No need for a power stroke in ISWI-mediated nucleosome sliding

Abstract

Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND-SANT-SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome. We inserted flexible linkers at strategic sites in ISWI to disrupt this putative power stroke and assess its functional importance by quantitative biochemical assays. Notably, the flexible linkers did not disrupt catalysis. Instead of engaging in a power stroke, the HSS module might therefore assist DNA to ratchet into the nucleosome. Our results clarify the roles had by the domains and suggest that the HSS domain evolved to optimize a rudimentary remodelling engine.

Figure 1

ISWI derivatives used in this study. (A) Schematic representation of ISWI derivatives. Glycine-rich inserts were introduced behind the indicated amino-acid positions. Numbers in subscript refer to the insertion position and the size of the insert. ISWI_ΔHSS spans the amino acids 26–648 and lacks the C-terminal HSS domains. (B) Coomassie-stained SDS–PAGE gel showing the purified ISWI derivatives from A. aa, amino acid; HSS, HAND, SANT and SLIDE; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; WT, wild type.

Figure 2

Insertion of flexible polypeptide linkers does not disrupt DNA- and nucleosome-stimulated ATP hydrolysis. ATPase rates were measured in the presence of saturating concentrations of ATP, DNA or nucleosomes. Errors are s.d. for ISWI_Wt (n=3) and minimal and maximal values of two independent measurements for all other enzyme derivatives. ATPase rates in the absence of DNA were <0.02 s⁻¹ under otherwise identical conditions (data not shown). ISWI_Wt, wild-type ISWI.

Figure 3

Insertion of flexible polypeptide linkers does not disrupt nucleosome remodelling. (A) Remodelling activity was probed by following the accessibility of a unique, central KpnI restriction site in a 13-mer nucleosomal array (see schematic). Exemplary time courses for remodelling by ISWI_Wt and ISWI_{658:10 aa} (both 3 μM). In mock-treated samples (−ISWI), the KpnI site was not accessible. The original unspliced gel picture containing the mock-treated sample and the ISWI_Wt time-course is provided as supplementary Fig S2 online. (B,C) The observed rate constants for remodelling, k_obs, were determined for ISWI derivatives at the indicated concentrations by fitting time courses as in A to single exponential functions. Errors are s.d. for ISWI_{589:10 aa} and ISWI_{658:10 aa} (n=4 for 0.1 μM and 1 μM; n=3 for 3 μM) and 1 μM ISWI_Wt (n=3). In all other instances, minimal and maximal values of two independent measurements are shown. aa, amino acid; ISWI_Wt, wild-type ISWI; kb, kilobases.

Figure 4

Insertion of flexible polypeptide linkers does not compromise nucleosome sliding. (A) Schematic depiction of the nucleosome sliding assay. 25-mer nucleosomal arrays containing exposed AvaI restriction sites in the linker DNA were used to follow nucleosome sliding. Accessibility to AvaI (arrows) changes upon remodelling. Nucleosomes (ovals) and AvaI sites (x) are indicated. (B) ISWI_Wt and the insertion mutants were incubated with nucleosomal arrays for 3, 13 and 48 min, whereas ISWI_ΔHSS was incubated for 6 h. Control reactions (−) were depleted of ATP with apyrase before addition of ISWI and incubated for 6 h (ISWI_ΔHSS) or 48 min (all other enzymes). ISWI_Wt, wild-type ISWI.

Figure 5

Models for nucleosome remodelling by ISWI. (A) Power stroke model. First, the ATPase engine of ISWI translocates 7 bp of DNA. The ATPase and HSS domains then undergo a power stroke that exerts force on the HSS domains. The power stroke pulls 3 bp of flanking DNA into the nucleosome. Hypothetical force-transducing elements are coloured red. (B) Ratchet model. As in A, the ATPase engine translocates 7 bp. This translocation strains the structure of the nucleosome, in particular the DNA delimitated by the ATPase and HSS domains (red). When the strain on the nucleosome structure becomes too large, 3 bp of DNA ratchet into the nucleosome. No direct coordination between the HSS and ATPase domain is required for this mechanism and flexible linkage between individual domains (curved lines) does not affect remodelling. Histones are light blue; DNA is grey.