Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by Rad51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein-dsDNA complexes. Rad54 and Rad51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, Rad51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of Rad51 in the product complex. Electron microscopy visualized the Rad51-Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the Rad51-dsDNA filament. Our data support a mechanism of processive dsDNA-Rad51 filament dissociation by the translocating Rad54 protein.recombination ͉ ATPase ͉ Snf2-like proteins H omologous recombination (HR) is a high-fidelity, templatedependent pathway involved in the nonmutagenic tolerance of DNA damage, the repair of complex DNA damage, and the recovery of stalled and collapsed replication forks (1, 2). The Rad52 group proteins define the HR pathway. Initially, RPA, the eukaryotic ssDNA-binding protein, binds to ssDNA exposed by doublestrand break processing or discoordination of leading and lagging strands at stalled replication forks. Mediator proteins (Rad52, Brca2, and Rad51 paralogs) orchestrate the assembly of Rad51 protein on ssDNA to form the nucleoprotein filament, which performs homology search and DNA-strand invasion to prime repair synthesis by using the undamaged sister chromatid or homolog as a template. After resolution of the pairing intermediates, HR has restored the contiguity of the chromosomes with a crossover or noncrossover outcome.Eukaryotes contain a large number of Snf2-related proteins (3, 4). This group of proteins shares structural and sequence similarity with SF2 DNA helicases, but they are unable to catalyze strand separation typical for DNA helicases (5). Instead, these proteins likely translocate on dsDNA inducing topological changes. Using the energy of ATP hydrolysis, their action leads to the remodeling or dissociation of protein-dsDNA complexes (4, 6). Prominent members of this group are the chromatin-remodeling factors Snf2, Isw1, Isw2, or Chd1. Each eukaryotic genome codes for a variety of Snf2-related proteins. Saccharomyces cerevisiae alone has 17 paralogs, and a number of these proteins appear to function outside a nucleosome remodeling context; for example, the Mot1 protein was found to dissociate the TATA box-binding protein from dsDNA (7). DNA repair pathways are richly endowed with specific, largely nonredundant Snf2-related proteins that function in transcriptioncoupled repair (Cockayne's Syndrome B͞Rad26 in S. cerevisiae; ref. Rad54 is a critical member of the Rad52 group, and its absence leads to catastrophic sensitivity to double-strand breaks in yeast (2,11,12). This protein displays robust dsDNA-specific ATPase ac...