Functional Interactions among Yeast Rad51 Recombinase, Rad52 Mediator, and Replication Protein A in DNA Strand Exchange
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m910244199
ISSN1083-351X
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoRad51-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. 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. single-stranded DNA double-stranded DNA dithiothreitol bovine serum albumin 4-morpholinepropanesulfonic acid break-induced replication nitrilotriacetic acid replication protein A In eukaryotic organisms, genetic recombination is mediated by genes of the RAD52 epistasis group. These genes were first identified in Saccharomyces cerevisiae and consist ofRAD50, 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.Paques F. Haber J.H. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar). The RAD51 gene product is structurally related to RecA (2.Aboussekhra A. Chanet R. Adjiri A. Fabre P. Mol. Cell. Biol. 1992; 12: 3224-3234Crossref PubMed Scopus (320) Google Scholar, 3.Basile G. Aker M. Mortimer R.K. Mol. Cell. Biol. 1992; 12: 3235-3246Crossref PubMed Scopus (239) Google Scholar, 4.Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar), which plays a central role in recombination processes inEscherichia 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 ssDNA1 to form a right-handed nucleoprotein filament that has a highly regular pitch (∼95 Å) and in which the DNA is held in a highly extended conformation (axial rise of ∼5.4 Å per base or base pair) (5.Ogawa T., Yu, X. Shinohara A. Egelman E.H. Science. 1993; 259: 1896-1899Crossref PubMed Scopus (561) Google Scholar, 6.Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar). The formation of heteroduplex DNA with the incoming duplex DNA partner occurs within the confines of this nucleoprotein filament (6.Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar). The assembly of this presynaptic Rad51-ssDNA nucleoprotein filament requires ATP binding but not its hydrolysis (7.Sung P. Stratton S.A. J. Biol. Chem. 1996; 271: 27983-27986Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and is stimulated by the heterotrimeric ssDNA binding factor RPA, which functions to remove secondary structure in the ssDNA (8.Sugiyama T. Zaitseva E.M. Kowalczykowski S.C. J. Biol. Chem. 1997; 272: 7940-7945Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 9.Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar). Human RAD51 also cooperates with human RPA to yield heteroduplex DNA (10.Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 11.Gupta R.C. Golub E.I. Wold M.S. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9843-9848Crossref PubMed Scopus (64) Google Scholar). 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 is seen if RPA is added together with or before Rad51 protein to the ssDNA (12.New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 13.Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar, 14.Sung P. Genes Dev. 1997; 11: 1111-1112Crossref PubMed Scopus (463) Google Scholar, 15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Rad52 (12.New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 13.Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar, 15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar) and the heterodimer of the Rad55 and Rad57 proteins (14.Sung P. Genes Dev. 1997; 11: 1111-1112Crossref PubMed Scopus (463) Google Scholar) have been found to promote heteroduplex formation when there is a need for Rad51 to compete with RPA for binding sites on the ssDNA. These ancillary protein factors, or mediators (15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 16.Kanaar R. Hoeijmakers J.H. Nature. 1998; 391: 335-338Crossref PubMed Scopus (26) Google Scholar), are functionally equivalent to the E. coli RecO-RecR complex (17.Umezu K.N. Chi W. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3875-3879Crossref PubMed Scopus (201) Google Scholar) and T4 UvsY protein (18.Harris L.D. Griffith J.D. J. Mol. Biol. 1989; 206: 19-28Crossref PubMed Scopus (53) Google Scholar, 19.Jiang H. Giedroc D. Kodadek T. J. Biol. Chem. 1993; 268: 7904-7911Abstract Full Text PDF PubMed Google Scholar, 20.Yonesaki T. Minagawa T. J. Biol. Chem. 1989; 264: 7814-7820Abstract Full Text PDF PubMed Google Scholar, 21.Sweezy M.A. Morrical S.C. J. Mol. Biol. 1997; 266: 927-938Crossref PubMed Scopus (33) Google Scholar), which allow their cognate recombinases RecA and UvsX to gain access to ssDNA already coated with the ssDNA binding factor. However, the manner in which Rad52 and the Rad55-Rad57 heterodimer overcome the competition by RPA is not known at the present time. Here we describe biochemical studies that enable us to begin understanding the biochemical properties and the mediator function of Rad52 in greater detail. E. coli strain M15(pREP4) harboring the plasmid expressing His6-tagged Rad52 under the control of the T5 promoter (gift from Rodney Rothstein) (22.Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar) was used. Extract was made from 15 g of cell paste (from 8 liters of culture) in 120 ml of cell breakage buffer (50 mm Tris-HCl, pH 7.5, 10% sucrose, 150 mm KCl, 3 mm EDTA, 1 mm 2-mercaptoethanol) in the presence of protease inhibitors using a French press (23.Sung P. Matson S.W. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6045-6049Crossref PubMed Scopus (90) Google Scholar). The lysate was clarified by centrifugation (100,000 × g, 90 min), and the supernatant (fraction I, 120 ml) was applied onto a column of SP-Sepharose (2.5 × 6.1 cm; 30 ml total) equilibrated in buffer K (20 mm KH2PO4, pH 7.4, 0.5 mm EDTA, 1 mm DTT) containing 150 mm KCl. The column was developed with a 300-ml gradient of 150–650 mm KCl in buffer K. Rad52 elutes from SP-Sepharose at ≈360 mm KCl, the pool of which (fraction II, 30 ml) was diluted with 2 volumes of K buffer and applied to a Q-Sepharose column (1.5 × 5.5 cm; 8 ml total), which was developed with a 100-ml gradient of 120–450 mm KCl in K buffer, collecting 2-ml fractions. The pool of Rad52 (fraction III; 10 ml), eluting from Q-Sepharose at about 300 mm KCl, was mixed with 1 ml of nickel-NTA-agarose (Qiagen) for 2 h at 4 °C. The nickel matrix was poured into a glass column (1 cm diameter) and washed with 10 volumes each of 10, 20, and 30 mm imidazole in buffer K containing 500 mm KCl. Rad52 was eluted with 4 ml of 200 mm imidazole in buffer K containing 500 mm KCl and then concentrated to 1 ml using a Centricon microconcentrator (Amicon). The concentrated nickel pool (fraction IV) was subject to sizing in a column of Sepharose 6B (1.6 × 40 cm; 80 ml matrix) in buffer K containing 150 mm KCl. The Rad52 pool (fraction V, 8 ml) was loaded directly onto a Mono S column (HR5/5), which was developed with a 30-ml gradient of 150–500 mm KCl in buffer K. Fractions containing the peak of Rad52 protein, eluting at ≈350 mm KCl, were pooled (fraction VI; 4 ml) and concentrated to 5 mg/ml and stored in small portions at −70 °C. The concentration of Rad52 protein was measured by densitometric comparison of multiple loadings of Rad52 protein against known amounts of bovine serum albumin and ovalbumin in a Coomassie Blue R-stained polyacrylamide gel. Rad51 was purified to near-homogeneity from yeast strain LP2749-9B harboring the plasmid pR51.3 (2 μm,PGK-RAD51), using a combination of ammonium sulfate precipitation and chromatographic fractionation steps in columns of Q-Sepharose, hydroxyapatite, Bio-Rex 70, and Mono Q (7.Sung P. Stratton S.A. J. Biol. Chem. 1996; 271: 27983-27986Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Rad51 was stored in K buffer containing 350 mm KCl. The concentration of Rad51 protein was measured using molar extinction coefficient of 1.29 × 104m−1cm−1 at 280 nm (33.Zaitseva E.M. Zaitsev E.N. Kowalczykowski S.C. J. Biol. Chem. 1999; 274: 2907-2915Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). RPA was purified from a yeast strain genetically tailored to co-overexpress the three subunits of RPA (a gift from Richard Kolodner). Extract was prepared and then subjected to fractionation in columns of Affi-Gel Blue, ssDNA cellulose, hydroxyapatite, and Mono Q as described (14.Sung P. Genes Dev. 1997; 11: 1111-1112Crossref PubMed Scopus (463) Google Scholar). The RPA purified this way was nearly homogeneous and was stored in K buffer containing 200 mm KCl. The concentration of RPA was measured by densitometric comparison of multiple loadings of RPA against known amounts of bovine serum albumin and ovalbumin in a Coomassie Blue R-stained polyacrylamide gel. Purified Rad51 (3 mg) and Rad52 (3.25 mg) were incubated in 5 ml of buffer K with 300 mm KCl for 12 h on ice and then mixed with 1 ml of nickel-NTA-agarose to immobilize the Rad51-Rad52 complex, which was eluted from the nickel matrix with 3 ml of 200 mm imidazole in buffer K containing 300 mm KCl. The eluate was concentrated to 0.3 ml in a Centricon-30 microconcentrator, diluted to 3 ml with buffer K containing 300 KCl, and reconcentrated to 0.3 ml. This filter-dialysis step was repeated in the same concentrator, and the final concentrate, containing Rad51-Rad52 complex at 150 μm, was stored in small portions at −70 °C. φX174 viral (+) strand was purchased from New England Biolabs, and the replicative form (about 90% supercoiled form and 10% nicked circular form) was from Life Technologies, Inc. The 83-mer oligonucleotides used in the strand exchange experiments were as follows: Oligo 1 with 16% GC content, 5′-AAA TGA ACA TAA AGT AAA TAA GTA TAA GGA TAA TAC AAA ATA AGT AAA TGA ATA AAC ATA GAA AAT AAA GTA AAG GAT ATA AA; Oligo 2, the exact complement of Oligo 1, was labeled at the 5′ end with [γ-32P]ATP by T4 polynucleotide kinase, and then annealed to Oligo 1. The resulting duplex was purified from 10% polyacrylamide gels by overnight diffusion at 4 °C into TAE buffer (40 mm Tris-HCl, pH 7.4, 20 mm NaOAC, 0.5 mm EDTA). A Sepharose 6B column (1 × 45 cm; 35 ml total) was used to monitor the migration of Rad51, Rad52, and the Rad51-Rad52 complex in the experiment described in Fig. 2 A. Rad51 protein (11.6 μm in panels I, III, and IV) and Rad52 protein (11.6 μm in panels II andIII and 2.3 μm in panel IV) were incubated in 100 μl of column buffer (buffer K containing 150 mm KCl and 1 mm DTT) on ice for 1 h, diluted with 400 μl of column buffer, and then filtered through the sizing column at 0.2 ml/min, collecting 0.5-ml fractions. The indicated column fractions were subject to immunoblot analyses to determine their content of the Rad51 and Rad52 proteins. For calibration of the column, thyroglobulin (669 kDa), catalase (232 kDa), and blue dextran (>2,000 kDa) were used, and their elution positions are marked on the chromatogram in Fig. 2 A. In Fig. 2 B, Rad51 and Rad52 proteins were incubated at various molar ratios (0.5 μm Rad51 and 2 μm Rad52 or 1:4; 1.5 μm Rad51 and 1.25 μm Rad52 or 1.2:1; 3 μm Rad51 and 1 μm Rad52 or 3:1; or 2 μm Rad51 alone) in 1 ml of K buffer containing 300 mm KCl and 0.01% Nonidet P-40 at 4 °C for 1 h. In Fig. 2 C, reaction mixtures containing 1.5 μm Rad51 or a combination of 1.5 μm Rad51 and 1.5 μm Rad52 were incubated in the same buffer with or without 2.5 mm ATP and 3 mm MgCl2 for 1 h. All the mixtures were gently mixed with 200 μl of nickel-NTA-agarose beads at 4 °C for 3 h. The beads were washed with 2 ml of 20 mmimidazole in the same buffer with or without ATP/MgCl2before eluting the bound proteins with 400 μl of 3% SDS by boiling for 1 min. The substrates used were φX174 viral (+) strand and the replicative form linearized with PstI. In Fig. 3, A–C, the reactions contained both ssDNA (30 μm nucleotides) and dsDNA (20 μmnucleotides) with the indicated amounts of Rad51 protein, Rad52 protein, or Rad51-Rad52 complex in 10 μl of reaction buffer (35 mm K-MOPS, pH 7.2, 1 mm DTT, and 100 μg/ml BSA, with or without 2.5 mm ATP and 3 mmMgCl2, as indicated). The reaction mixtures were incubated at 25 °C for 10 min, mixed with 2 μl of loading buffer (0.1% Orange G in 30 mm Tris-HCl, pH 7.5, containing 50% glycerol), and then subjected to electrophoresis in 0.9% agarose gels at 100 mA in TAE buffer at 25 °C until the dye front had migrated 4 cm. The gels were stained with ethidium bromide to reveal the DNA species. In Fig. 3 D, Rad51 (0.45 μm), Rad52 (0.45 μm), or a mixture of these two proteins in 150 μl of buffer T (20 mm Tris-HCl, pH 7.5, 10% glycerol, 0.5 mm EDTA, 1 mm DTT, and 0.01% Nonidet P-40) containing 150 mm KCl and 100 μg/ml BSA was incubated on ice for 45 min and then mixed with 15 μl of ssDNA cellulose beads (1 μg of denatured calf thymus DNA/μl beads; purchased from United States Biochemical Corp.) for 45 min at 25 °C. The beads were collected by a 5-s centrifugation in a microcentrifuge, washed with 150 μl of buffer T containing 300 mm KCl, and treated with 35 μl of 3% SDS at 37 °C for 15 min to elute bound proteins. Equivalent amounts of the input material, the supernatant that contained unbound proteins, the KCl wash, and SDS eluate were subject to immunoblotting to examine the Rad51 and Rad52 contents. Rad51 protein (10 μm) with or without Rad52 protein (4 μm) was incubated with or without φX ssDNA (30 μm nucleotides) and 1 mm[γ-32P]ATP using the buffer conditions employed in the strand exchange assay. At the indicated times, 1-μl aliquots were removed and directly spotted on a polyethyleneimine-cellulose sheet, which was developed in 0.75 m potassium phosphate. The polyethyleneimine-cellulose sheets were analyzed in the PhosphorImager. The indicated amounts of Rad51 protein in 1 μl of storage buffer were incubated with ssDNA (30 μm) added in 1 μl of TE buffer (10 mmTris-HCl, pH 7.5, 0.2 mm EDTA) in 10 μl of buffer R (35 mm K-MOPS, pH 7.2, 1 mm DTT) containing 50 mm KCl, 2.5 mm ATP, and 3 mmMgCl2 for 5 min at 37 °C. After the addition of the indicated amounts of RPA in 0.5 μl of storage buffer, reaction mixtures were incubated at 37 °C for another 5 min before the incorporation of dsDNA (30 μm) in 1 μl of TE and 1 μl of 50 mm spermidine hydrochloride. The reaction mixtures were incubated at 37 °C and stopped by the addition of an equal volume of 1% SDS containing 1 mg/ml proteinase K. Deproteinization of the reaction mixtures was carried out at 37 °C for 20 min. After the addition of 0.2 volume of gel loading buffer, samples were run in 0.9% agarose gels in TAE buffer, stained with ethidium bromide for 60 min, and then destained for at least 4 h in a large volume of H2O. Images were recorded in a NucleoTech Gel documentation system and analyzed with the software provided. In the time course experiments, the reaction mixtures were scaled up accordingly, and 6-μl portions of the mixtures were withdrawn for analysis at each time point. Reaction mixtures (12.5 μl final volume) containing the indicated amounts of Rad51, Rad52, and RPA were incubated on ice for 45 min, followed by the addition of φX ssDNA. The reaction mixtures were then incubated at 37 °C for 10 min, followed by the incorporation of the linear dsDNA and spermidine, as described in the standard reaction above. In Fig. 6 A, 1.0 μm Rad51 or Rad51-Rad52 complex was incubated with Oligo 2 (3 μm) in 10.5 μl of buffer R containing 20 mm KCl, 2.5 mm ATP, and 3 mmMgCl2 at 37 °C for 5 min, followed by the addition of 1 μl of 50 mm spermidine and the homologous duplex (6 μm) consisting of unlabeled Oligo 1 and32P-labeled Oligo 2 in 1 μl. At the indicated times, 4 μl of the reaction mixture was deproteinized and resolved in a 10% polyacrylamide gel in TAE buffer, which was dried onto a sheet of DEAE paper, and the DNA species was quantified in a PhosphorImager. In the experiment in Fig. 7, A and B, unlabeled Oligo 2 (3 μm) was incubated with Rad51 or Rad51-Rad52 complex in 10 μl of buffer R at 37 °C for 5 min before an increasing concentration of RPA (0.07, 0.14, 0.28, and 0.56 μm) was added in 0.5 μl. After a further 5 min, 1 μl of 50 mmspermidine hydrochloride and the homologous 32P-labeled duplex in 1 μl were incorporated to complete the reactions (12.5 μl final volume). Alternatively, Oligo 2 was co-incubated in 10.5 μl of buffer R with RPA and Rad51 or Rad51-Rad52 complex for 5 min or preincubated with RPA for 5 min before Rad51 or Rad51-Rad52 complex was added and then followed by an additional 5 min of incubation. Subsequent to the addition of labeled duplex DNA and spermidine, all the reaction mixtures (12.5 μl final volume) were incubated at 37 °C for 15 min before being deproteinized and analyzed in 10% polyacrylamide gels in TAE buffer. The gels were dried and the DNA species quantified in the PhosphorImager.Figure 7Rad51-Rad52, but not Rad51, can utilize an RPA-coated ssDNA template for homologous pairing. A,homologous pairing reactions that used oligonucleotides (3 μm ss and 6 μm homologous duplex) with Rad51 protein (1 μm), in which increasing concentrations of RPA (0.07, 0.14, 0.28, and 0.56 μm) were either preincubated with the ss oligonucleotide (●), added with Rad51 to the ss oligonucleotide (○), or added after the ss oligonucleotide had been preincubated with Rad51 (■). B, Rad51-Rad52 complex (1 μm) and oligonucleotide substrates (3 μmss and 6 μm homologous duplex) were used in the strand exchange reactions. In these reactions, increasing concentrations of RPA (0.07, 0.14, 0.28, and 0.56 μm) were either preincubated with the ss oligonucleotide (●), added with Rad51-Rad52 complex to the ss oligonucleotide (○), or added after the ss oligonucleotide had been preincubated with Rad51-Rad52 complex (■). After the addition of the 32P-labeled duplex, reaction mixtures were further incubated at 37 °C for 15 min. The reaction mixtures were resolved in 10% polyacrylamide gels, and the reaction products were quantified in the PhosphorImager.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All the reaction mixtures in the experiment in Fig. 5 had a final volume of 37.5 μl. In the experiments in panels Iand II of Fig. 5 A, Rad51 protein (10 μm in panel I and 20 μm inpanel II) was preincubated with φX ssDNA (30 μm nucleotides) and then with RPA (1.5 μm) as in the standard reaction. Following the preincubations, 1 μl of either TE or TE containing BsaI-linearized pBluescript DNA (75 μm nucleotides) was added to the reaction mixtures, which were incubated at 37 °C for another 2 min, before the φX duplex (30 μm nucleotides) and spermidine were incorporated to complete the reaction. In the experiment in panel III of Fig. 5 A, Rad51 (10 μm) and Rad52 proteins (2.5 μm) were preincubated with φX ssDNA (30 μm nucleotides) and then with RPA (1.5 μm). Following these preincubations, 1 μl of either TE or TE containing linear pBluescript duplex DNA (75 μm nucleotides) was added to the reaction mixtures, which were incubated at 37 °C for 2 additional min before φX duplex DNA (30 μm nucleotides) and spermidine were incorporated to complete the reaction. A 6-μl portion of the reaction mixtures was withdrawn at the indicated times and processed for gel electrophoresis. Self-aggregation reaction (25 μl final volume) was carried out by following the order of addition of reaction components and using the buffer described for the standard strand exchange reaction. After the addition of dsDNA, reaction samples were incubated at 37 °C for 2 min and immediately spun at 12,000 × g for 2 min at 25 °C. After centrifugation, 20 μl of the supernatant was mixed with 20 μl of 1% SDS, and 35 μl of 0.5% SDS was added to the pellet fraction to dissolve the precipitated protein-DNA complex. The supernatant and pellet fractions, 8 μl each, were analyzed for their protein contents by SDS-PAGE and Coomassie Blue staining. To examine the DNA contents, a 10-μl portion of the supernatant and pellet fractions was treated with 0.5 mg/ml proteinase K at 37 °C for 20 min and then subjected to agarose gel electrophoresis as described for the strand exchange experiments. Rad51, RPA, and DNA (ss and ds) were omitted from some of the experiments (Fig. 6, C andD), as indicated, and Oligo 2 replaced the φX ssDNA in Fig. 6 E. We examined the level of strand exchange reaction products (Fig.1 A) by fixing the amount of Rad51 (10 μm), and we varied the concentration of RPA (0.4–2.8 μm), added either with Rad51 (Fig. 1 B, panel I) or after Rad51 has already nucleated onto the ssDNA (Fig.1 B, panel II), as in the standard reaction. At levels of RPA of 2 μm and above, pronounced inhibition of the reaction was observed with the co-addition of components (Fig. 1, Band C). However, at amounts of RPA lower than 2 μm, the extent of suppression of reaction products was much less severe (Fig. 1, B and C). It seems likely that at lower concentrations of RPA, enough Rad51 can still nucleate onto the ssDNA to prime the assembly of the presynaptic nucleoprotein filament. We have previously described expression of Rad52 in yeast and its purification to near-homogeneity (15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Since much larger amounts of functionally active and nearly homogeneous Rad52 can be obtained by expression in E. coli (12.New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 13.Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar, 22.Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar), we have since used theE. coli system for purifying Rad52 (22.Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar). As indicated from recent work (12.New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 13.Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar, 15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar) and reiterated here (Fig. 1 D), the addition of Rad52 at an amount substoichiometric (1.2 μm) to that of Rad51 (10 μm) restored strand exchange to a level comparable to what was obtained in the standard reaction (Fig.1 D). Rad52 by itself, with or without RPA, is devoid of homologous DNA pairing activity (12.New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 13.Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar, 15.Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). As shown in Fig. 2 A, panel I, Rad51 (43 kDa) eluted from a Sepharose 6B column with an average V e/V t (elution volume/total column volume) of ∼0.7, which is slightly before the elution position of catalase (232 kDa), suggesting a multimeric structure under the conditions used. Rad52 (56 kDa) eluted from Sepharose 6B with an averageV e/V t of ∼0.6, slightly before the elution volume of thyroglobulin (669 kDa), indicating that Rad52 also exists as a multimeric structure (Fig. 2 A, panel II); the same results were obtained with Rad52 purified from yeast cells (data not shown). When mixed with an equimolar amount of Rad52, the majority of Rad51 (>85%) emerged from the sizing column at a much earlier position (average V e/V t of ∼0.55) than free Rad51 (V e/V t of ∼0.7). Consistent with the formation of a stable complex of Rad51 and Rad52, the Rad52 peak was also shifted slightly (Fig. 2 A, panel III). When the amount of Rad52 was lowered to one-fifth of the previous level, the portion of the Rad51 shifted to the earlier elution position dropped accordingly to about 20% of the total (see Fig.2 A, panel IV). Similarly, when Rad52 was fixed at the previous level but with the amount of Rad51 being increased five times, the portion of Rad51 found in association with Rad52 was once again about 20% of the total (data not shown). Thus, Rad51 and Rad52 form a stable complex consisting of approximately equimolar amounts of the two proteins. Judging from the elution position of the Rad51-Rad52 complex, it appears that the complex contains multiple molecules of the two proteins. Complex formation between Rad51 and Rad52 was also examined by mixing different molar amounts of the two proteins and then immobilizing the complex on nickel-NTA-agarose through the histidine tag on Rad52. The results shown in Fig. 2 B are again consistent with an approximately equimolar complex between Rad51 and Rad52. The addition of ATP and magnesium did not alter the amount or the stoichiometry of Rad51-Rad52 complex formed, assessed both by immobilizing the complex via the histidine tag on Rad52 (Fig. 2 C) or by sizing in Sepharose 6B (data not shown). The same molar concentrations of Rad51, Rad52, and Rad51-Rad52 complex were incubated with a mixture of ssDNA and dsDNA in the presence of ATP and magnesium, and nucleoprotein complexes were separated from free DNA in an agarose gel and visualized by staining with ethidium bromide. Fig.3 A shows that both Rad52 and the Rad51-Rad52 complex bound specifically to the ssDNA. The experiment presented in Fig. 3 A was done in the presence of ATP and magnesium, but we have detected no difference in terms of the amount of ssDNA shifted or the binding specificity of the Rad51-Rad52 complex when ATP was omitted from the reaction mixture (Fig. 3 B). No significant shifting of either the ss or ds form of DNA was observed with the concentrations of Rad51 used (Fig. 3 A). Much higher concentrations of Rad51 are needed to see ATP-dependent binding to the ssDNA and dsDNA (Fig. 3 C). In the DNA mobility shift experiments (Fig. 3, A andB), we could not ascertain that Rad51 was present in the nucleoprotein complex. To demonstrate directly that Rad51 is being targeted to ssDNA by Rad52, we co-incubated Rad51 and Rad52 with ssDNA cellulose and then analyzed the bound proteins by immunoblot analyses after their elution from the matrix by SDS treatment. Under the same conditions, Rad51 alone did not bind to ssDNA cellulose, but Rad52 alone did (Fig. 3 D). Importantly, very similar amounts of
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