In order to ensure faithful DNA replication, DNA repair, and gene expression, eukaryotic organisms must contend with the packaging of genomic DNA into chromatin. Dynamic changes in chromatin organization can be catalyzed by ATP-dependent chromatin-remodeling enzymes that use the energy derived from ATP hydrolysis to alter histone-DNA interactions (9
). All members of this class of remodeling enzyme contain a catalytic subunit that harbors a conserved ATPase domain related to yeast (Saccharomyces cerevisiae
) Swi2/Snf2, the founding member of a subfamily of the SF2 superfamily of DNA and RNA helicases (7
). Although no member of the Swi2/Snf2 family exhibits bona fide DNA helicase activity, all show DNA-stimulated ATPase activity that is required for chromatin-remodeling function in vitro and/or in vivo (6
The diagnostic feature of Snf2/Swi2 ATPases is a set of seven ATPase/helicase sequence motifs (7
). Structural studies of several bacterial SF2 family members indicate that this ATPase domain is assembled as two subdomains—a conserved N-terminal subdomain I (motifs I, Ia, II, and III) that is required for ATP binding and hydrolysis, and a C-terminal subdomain II (motifs IV to VI) which may play a role in energy transduction (4
). More biochemical information exists for motifs within the N-terminal subdomain I, due in part to the fact that it is found in a more diverse group of ATPases, which includes the recombination protein RecA (4
). Residues within each of the seven Swi2/Snf2 ATPase/helicase motifs are essential for the functions of the yeast SWI/SNF chromatin-remodeling enzyme in vivo, and single amino acid changes within motif I (Walker A box) eliminate the ATPase activity of SWI/SNF in vitro (15
). In contrast, the biochemical roles for the remaining six Swi2/Snf2 ATPase motifs have not been previously investigated.
The ~1 MDa yeast SWI/SNF complex was one of the first ATP-dependent chromatin-remodeling enzymes to be identified, and consequently many in vivo and in vitro studies have focused on this enzyme (28
). SWI/SNF is required for expression of a subset of highly inducible yeast genes as well as for gene expression during late mitosis (14
). Furthermore, SWI/SNF can interact directly with a variety of gene-specific transcriptional activators, and these contacts can lead to the recruitment of SWI/SNF remodeling activity to target nucleosomes both in vivo and in vitro (23
). How does SWI/SNF alter chromatin? In vitro assays have demonstrated that SWI/SNF-like enzymes can promote the accessibility of nucleases or DNA-binding transcription factors to nucleosomal sites. This enhanced accessibility of nucleosomal DNA may be due to the ATP-dependent movement of histone octamers in cis
along the DNA, the transfer of histone octamers from one nucleosomal array to another (transfer in trans
), the removal or replacement of nucleosomal histones, or the creation of alternative histone-DNA contacts that might include accessible loops of DNA on the nucleosomal surface (reviewed in reference 28
). The degree to which each of these biochemical activities contributes to chromatin-remodeling reactions in vivo remains unclear.
We are interested in understanding how ATP hydrolysis is utilized during the process of chromatin remodeling. To this end we purified SWI/SNF complexes that harbor Swi2/Snf2 subunits containing amino acid substitutions within the conserved ATPase/helicase motifs. Swi2/Snf2 residues were identified that disrupt the various kinetic parameters for ATP hydrolysis (e.g., Km or kcat), and these studies are consistent with previous functional analyses of distantly related DNA or RNA helicases. In contrast, we find that alterations within conserved motif V have little to no effect on ATPase activity but cripple the remodeling activity of SWI/SNF. The data presented here suggest that motif V plays an important role in coupling the hydrolysis of ATP to the mechanism of chromatin remodeling. The evidence also suggests a model whereby residues within motif V play a role in nucleosomal substrate recognition.