SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.

In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Here we show that Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, we propose that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination.

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with singlestranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.Homologous recombination plays an essential role in repair of DNA double-stranded breaks, a lethal type of DNA damage. The mechanisms of double-stranded break repair by recombination have been extensively investigated at the genetic and biochemical levels (1-6). In yeast, genetic analysis reveals a set of genes, called the RAD52 epistasis group, that are directly involved in double-stranded break repair (7). This group includes the RAD50, RAD58/MRE11, XRS2, RPA1, RAD51, RAD52, RAD54, RAD55, RAD57, and RAD59 genes. Among the proteins encoded by these genes, the Rad51 protein is the most evolutionarily conserved; its homologues are spread from bacteria to mammals (8). The Rad51 protein family members possess a unique property; they form helical nucleoprotein filaments with DNA (9 -11) and promote DNA pairing and strand transfer, a basic step of homologous recombination (11-16).More recently, it was demonstrated that Rad52 protein, Rad54 protein, and the Rad55/Rad57 heterodimer stimulate DNA pairing promoted by Rad51 protein. The mechanistic studies revealed that stimulation by Rad52 protein and the Rad55/Rad57 proteins is at the early (presynaptic) stage of DNA strand exchange. Rad52 protein enhances the ability of Rad51 protein to displace RPA from ssDNA 1 (17)(18)(19)(20), as do the Rad55/Rad57 proteins, but by a different mechanism (21).The mechanism by which Rad54 protein stimulates the DNA pairing activity of Rad51 protein is different (22-28). It was found that, in contrast to other proteins that stimulate the DNA pairing activity of Rad51 protein, Rad54 protein acts at a later (synaptic) stage of DNA strand exchange (24, 29). Rad54 protein belongs to the Snf2/Swi2 group of proteins, which are involved in ATP-dependent remodeling of protein complexes, in...

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