The ATPase domain of Swi2p/Snf2p is the founding member of the SNF2 family of ATPases, which belongs to the helicase superfamily 2 (SF2) (Eisen et al., 1995). SF2-type ATPase domains consist of a set of seven ATPase/helicase motifs, which are organised in two subdomains. The N-terminal subdomain I includes motifs I, Ia, II, and III and has been implicated in ATP-binding and hydrolysis, whereas the C-terminal subdomain II contains motifs IV to VI and is thought to translate the ATP-derived energy into DNA rearrangements. Subdomain I is also present in the bacterial recombination protein RecA (Caruthers and McKay, 2002).
Rad54 is a SF2-type ATPase that functions in concert with the recombinase Rad51 in DNA double strand break repair. Its ATPase activity is stimulated by double stranded DNA, and like other members of the Swi2p/Snf2p family, it is able to translocate on DNA, to create negative superhelical torsion and to enhance accessibility to nucleosomal DNA (Jaskelioff et al., 2003).
Recent crystal structures of two Rad54 ATPase domains from Sulfolobus solfataricus and zebrafish provide valuable insights in the mechanism of DNA translocation and energy transduction by Swi2p/Snf2p-type ATPases (Dürr et al., 2005; Thomä et al., 2005).
Both crystal structures confirm that the overall organisation of the Rad54 ATPase domain closely resembles other SF2-class ATPases. However, two helical subdomains, which are fused to the ATPase core, seem to be specific for the Swi2p/Snf2p type. This allows speculating that these domains might crucial for Swi2p/Snf2p function.
The structure of Sulfolobus solfataricus Rad54 in complex with DNA shows that Rad54 binds the DNA double helix at the backbone and suggests that it travels along the minor groove. During the translocation, the DNA double helix is rotated along its helical axis, or, in other words, negative superhelical torsion is created. A multi-subunit chromatin remodelling complex with further substrate binding sites in addition to the DNA binding site could therefore use both the translocation and the twisting of DNA to disrupt protein-DNA interactions (Dürr et al., 2005).
The Swi2p/Snf2p ATPases are structurally related to DExx box helicases, although they do not display helicase activity. In contrast to the helicases, Rad54 does not possess any single strand binding domain and no wedge-like structures that could separate the two DNA strands. The DNA-dependent stimulation of the ATPase activity can be explained with the help of
the Rad54 structure. DNA binding triggers a conformational change within the Mg2+-binding
and ATP-hydrolysing motif II. Due to these structural rearrangements, a conserved glutamate residue adopts a conformation that allows ATP hydrolysis (Dürr et al., 2005).
A detailed mutational analysis of several Swi2p/Snf2p family members has recently shown that mutations in the ATPase motif V have only minor effects on the DNA-stimulated ATPase activity, but destroy the remodelling activity. As a consequence, motif V is thought to couple the energy of ATP hydrolysis to the mechanical force required for chromatin remodelling (Smith and Peterson, 2005). Consistent with these observations, several mutations in this motif have also been reported to be implicated in various cancers (Medina et al., 2004; Wong et al., 2000).
2.4.4
SWI/SNF complexes
Several different remodelling activities have been reported for SWI/SNF and related complexes. SWI/SNF- and RSC complexes peel off the DNA from the histone octamer surface and have been shown to displace nucleosomes in trans, but also induce nucleosome sliding in cis (Jaskelioff et al., 2000; Lorch et al., 2001; Phelan et al., 2000; Vicent et al., 2004). Moreover, SWI/SNF disrupts regular nucleosomal arrays and forms noncovalently linked dinucleosome structures (Phelan et al., 2000; Schnitzler et al., 2001). It has also been reported that these complexes are able to displace H2A/H2B dimers from or exchange them between nucleosomes (Bruno et al., 2003; Vicent et al., 2004).
The subunit stoichiometry of the eleven-subunit SWI/SNF has been investigated in detail. For six out of the eleven subunits, including the ATPase subunit Swi2p/Snf2p, it could be shown by differential epitope tagging and quantitative tyrosine-iodination that only one copy is present in the complex. The rest of the subunits are present in duplicate or triplicate. The calculated molecular weight of SWI/SNF is therefore approximately 1.15 MDa, in agreement with scanning transmission electron microscopy studies (Smith et al., 2003).
The structure of both SWI/SNF and RSC has been studied by electron microscopy (Asturias et al., 2002; Schnitzler et al., 2001; Smith et al., 2003). The low resolution structures of both complexes show a disc-like shape with several prominent lobes and a central cavity large enough to accommodate a single nucleosome. Naturally, this pocket is the prime candidate for the nucleosome binding site (Asturias et al., 2002; Smith et al., 2003), see Figure 2.6.
Recently, Cairns and colleagues proposed a ‘wave-ratchet-wave’ mechanism for RSC- mediated remodelling, based on detailed biochemical studies (Saha et al., 2005). According to this model, the ATPase Sth1p binds to nucleosomal DNA at an internal site, approximately two helical turns from the dyad axis. Upon ATP hydrolysis, the DNA is then translocated towards the dyad axis by pulling and twisting, which causes a first wave of one-dimensional diffusion of the DNA towards the Sth1p-binding site. The DNA is then released through a uni-directional ratchet and propagated in a second wave of DNA diffusion towards the distal linker.
Figure 2.6: 3D structure of the S. cerevisiae SWI/SNF complex at 30 Å resolution. Numbers represent individual centres of mass. Panels C and D are shown in the same orientation, and the semi-transparent view in D shows the twelve centres of mass (white spheres). Panels A and B are 90° rotations around the horizontal axis in C and D and panels E and F are 90° rotations around the vertical axis in C and D. The rim of the conical depression, which is the putative nucleosome binding site, is surrounded by the mass centres 1 to
6. Centre 8 is located close to the base of the depression. From (Smith et al., 2003).