bars represent the calculated errors of the fits. (D) Deletion of the DBD linker does not disrupt nucleosome binding, as shown by native PAGE. Super-shifted bands indicate Chd1–nucleosome complexes. (E) Deletion of the DBD linker still allows the Chd1 ATPase motor to be stimulated by nucleosomes. Shown are the averages and s.d. (n≥3) of nucleosome-stimulated ATPase activities with increasing concentrations of nucleosome substrate. Nucleosome-free rates were subtracted from all values, and the resultant activities were fit to the Michaelis–Menton equation (Chd1ΔNC and Chd1ΔNCΔ961–1,005, solid lines) or linearly (Chd1ΔDBD, broken line). Chd1ΔDBD contained residues 118–1,005. DBD, DNA-binding domain; PAGE, polyacrylamide gel electrophoresis.

Figure 3
Figure 3

Insertion of a long flexible segment into the DBD linker allows the Chd1–AraC remodeller to reach farther away from the nucleosome. End-positioned nucleosomes were generated containing the 17 bp araI1 site located +3, +13, +23, +33 or +43 bp away from the nucleosome edge. For each remodeller variant, a rate constant was calculated for each nucleosome substrate from single exponential fits to the data (n≥3). The bar graph shows rate constants, along with calculated errors of fitting, relative to Chd1ΔNC as in Fig 2. For Chd1ΔNC, the average kobs for nucleosomes with araI1 at +3 and +13 was 0.32±0.02 min−1. Asterisks denote relative rate constants≤0.004. araI1, AraC binding site; DBD, DNA-binding domain.

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