S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA doublestrand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50⅐Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3 to 5 exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50⅐Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3 and 5 single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3 single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5 strand at secondary structures formed upon DNA strand separation.DSBs are induced by ionizing radiation and are also formed during initiating events in various modes of homologous recombination (1). Genetic studies in Saccharomyces cerevisiae have been instrumental in the discovery of genes required for recombination and DSB 1 repair through homologous recombination. These genes, RAD50, MRE11, XRS2, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, and RDH54/TID1, are collectively referred to as the RAD52 epistasis group (2).A number of studies in S. cerevisiae indicate that DSBs in homologous recombination events are processed in a nucleolytic fashion to generate an intermediate with overhanging 3Ј ssDNA tails. The ssDNA tails are bound by Rad51 and other recombination factors, which function in concert to locate regions of homology on a corresponding DNA duplex (a homologous chromosome or sister chromatid) and form heteroduplex DNA joints (1, 2). RAD50, MRE11, and XRS2 are involved in the nucleolytic processing of DSBs. Analysis of the Rad50 and Mre11 sequences reveals homology of these two proteins to Escherichia coli SbcC and SbcD, respectively (3), which combine to form a complex that exhibits both exo-and endonuclease activities, including the capacity to cleave hairpin structures (4). Mre11 from human and yeast possesses exo-and endonuclease activities (5-9). Interestingly, overexpression of Rad50 and Mre11 can allow DNA synthesis to efficiently progress through DNA sequences with a propensity to form secondary structures, suggesting that the Mre11 complex in yeast might also have the ability to cleave such DNA structures (10).As evidenced by co-immunoprecipitation and two-hybrid experiments, Rad50, Mre11, and Xrs2 are associated in a complex (7, 11). Likewise, the human counterparts of these proteins, human Rad50, Mre11, and NBS1 (the Xrs2 equiv...

Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganesedependent single-stranded DNA endonuclease and 3 to 5 exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability.Genetic studies on Saccharomyces cerevisiae mutants sensitive to ionizing radiation and to other agents that cause DNA double-stranded breaks have identified a large number of genetic loci required for the repair of such breaks. Many of these genes, including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, MRE11, and XRS2, show epistasis and are collectively known as the RAD52 epistasis group. Mutants of the RAD52 group also have defects of varying degrees in mitotic and meiotic recombination, which are initiated via DNA double-stranded break formation. Because meiotic recombination is essential for the proper segregation of homologous chromosomal pairs during meiosis I, the RAD52 group mutants often exhibit severe meiotic abnormalities, including inviability (see Refs. 1 and 2 for discussions and references).Extensive genetic evidence in yeast indicates that DNA double-stranded breaks are processed exonucleolytically, yielding 3Ј overhanging single-stranded (ss) 1 tails of about 600 bases in length (3, 4). According to the double-stranded break repair model for recombination (5), the 3Ј ssDNA tails formed as a result of break processing are bound by recombination proteins, which then mediate a search for the chromosomal homolog and heteroduplex DNA formation with the homolog (5). The RAD52 group genes may be divided into two categories. The first class consists of the RAD50, MRE11, and XRS2 genes, whose protein products are thought to be involved in the nucleolytic processing of DNA double-stranded breaks (6). Consistent with this classification, the Rad50 and Mre11 proteins have been shown to be homologous to the Escherichia coli SbcC and SbcD proteins, which combine to form a complex with endonuclease and exonuclease activities (7). The second category of the RAD52 group genes includes RAD51, RAD52, RAD54, RAD55, RAD57, and RDH54, whose products nucleate onto the ssDNA tails generated from break processing and then mediate the formation of heteroduplex DNA between the recombining chromosomes (1, 2). Whether the Rad59 protein, which is homologous to Rad52 (8), also has a role in heteroduplex DNA formation remains to be established.Important insights concerning the mechanism by which the RAD52 group proteins form heteroduplex DNA have been garnered through biochemical studies of purified human and yeast proteins (9 -11). However, no information as to the bi...

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...

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