Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown here to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors.Genetic studies in various eukaryotic organisms have indicated that homologous recombination processes are mediated by a group of evolutionarily conserved genes known as the RAD52 epistasis group. As revealed in studies on meiotic recombination and mating type switching in Saccharomyces cerevisiae, DNA double-strand breaks are formed and then processed exonucleolytically to yield long single-stranded tails with a 3Ј extremity. Nucleation of various RAD52 group proteins onto these ssDNA 1 tails renders them recombinogenic, leading to the search for a homologous DNA target (sister chromatid or homologous chromosome), formation of DNA joints with the target, and an exchange of genetic information with it. The repair by recombination of DNA double-strand breaks induced by ionizing radiation and other DNA damaging agents very likely follows the same mechanistic route, as it too is dependent on genes of the RAD52 epistasis group (reviewed in Refs. 1 and 2).Among members of the RAD52 group, the RAD51-encoded product is of particular interest because of its structural and functional similarities to the Escherichia coli recombination protein RecA (2-5). RecA promotes the pairing and strand exchange between homologous DNA molecules to form heteroduplex DNA (4, 5), an enzymatic activity believed to be germane for the central role of RecA in recombination and DNA repair processes. Likewise, homologous DNA pairing and strand exchange activities have been shown for S. cerevisiae Rad51 (yRad51) (6). Under optimized conditions, the length of heteroduplex DNA joints formed by yRad51 and RecA can extend over quite a few kilobase pairs (4, 5, 7).In published studies, human Rad51 (hRad51) was found to have the ability to make DNA joints but the maximal potential for forming only about 1 kilobase pairs of heteroduplex DNA (8 -11). Furthermore, while yRad51 and RecA require their cognate single-strand DNA binding factors, SSB and yRPA, for optimal recombinase activity, hRPA has been suggested to stimulate the hRad51-mediated homologous pairing and strand exchange reaction only when the hRad51 concentration is suboptimal (9, 10).Given the central role of hRad51 in recombination processes and the fact that the activities of hRad51 are apparently subject to modulation...

Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate. Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA. Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA. Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA. Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52. The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used. Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA.In eukaryotic organisms, genetic recombination is mediated by genes of the RAD52 epistasis group. These genes were first identified in Saccharomyces cerevisiae and consist of RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/ TID1, MRE11, and XRS2. Mutations in these genes very often result in severe meiotic phenotypes including an arrest in meiotic prophase, low sporulation efficiency, and spore inviability, which arise because of a requirement for the recombination machinery in ensuring the proper disjunction of chromosomal homologs in meiosis I. The RAD52 group genes also mediate the repair of DNA strand breaks by homologous recombination (reviewed in Ref. 1).The RAD51 gene product is structurally related to RecA (2-4), which plays a central role in recombination processes in Escherichia coli. Studies on RecA, its bacteriophage T4 counterpart UvsX, and eukaryotic Rad51 have indicated that they mediate the homologous DNA pairing and strand exchange reaction that forms heteroduplex DNA during recombination. In the earliest phase of this reaction, referred to as presynapsis, Rad51 polymerizes on ssDNA 1 to form a right-handed nucleoprotein filament that has a highly regular pitch (ϳ95 Å) and in which the DNA is held in a highly extended conformation (axial rise of ϳ5.4 Å per base or base pair) (5, 6). The formation of heteroduplex DNA with the incoming duplex DNA partner occurs within the confines of this nucleoprotein filament (6). The assembly of this presynaptic Rad51-ssDNA nucleoprotein filament requires ATP binding but not its hydrolysis (7) and is stimulated by the heterotrimeric ssDNA binding factor RPA, which functions to remove secondary structure in the ssDNA (8, 9). Human RAD51 also cooperates with human RPA to yield heteroduplex DNA (10, 11).Maximal level of strand exchange is obtained when RPA is introduced after Rad51 has already nucleated onto the ssDNA substrate. Paradoxically, a pronounced suppression of the reaction i...

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