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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumRad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchangeJachen Armon Solinger* and Wolf-Dietrich Heyer*†‡ Division of Biological Sciences, *Section of Microbiology and †Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51 Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins. heteroduplex DNA extension double-strand break repair | recombination | yeast Accurate repair of DNA double-strand breaks (DSB) is important for the survival and genomic stability of all organisms. Homologous recombination is an evolutionarily conserved process that is involved in DSB repair in all life forms (1). Central to this process is the homologous DNA pairing and DNA strand exchange that is performed by the Escherichia coli RecA protein or its eukaryal and archaeal homologs, Rad51 and RadA proteins, respectively (2). These proteins form nucleoprotein filaments with single-stranded DNA (ssDNA) of highly similar structure and function. The formation of the Rad51 (RecA, RadA) nucleoprotein filament is referred to as the presynaptic phase of homologous recombination and is stimulated by ssDNA binding proteins, like the eukaryotic Rpa (Replication protein A) (3, 4). The nucleoprotein filament performs the critical functions in recombination in the synaptic phase of the reaction: homology search and DNA strand exchange between the bound ssDNA and the homologous double-stranded DNA (dsDNA) partner (5). Heteroduplex DNA (hDNA) extension and branch migration occurs in the postsynaptic phase of the reaction (2). In E. coli, hDNA extension and branch migration is catalyzed by the RuvAB proteins (6). Finally, resolution of Holliday junctions is achieved in E. coli by the junction-specific RuvC endonuclease (6). A mechanism of hDNA extension that differs from the RuvAB paradigm has been described in bacteriophage T4 (7). Biochemical experiments have revealed activities that resemble bacterial RuvABC in fractionated mammalian cell extracts (8–10), but the responsible gene products have not been identified yet. Sequence analysis has failed to identify proteins with significant sequence homology to RuvABC proteins in eukaryotes (refs. 1 and 11; see Fig. 6). The mechanisms of hDNA extension, branch migration, and Holliday junction resolution in eukaryotes are poorly understood presently. The yeast Saccharomyces cerevisiae is an excellent system to study DSB repair by homologous recombination. The genes of the RAD52 epistasis group (RAD50, XRS2, MRE11, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RFA1) define this recombinational repair pathway (1). Numerous interactions occur between the encoded proteins, and they have provided a basis for understanding the specific function(s) of each protein during homologous recombination. The central role of Rad51 protein is supported by its numerous interactions with itself (12, 13), Rad52 (12, 14), Rad55 (13, 15), and Rad54 proteins (16, 17). In addition to its interaction with Rad51 protein, Rad52 protein also interacts with Rpa (18, 19). Rad55 and Rad57 proteins form a stable heterodimer (20). In addition, Rad50, Mre11, and Xrs2 proteins form a heterotrimer with nuclease activity, believed to be involved in DSB processing (1). Functional studies with the eukaryotic RAD52 group proteins have provided insights into the mechanism of recombinational repair. Rpa, Rad55/57 heterodimer, Rad52, Rad54, and its homolog Tid1/Rdh54 proteins have been found to stimulate Rad51 protein-mediated in vitro recombination. By eliminating secondary structures in ssDNA, Rpa stimulates the presynaptic phase and enhances the formation of the presynaptic filament (3, 4). Rad52 protein and the Rad55/57 heterodimer stimulate the presynaptic phase by mediating the exchange of an Rpa-ssDNA filament for a Rad51 protein-ssDNA filament (21–23). Human Rad52 protein was shown to stimulate the human Rad51 protein in an Rpa-independent mode (24). Finally, Rad54 protein was found to stimulate Rad51 protein-mediated in vitro recombination reactions (25, 26) in the synaptic phase of the reaction by specifically interacting with the established Rad51 nucleoprotein filament (27–29). Topological remodeling of the dsDNA by Rad54 was proposed as a mechanism for the observed stimulation (26–28, 30). The Rad54-related Tid1 protein was also found to stimulate Rad51-mediated in vitro recombination in a fashion similar to that of Rad54 protein, probably involving topological remodeling of the duplex DNA as well (31). Mutations in the RAD54 gene in S. cerevisiae confer a strong DSB-repair defect and also affect other aspects of DNA metabolism, consistent with an important function during homologous recombination (1). The gene is evolutionary conserved and plays a similar role in vertebrates (32–34). Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases that modulate protein:DNA interactions in transcription, DNA repair, and recombination (35). Rad54 protein possesses a dsDNA-specific ATPase activity that is important for its in vivo and in vitro functions (25–29, 36, 37). The energy of ATP-hydrolysis is required for Rad54 protein to topologically remodel duplex DNA (26, 30) by introducing unconstrained negative and positive supercoils (28). This activity is probably responsible for the stimulation of Rad51 protein-mediated in vitro recombination (27, 28). This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: DSB, double-strand break; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; Rpa, Replication protein A; hDNA, heteroduplex DNA.
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