Disparity in the DNA translocase domains of SWI/SNF and ISW2

Abstract

An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17–18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone–DNA interactions.

The SnAC Domain of SWI/SNF Is a Histone Anchor Required for Remodeling

“…Previously this approach has shown that the ATPase and HSA (helicase SANT-associated) domains of Snf2 associate with free DNA when SWI/SNF is bound and identified the specific orientation of the ATPase domain relative to DNA (8). We now find that the SnAC and ATPase domains are associated with the histone proteins when SWI/SNF is bound to nucleosomes.…”

“…Fe-BABE-modified nucleosomes were bound to WT SWI/SNF, and cleavage was initiated by adding H 2 O 2 and ascorbate. The SWI/SNF used in these experiments had a hemagglutinin (HA) epitope tag at the C terminus of Snf2, and immunoblotting detected full-length and proteolytic fragments containing the C-terminal end of Snf2 (8). Accurate determination of the cleavage sites was done using C-terminal HA epitope-tagged Snf2 polypeptides prepared by in vitro translation for size markers.…”

“…Mapping of Fe-BABE-mediated cleavage of Snf2 was performed as described previously (8). The cleavage products were analyzed by SDS-PAGE and immunoblotting using antihemagglutinin (anti-HA) antibody (Pierce, Rockford, IL) against the C-terminal HA tag of Snf2.…”

“…Nucleosome movement by SWI/SNF-and ISWI-type complexes requires the ATPase domain to translocate along nucleosomal DNA near the dyad axis (7)(8)(9)(10). DNA gaps near superhelical location 2 (SHL2) of the nucleosome block movement without interfering with binding of the remodeler.…”

“…The interactions of SWI/SNF with nucleosomes have been probed by site-directed cross-linking to specific sites in DNA and histones. DNA cross-linking has shown that Snf2, the catalytic subunit of SWI/SNF, is exclusively associated with nucleosomal DNA near SHL2 and other subunits are not seen elsewhere with extranucleosomal or nucleosomal DNA (8,13). The only excep-tion was the Snf6 subunit, which is colocalized with the transcription activator Gal4-VP16 to its binding site in the extranucleosomal DNA region.…”