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• W2089841991 abstract "In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409–420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52Δ409–412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52Δ409–412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination. In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409–420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52Δ409–412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52Δ409–412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination. single-stranded DNA replication protein A Saccharomyces cerevisiae RPA dithiothreitol glutathione S-transferase oligonucleotide amino acid 4-morpholinepropanesulfonic acid Homologous recombination in eukaryotic organisms is conserved in mechanism and mediated by a group of genes known as theRAD52 epistasis group. The RAD52 group members were first identified in the baker's yeast, Saccharomyces cerevisiae, and include RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11, and XRS2(1Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S59Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar, 3Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 4Cox M.M. Annu. Rev. Genet. 2001; 35: 53-82Crossref PubMed Scopus (197) Google Scholar). In S. cerevisiae and in other eukaryotes, homologous recombination is also an important means of eliminating DNA double-stranded breaks induced by ionizing radiation and other lesions that arise during the normal course of DNA replication (4Cox M.M. Annu. Rev. Genet. 2001; 35: 53-82Crossref PubMed Scopus (197) Google Scholar). In mammals, homologous recombination also appears to be indispensable for cell viability and tumor suppression (1Pierce A.J. Stark J.M. Araujo F.D. Moynahan M.E. Berwick M. Jasin M. Trends Cell Biol. 2001; 11: S52-S59Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 4Cox M.M. Annu. Rev. Genet. 2001; 35: 53-82Crossref PubMed Scopus (197) Google Scholar). A DNA double strand break can be repaired by pathways that are based on either end-joining or homologous recombination. In the latter case, the ends of the break are processed by a nuclease to yield 3′ ssDNA tails. These ssDNA1 tails attract recombination proteins, and the resulting nucleoprotein complex conducts a search for a homologous DNA sequence. Next, one of the ssDNA tails invades the homologous DNA target to form a DNA joint wherede novo DNA synthesis can take place, eventually leading to an exchange of genetic information between the recombining chromosomes and to restoration of the integrity of the broken chromosome (2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar,3Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). The enzymatic process responsible for the formation of heteroduplex DNA joints in recombination is called homologous DNA pairing and strand exchange (2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar). The RAD51 encoded product, the equivalent of the Escherichia coli recombinase RecA, mediates the homologous DNA pairing and strand exchange reaction (5Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (744) Google Scholar). Electron microscopic analyses have indicated that Rad51, like E. coliRecA protein, forms a highly ordered nucleoprotein filament on DNA (6Bianco P.R. Tracy R.B. Kowalczykowski S.C. Front. Biosci. 1998; 3: D570-D603Crossref PubMed Google Scholar). Biochemical studies have suggested that pairing and exchange of DNA strands in recombination processes occur within the confines of the Rad51-ssDNA nucleoprotein filament. The reaction phase in which the Rad51-ssDNA nucleoprotein filament is assembled is commonly referred to as the presynaptic phase, and the nucleoprotein filament as the presynaptic filament (2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar, 6Bianco P.R. Tracy R.B. Kowalczykowski S.C. Front. Biosci. 1998; 3: D570-D603Crossref PubMed Google Scholar, 7Roca A.I. Cox M.M. Prog. Nucleic Acids Res. Mol. Biol. 1997; 56: 129-223Crossref PubMed Google Scholar). Formation of the presynaptic filament requires ATP binding by Rad51 (2Sung P. Trujillo K.M. Van Komen S. Mutat. Res. 2000; 451: 257-275Crossref PubMed Scopus (158) Google Scholar). When plasmid length ssDNA substrates are used, presynaptic filament assembly is facilitated by the heterotrimeric single-stranded DNA binding factor, replication protein A (RPA), which functions to remove secondary structure in the ssDNA (5Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (744) Google Scholar, 8Sugiyama T. New J.H. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6049-6054Crossref PubMed Scopus (254) Google Scholar, 9Sigurdsson S. Trujillo K. Song B. Stratton S. Sung P. J. Biol. Chem. 2001; 276: 8798-8806Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The beneficial effect of RPA is seen most clearly when it is incorporated after Rad51 has been given an opportunity to nucleate onto the ssDNA template. In contrast, if RPA is added together with Rad51, it interferes with the filament assembly process by competing for binding sites on the ssDNA molecule. However, the inhibitory behavior of RPA can be alleviated by the addition of either of two recombination mediators (10Beernink H.T. Morrical S.W. Trends Biochem. Sci. 1999; 24: 385-389Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), Rad52 or the Rad55-Rad57 heterodimer (11Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (457) Google Scholar, 12Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 13New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (493) Google Scholar, 14Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (230) Google Scholar). We are interested in the molecular basis of the mediator function of Rad52 and the Rad55-Rad57 heterodimer in the above mentioned reaction. Both Rad52 and the Rad55-Rad57 complex bind ssDNA and physically interact with Rad51 (15Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (379) Google Scholar). 2L. Krejci and P. Song, unpublished observation. 2L. Krejci and P. Song, unpublished observation. In the present study, we have performed a fine mapping of the domain in Rad52 that is responsible for the interaction with Rad51. Furthermore, we have used this information to introduce a small deletion mutation into Rad52 to ascertain the significance of Rad51-interaction in Rad52 mediator function. The combination of genetic and biochemical analyses of the mutant rad52 protein unequivocally demonstrate the requirement for a physical association of Rad52 with Rad51 to effect its mediator function. Yeast extract-peptone-dextrose (YPD) medium, synthetic complete (SC) medium, and synthetic complete medium without leucine (SC−Leu) and without uracil (SC−Ura) were prepared as described previously (16Sherman F. Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA1991: 3-21Google Scholar) except that the synthetic media contained twice the amount of leucine (60 mg/liter). Yeast extract-peptone-acetate (YEPA) contained 10 g/liter yeast extract, 20 g/liter peptone, and 20 g/liter potassium acetate. Sporulation medium contained 2.5 g/liter yeast extract and 15 g/liter potassium acetate supplemented with 62 mg/liter Leu and 20.6 mg/liter each of adenine, His, Trp, and uracil. All strains are derivatives of Trp-303 (17Thomas B.J. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1327) Google Scholar) except that they are wild type for RAD5 (18Fan H.Y. Cheng K.K. Klein H.L. Genetics. 1996; 142: 749-759Crossref PubMed Google Scholar, 19Zou H. Rothstein R. Cell. 1997; 90: 87-96Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Standard genetic techniques were used to manipulate yeast strains (20Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). The rad52Δ409–412 allele was integrated into the yeast genome at the RAD52 locus by a cloning-free PCR-based allele replacement method (21Erdeniz N. Mortensen U.H. Rothstein R. Genome Res. 1997; 7: 1174-1183Crossref PubMed Scopus (139) Google Scholar). Specifically, gene-targeting substrates were made by amplifying a region of the rad52 allele,which comprises the Δ409–412 mutation, from the vector pR52Δ409–412.1 by PCR using the primers and Pr-Rad52-C-Adap-B (5′-GATCCCCGGGAATTGCCATGTGGTCTTCCAACTTCTCTTCG-3′) and Pr-C-adap-A (5′-AATTCCAGCTGACCACCATGAAGGATCCCGTTGTAGCTAAG-3′). The underlined sections of the primers correspond to unique tags that match sequences upstream and downstream of Kluyveromyces lactis URA3, respectively. Next, two PCR fragments containing the upstream and the downstream two-thirds of the K. lactis URA3gene were fused individually to the rad52Δ409–412 PCR fragment as described in Erdeniz et al. (21Erdeniz N. Mortensen U.H. Rothstein R. Genome Res. 1997; 7: 1174-1183Crossref PubMed Scopus (139) Google Scholar). GST fusion fragments of Rad52 were constructed as follows: GST-Rad52N (aa 1–168), GST-Rad52 M (aa 169–327), and GST-Rad52C (aa 328–504), encoded within theHpaII/BglII, BglII/BamHI, and BamHI/DraI fragments from theRAD52 open reading frame were subcloned intoSmaI-digested pGEX-3X, BamHI-digested pGEX-2T, and SmaI-digested pGEX-3X vector, respectively. For the expression of other GST fusion proteins, specific primers withEcoRI sites were used for the PCR reactions. The PCR products were digested with EcoRI, purified by phenol extraction and ethanol precipitation, and then ligated intoEcoRI-linearized pGEX-3X vector to fuse the Rad52 fragments to the GST protein. The ligation products were transformed intoE. coli strain BL21(DE3) for protein expression. The φX 174 viral (+) strand was purchased from New England Biolabs, and the replicative form (about 90% supercoiled form and 10% nicked circular form) was from Invitrogen. Oligonucleotide 1 used in the ssDNA annealing and DNA binding experiments had the sequence 5′-AAATGAACATAAAGTAAATAAGTATAAGGATAATACAAAATAAGTAAATGAATAAACATAGAAAATAAAGTAAAGGATATAAA-3′. Oligonucleotide 2 is the exact complement of oligonucleotide 1. These oligonucleotides were purified from a 15% polyacrylamide gel as described previously (22Trujillo K.M. Sung P. J. Biol. Chem. 2001; 276: 35458-35464Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The two oligonucleotides were labeled with [γ-32P]ATP and T4 polynucleotide kinase for use in DNA binding and single strand annealing experiments. Varying amounts of Rad52 or rad52 Δ409–412 protein was incubated with 32P-labeled Oligo-1 (1.36 μm nucleotides) at 37 °C in 10 μl of buffer D (40 mm Tris-HCl, pH 7.8, 50 mm KCl, 1 mm DTT, and 100 μg/ml bovine serum albumin) for 10 min. After the addition of gel loading buffer (50% glycerol, 20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 0.05% orange G), the reaction mixtures were resolved in 12% native polyacrylamide gels at 4 °C in TAE buffer (40 mm Tris-HCl, pH 7.4, 0.5 mm EDTA) and dried, and the DNA species were quantified using Quantity One software in the phosphorimaging device (Personal Molecular Imager FX from Bio-Rad). To release the bound DNA, the reaction mixture was deproteinized with 0.5% SDS and 500 μg/ml proteinase K at 37 °C for 10 min before being loading onto the polyacrylamide gel. Oligo-1 (3.6 μm nucleotides) and radiolabeled Oligo-2 (3.6 μm nucleotides) were incubated in separate tubes at 37 °C for 2 min in the absence or presence of RPA (0.55 μΜ) in 24 μl of buffer D. Rad52 or rad52 Δ409–412 (0.36 μm) was added in 2 μl to the tube containing Oligo-1 and then mixed with Oligo-2. The completed reactions (50 μl) were incubated at 25 °C, and at the indicated times, 9 μl of the annealing reactions was removed and treated with 0.5% SDS, 500 μg/ml proteinase K, and an excess of unlabeled Oligo-2 (20 μm) at 25 °C for 5 min in a total volume of 15 μl. The various samples (6 μl) were resolved in 12% native polyacrylamide gels run in TAE buffer. DNA annealing was quantified as the portion of the 32P-labeled Oligo-2 that had been converted into the double-stranded form. All of the GST fusion proteins were expressed in E. coli strain BL21(DE3), and all of the protein purification steps were carried out at 4 °C. For the purification of the GST fusion proteins, lysate was prepared fromE. coli cell paste using a French press in buffer G (20 mm NaH2PO4, pH 7.4, 0.5 mm EDTA, 1 mm DTT, and 150 mm NaCl that also contained the protease inhibitors aprotinin, chymostatin, leupeptin, and pepstatin A at 5 μg/ml each, as well as 1 mm phenylmethylsulfonyl fluoride). The crude lysate was clarified by centrifugation (100,000 ×g, 90 min), and the supernatant (20 ml) from the centrifugation step was mixed with 1 ml of glutathione-Sepharose 4B (Amersham Biosciences) for 3 h at 4 °C. The beads were washed three times with 20 ml of buffer G containing 150 mm KCl. The bound GST fusion protein was eluted with 5 ml of 10 mm reduced glutathione in buffer G. The eluate was dialyzed against buffer G and concentrated to 10 mg/ml in a Centricon-30 microconcentrator. Plasmids encoding untagged versions of Rad52 protein (pR52.2) and rad52Δ409–412 mutant protein (pR52Δ409–412.1) under the control of the GAL-PGK promoter were introduced into yeast strain BJ5464-6B. For the purification of these proteins, extract was prepared from 300 g of cell paste from 100 liters of culture in high salt buffer (23Song B. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The extract was clarified by centrifugation and then subjected to the chromatographic fractionation procedure described before (12Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar) except that Sephacryl 400 was used instead of Sepharose 6B in the gel filtration step. The purified GST fusion proteins (0.4 μm) were incubated with purified Rad51 (0.2 μm) in 30 μl buffer G and incubated at 4 °C for 1 h before the reaction mixture was mixed with 10 μl of glutathione-Sepharose-4B beads in 150 μl of buffer K (20 mm KH2PO4, pH 7.4, 150 mm KCl, 10% glycerol, 0.01% Nonidet P-40) at 4 °C for an additional hour. The beads were then washed twice with 150 μl of buffer K with 300 mm KCl, and the bound proteins were eluted with 30 μl of 3% SDS. The supernatant that contained unbound protein, the KCl wash (10 μl each), and also the SDS eluate (3 μl) were subjected to immunoblotting analysis with anti-Rad51 antibodies to determine their Rad51 content. Coomassie Blue staining of the SDS eluates revealed that ∼70% of GST and all of the GST fusion proteins were immobilized on glutathione-Sepharose. Affi-gel 15 beads containing Rad51 (Affi-Rad51; 5 mg/ml) and bovine serum albumin (Affi-BSA, 12 mg/ml) were prepared as described previously (24Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (340) Google Scholar). Purified Rad52 or rad52 Δ409–412 (5 μg) was mixed with 5 μl of Affi-Rad51 or Affi-BSA in 50 μl of buffer K for 30 min at 4 °C. The beads were washed once with 150 μl of buffer K before being treated with 50 μl of 2% SDS to elute-bound proteins. The starting material, supernatant that contained unbound Rad52 or rad52 protein, and the SDS eluate (10 μl each) were analyzed by SDS-PAGE in a 10% gel. A Sephacryl S400 column (1 × 30 cm, 20 ml total) was used to monitor the migration of Rad51 (30 μg), Rad52 (40 μg) and rad52Δ409–412 (40 μg) and to examine complex formation between Rad51 (30 μg) and Rad52 (40 μg). The mixtures of Rad51/Rad52 and Rad51/rad52Δ409–412 were incubated on ice for 1 h in 100 μl of buffer K and then filtered through the sizing column at 0.1 ml/min in the same buffer, collecting 0.5 ml fractions. The indicated column fractions were separated by SDS-PAGE electrophoresis to determine their content of the Rad51, Rad52, or rad52Δ409–412 proteins, respectively. For calibration of the column, thyroglobulin (669 kDa), catalase (232 kDa), and dextran blue (>2000 kDa) were used. All steps were carried out at 37 °C unless stated otherwise. The standard DNA strand exchange reaction was performed as described previously (23Song B. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Briefly, Rad51 (10 μm) was incubated with ssDNA (30 μmnucleotides) in 10 μl of buffer R (35 mm K-MOPS, pH 7.2, 1 mm DTT, 50 mm KCl, 2.5 mmATP, and 3 mm MgCl2) for 5 min. After the addition of the indicated amounts of RPA in 0.5 μl, the reaction mixtures were incubated for another 5 min before the incorporation of 1 μl of double-stranded DNA (25 μm nucleotides) and 1 μl of 50 mm spermidine hydrochloride. At the indicated times, a 4.5-μl portion of the reaction mixtures was withdrawn, deproteinized, and then analyzed in 0.9% agarose gels in TAE buffer. The gels were treated with eithidium bromide to stain the DNA species. Gel images were recorded in a NucleoTech gel documentation system and analyzed with GelExpert software. To examine the Rad52 mediator function, reaction mixtures (12.5 μl, final volume) containing the indicated amounts of Rad51, Rad52, and RPA were incubated on ice for 45 min in 10.5 μl of buffer R followed by the addition of ssDNA and a 10-min incubation. After the incorporation of linear duplex and spermidine, the completed reactions were incubated and analyzed as described for the standard reaction. For the time course experiments in Fig. 7, the reactions were scaled up four times to 50 μl but were otherwise assembled in the same fashion. Three independent haploid spores from each strain were picked and analyzed for their sensitivity to γ-irradiation, and the average values were reported. Yeast cultures were grown in YPD at 30 °C to the mid-log phase. At this point, the cultures were sonicated using a W-385 device (Heat Systems-Ultrasonics, Farmingdale, NY), and the appropriate number of cells were plated on YPD plates and irradiated in a Gammacell-22060Co irradiator (Atomic Energy of Canada). In Fig.8 B, cells transformed with pYESS10-Rad51 (2μ, RAD51) and with the empty vector pRS426 (25Jiang H. Xie Y. Houston P. Stemke-Hale K. Mortensen U.H. Rothstein R. Kodadek T. J. Biol. Chem. 1996; 271: 33181-33186Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) were grown on selective medium (SC−Ura) containing galactose as the sole carbon source at all stages of the experiment. The yeast cultures were analyzed as described above, except that for each strain, serial 10-fold dilutions were made and 5 μl of the diluted cell suspensions were spotted in duplicate on solid media prior to irradiation. Mitotic rates of interchromosomal heteroallelic recombination were determined as described previously (26Smith J. Rothstein R. Genetics. 1999; 151: 447-458PubMed Google Scholar). For each strain, nine independent trials were performed. The meiotic interchromosomal heteroallelic recombination frequency, sporulation efficiency, and spore viability were determined as described in Lisby et al. (27Lisby M. Rothstein R. Mortensen U.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8276-8282Crossref PubMed Scopus (341) Google Scholar) except that strains were grown at 30 °C. Three trials were performed for each strain. Rad52 possesses 504 amino acid residues (28Adzuma K. Ogawa T. Ogawa H. Mol. Cell. Biol. 1984; 4: 2735-2744Crossref PubMed Scopus (90) Google Scholar). Results from yeast two-hybrid analyses have suggested that the carboxyl terminus of Rad52 encompassing residues 328 to 504 can interact with Rad51 (29Milne G.T. Weaver D.T. Genes Dev. 1993; 7: 1755-1765Crossref PubMed Scopus (204) Google Scholar). Exploiting this information, we divided Rad52 into three fragments: Rad52N (aa 34–168), Rad52 M (aa 169–327), and Rad52C (aa 328–504), which were fused individually to glutathione-S-transferase (GST) as depicted in Fig.1 A. These GST fusion proteins were expressed in E. coli and purified using affinity chromatography on glutathione-Sepharose (Fig. 1 B). We also expressed and purified a GST fusion protein, termed GST-NMC, which contain the Rad52 protein sequence starting from the third ATG codon (aa 34); the region corresponding to amino acid 1–34 is not required for in vivo Rad52 function. 3R. Rothstein and U. H. Mortensen, unpublished observation. To determine which portion of Rad52 contains the Rad51 binding domain, the purified GST fusion proteins were mixed with Rad51 and then immobilized on the glutathione-Sepharose beads. After washing with high salt buffer, the GST fusion proteins and associated Rad51 were eluted from the glutathione beads by treatment with SDS and then analyzed by immunoblotting with anti-Rad51 antibodies (Fig. 1 C). The results show that Rad51 binds GST-NMC and GST-C, but not GST-N, GST-M, or GST alone. We then asked whether the purified GST fusions could be retained on Affi-gel 15 beads that contained covalently coupled Rad51 protein (24Petukhova G. Stratton S. Sung P. Nature. 1998; 393: 91-94Crossref PubMed Scopus (340) Google Scholar). As expected, the Affi-Rad51 beads were able to bind GST-NMC and GST-C but not GST-M, GST-N, or GST (data not shown). None of the GST-Rad52 fusion proteins was retained on Affi-gel 15 beads containing bovine serum albumin (data not shown). Thus, in agreement with yeast two-hybrid studies (29Milne G.T. Weaver D.T. Genes Dev. 1993; 7: 1755-1765Crossref PubMed Scopus (204) Google Scholar), the results from our in vitro analyses with purified Rad52 protein fragments revealed that the Rad51 interaction domain is located within the last 177 amino acid residues of Rad52 protein. To delimit the region in Rad52 required for interaction with Rad51, additional GST-tagged fragments of Rad52 derived from the carboxyl-terminal residues were generated (Fig2 A). These GST fusions were again purified by affinity chromatography and tested for Rad51 interaction by pull-down using glutathione-Sepharose beads as described before (Fig. 1 C). The binding of the various GST-Rad52 fusions to Affi-gel-Rad51 beads was also examined. The results from these combined analyses, as summarized in Fig. 2, revealed that amino acids 407–419 of the Rad52 protein are likely involved in binding Rad51. Overexpression of the Rad52 protein from another yeast, K. lactis, confers a dominant negative phenotype in S. cerevisiae that can be overcome by overexpression of the S. cerevisiae Rad51 (29Milne G.T. Weaver D.T. Genes Dev. 1993; 7: 1755-1765Crossref PubMed Scopus (204) Google Scholar). The authors of this study (29Milne G.T. Weaver D.T. Genes Dev. 1993; 7: 1755-1765Crossref PubMed Scopus (204) Google Scholar) suggested that the negative dominance of the K. lactis Rad52 inS. cerevisiae cells is due to the formation of a biologically inactive complex between KlRad52 and ScRad51. Even though the carboxyl terminus of the S. cerevisiae and K. lactis Rad52 counterparts display only a low level of identity (29%), the KlRad52 protein contains a sequence that is highly similar to the one in ScRad52 protein found here to be involved in Rad51 binding (Fig 2 B). Consistent with the suggestion that the sequence encoded within amino acid residues 407–419 of Rad52 is critical for Rad51 binding, we found that a small deletion spanning amino acid residues 409–412 within this region completely ablates the ability of Rad52 to interact with Rad51 (Fig. 2 A,panel II), as determined by the GST pull-down assay, binding of the GST fusion proteins to Affi-Rad51 beads, and other criteria (see below). The results presented above show that amino acid residues 409–412 are likely to be required for Rad51 binding. To further demonstrate the importance of these four amino acid residues, we introduced the same deletion mutation (Δ409–412) into the untagged Rad52 protein. For biochemical analyses, we overexpressed both the rad52Δ409–412 mutant and the wild-type protein by using the GAL-PGK promoter and galactose induction in the protease-deficient yeast strain BJ5464-6B. The level of expression of the wild-type and mutant proteins was very similar (data not shown), and they could be purified to near homogeneity by the same chromatographic procedure (see “Materials and Methods”; Fig. 3 A). Approximately 1 mg of each of the wild-type and mutant proteins was obtained from 300 g of starting yeast paste. This represents a 5–10-fold improvement compared with protein yield obtained when thePGK promoter is used for protein expression, as described in our previously published study (12Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Unlike wild-type Rad52 protein, the purified rad52Δ409–412 mutant protein did not bind Affi-Rad51 beads (Fig. 3 B), indicating that the four-amino acid deletion indeed eliminates the ability of Rad52 to associate with Rad51. Both Rad51 and Rad52 self-associate to form oligomeric molecules (14Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (230) Google Scholar, 23Song B. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 30Passy S.I., Yu, X., Li, Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (81) Google Scholar). A very large complex of these two proteins can be isolated in a sizing column (23Song B. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Accordingly, we subjected the purified rad52Δ409–412 mutant protein to sizing analysis in Sephacryl 400 to obtain independent verification that it does not associate with Rad51 and also to determine whether the Δ409–412 mutation affects self-association. When a mixture of Rad51 and wild-type Rad52 was analyzed, they formed a complex that emerged from the gel filtration column at an earlier position than either Rad51 or Rad52 alone (Fig. 4, comparepanel IV with panels I and III). In contrast, when the rad52Δ409–412 mutant was mixed with Rad51, no apparent shift of the elution profile of either protein was observed (Fig. 4, compare panel V with panels II andIII). Importantly, the peak of the rad52Δ409–412 mutant protein migrated at the same position as wild-type Rad52 (Fig. 4, compare panels I and II), strongly suggesting that the mutant rad52 protein has the same oligomeric composition as the wild-type protein. Thus, the results from the gel filtration analyses demonstrated that the rad52Δ409–412 mutant is defective in Rad51 interaction but maintains the ability to self-associate. As first reported by Mortensen et al. (15Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (379) Google Scholar) and later confirmed by others (13New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (493) Google Scholar, 14Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (230) Google Scholar, 23Song B. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), Rad52 possesses an ssDNA binding function. We therefore addressed the possibility that the four-amino acid deletion affects" @default.
• W2089841991 created "2016-06-24" @default.
• W2089841991 creator A5028622476 @default.
• W2089841991 creator A5041292304 @default.
• W2089841991 creator A5054962508 @default.
• W2089841991 creator A5062728565 @default.
• W2089841991 creator A5079626347 @default.
• W2089841991 creator A5090899284 @default.
• W2089841991 date "2002-10-01" @default.
• W2089841991 modified "2023-10-10" @default.
• W2089841991 title "Interaction with Rad51 Is Indispensable for Recombination Mediator Function of Rad52" @default.
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