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. 2002 Aug 15;16(16):2120-34.
doi: 10.1101/gad.995002.

Chromatin remodeling by RSC involves ATP-dependent DNA translocation

Anjanabha Saha  1 , Jacqueline Wittmeyer, Bradley R Cairns
Affiliations

Chromatin remodeling by RSC involves ATP-dependent DNA translocation

Anjanabha Saha et al. Genes Dev. .

Abstract

Chromatin-remodeling complexes couple ATP hydrolysis to alterations in histone-DNA interactions and nucleosome mobility, allowing transcription factors access to chromatin. Here, we use triple-helix strand-displacement assays, DNA length-dependent ATPase assays, and DNA-minicircle ATPase assays to establish that RSC, as well as its isolated ATPase subunit Sth1, are DNA translocases. RSC/Sth1 ATPase activity is stimulated by single-stranded DNA, suggesting that Sth1 tracks along one strand of the DNA duplex. Each RSC complex appears to contain a single molecule of Sth1, and isolated Sth1 is capable of nucleosome remodeling. We propose that the remodeling enzyme remains in a fixed position on the octamer and translocates a segment of DNA (with accompanying DNA twist), which breaks histone-DNA contacts and propagates as a wave of DNA around the octamer. The demonstration of DNA translocation presented here provides a mechanistic basis for this DNA wave. To test the relative contribution of twist to remodeling, we use nucleosomes containing nicks in precise locations to uncouple twist and translocation. Nucleosomes bearing nicks are remodeled less efficiently than intact nucleosomes. These results suggest that RSC and Sth1 are DNA translocases that use both DNA translocation and twist to remodel nucleosomes efficiently.

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Figures

Figure 1
Figure 1
Purification and ATPase activities of RSC and Sth1. (A) Purified RSC. RSC was purified from strain BCY211, which expresses TAP-tagged Rsc2 from the chromosomal RSC2 locus. Harvesting and purification were performed as described in Puig et al. (2001) and Materials and Methods. Small RSC subunits Rsc12–15, which stain weakly with silver, are present but not shown. (*) Degradation product of Sth1. (B) Purified Sth1. Sth1 was purified from strain BCY243, which bears a chromosomally integrated Gal4 overproduction system (B. Cairns, unpubl.) and a high-copy plasmid directing the synthesis of Flag-tagged Sth1 from the GAL1 promoter. Soluble Sth1 was purified by ion exchange (SP), Ni-NTA, and M2-Flag affinity columns. Western analysis verified the absence of RSC subunits in the purified material (data not shown). (C) ATPase properties of RSC and Sth1. Turnover numbers (Vmax) for RSC and Sth1 with various nucleic acid substrates: (ds DNA) double-stranded plasmid (BSCR, 3 kb); (ss DNA) single-stranded phagemid (BSCR, 3 kb); (ss or ds RNA) polyI · polyC double-stranded RNA, polyG single-stranded RNA, or polyU single-stranded RNA; (Nucleosomes) intact mononucleosomes (167 bp) as described in Fig. 6. Values are the average of at least three experiments, and all are within a standard error of ±5%.
Figure 2
Figure 2
Oligomeric state of Sth1 in RSC. Coimmunoprecipitation analysis was performed using whole-cell extracts and either anti-Flag agarose or IgG-Sepharose (for TAP tag). (A) Western analysis using anti-Sth1 antisera of extracts from three diploid strains bearing different tagged STH1 alleles. All three strains contain a wild-type STH1 allele. BCY241 (lane 1) also bears an integrated TAP-tagged STH1 allele, BCY240 (lane 2) also contains a Flag-tagged allele present on a centromeric plasmid (p1170.STH1), and BCY242 (lane 3) contains both the integrated TAP-tagged allele and p1170.STH1. (B) Precipitation of Flag-tagged Sth1. (Control) Anti-Flag beads with BCY241 extract. Anti-Flag precipitation was performed with BCY242 extract. (C) Precipitation of BCY242 extract with IgG. (Control) IgG with BCY240 extract.
Figure 3
Figure 3
Dependence of RSC and Sth1 ATPase activity on DNA length. The maximal velocity of ATPase activity was determined with RSC or Sth1 as described in Materials and Methods. Values are the average of at least three experiments and are reported relative to the Vmax observed with double-stranded plasmid DNA (BSCR, 3 kb). The standard error with double-stranded DNAs is ±5%; with single-stranded DNAs, ±3%. (A) Length dependence of RSC with double-stranded DNA. (B) Length dependence of RSC with single-stranded DNA. (C) Length dependence of Sth1 with double-stranded DNA. (D) Length dependence of Sth1 with single-stranded DNA.
Figure 4
Figure 4
ATPase activity with single-stranded DNA minicircles. (A) Integrity of the DNA minicircle. Integrity was verified by resistance to ExoVII. Linear or circular 45-mer DNA (300 ng) was treated with ExoVII, separated on a 7.5% polyacrylamide gel, and visualized by ethidium bromide staining. A negative of the image is provided. Identical susceptibility and resistance were obtained with the linear and circular 30-mer, respectively (data not shown). (B,C) ATPase activity of Sth1 (B) or RSC complex (C) with linear (closed diamonds) or circular (closed circles) 45-mers. Values are the average of at least three experiments and are reported relative to the Vmax elicited by double-stranded plasmid DNA (BSCR, 3 kb). Standard errors are reported in the text.
Figure 5
Figure 5
RSC and Sth1 displace a triple helix. Triple-helix substrates consisting of a 40-base triple helical region were prepared as described in Materials and Methods. Triple helices were center-positioned on a 190-bp duplex DNA, end-positioned on a 114-bp duplex DNA, or prepared without duplex extension. Substrates were treated as indicated at 30°C for 30 min (or heated briefly at 90°C, labeled Heat) and separated in a 15% polyacrylamide gel. Displacement of the 32P-labeled third strand as a percentage of total displacement (%Disp.) was quantified by PhosphorImager analysis. (A) Displacement of a center-positioned triple helix by Sth1. (B) Displacement of a center-positioned triple helix by RSC. (C) Sth1 cannot displace an isolated triple helix. Identical results were obtained with 8 nM RSC (data not shown). (D) Displacement of an end-positioned triple helix by Sth1 is not affected by the presence of a nick near the duplex/triplex junction. Intact substrate or an identical substrate bearing a nick 4 bp from the duplex/triplex junction was used. Here, reactions were performed at 30°C for 1 h to compensate for lower efficiency compared with center-positioned substrates. Identical results were obtained with 8 nM RSC (data not shown).
Figure 6
Figure 6
Effect of DNA nicks on nucleosome remodeling efficiency. Recombinant yeast octamers were assembled into radiolabeled intact or nicked mononucleosomes (167 bp) and purified as described in Materials and Methods. The 5S positioning sequence leaves 17 bp outside the nucleosome on the labeled end, and 4 bp on the unlabeled end. The nicks are positioned 23 and 33 bases upstream and downstream of the center of the DraI site (6 bp). (A) Reconstitution strategy and location of the DraI cleavage site relative to the DNA nicks. (B) Purified nucleosome substrates. Purified nucleosomes or DNA alone were separated on a 4% polyacrylamide gel and visualized by autoradiography. (C–F) Remodeling efficiency of intact and nicked nucleosomes. Where indicated, reactions contain DraI (20 units), ATP (1 mM), and nucleosomes (30 ng; 12 nM). All ATPase values provided are relative to activity with the RSC complex and intact nucleosomes (panel C, lane 8) so that direct comparisons may be made. Shown is a representative experiment, but the ATPase and cleavage values are the average of three experiments.
Figure 7
Figure 7
A model for DNA translocation in nucleosome remodeling and mobility. RSC engages the nucleosome (for clarity, only half is shown) at or near the entry/exit site and uses the energy of ATP hydrolysis to translocate a segment of DNA, depicted by the movement of a fixed point on the DNA (yellow sphere). This segment bears both a translational and a twist component that breaks histone–DNA contacts, and may propagate through the nucleosome by one-dimensional diffusion. DNA-binding factors may have access to and bind the DNA wave during propagation. Resolution of the wave at the other entry/exit site results in a new translational position, causing octamer sliding (not shown).
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