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• W2078932716 abstract "The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (H omosapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1–237, HsRad521–237) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad521–237 both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway. The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (H omosapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1–237, HsRad521–237) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad521–237 both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway. homologous recombination single-stranded DNA double-stranded DNA displacement loop nickel-nitrilotriacetic acid polyacrylamide gel electrophoresis The ability of cells to repair double strand breaks that occur on chromosomal DNA is critical to maintain the integrity of their genomic DNA. Double strand breaks can result from several events: ionizing radiation, DNA-damaging agents, replication errors, and specific enzymes that act in meiosis, mating-type switching, and V(D)J recombination (1Lett J.T. Prog. Nucleic Acids Res. Mol. Biol. 1990; 39: 305-352Crossref PubMed Scopus (26) Google Scholar, 2Gellert M. Genes Cells. 1996; 1: 269-275Crossref PubMed Scopus (21) Google Scholar, 3Michel B. Ehrlich S.D. Uzest M. EMBO J. 1997; 16: 430-438Crossref PubMed Scopus (382) Google Scholar, 4Haber J.E. Annu. Rev. Genet. 1998; 32: 561-599Crossref PubMed Scopus (323) Google Scholar, 5Haber J.E. Curr. Biol. 1998; 19: 832-835Abstract Full Text Full Text PDF Google Scholar). To repair such lesions, cells have developed homologous recombination (HR)1 and nonhomologous DNA end joining. HR is an important process for the preservation of the DNA sequence in chromosomal DNA. The homologous pairing step of the HR pathway, which is the process of searching for sequence homology in either homologous chromosomes or sister chromatids, is an essential step. Therefore, the identification of the enzymes that promote homologous pairing has been one of the central drives toward an understanding of the HR pathway (6West S.C. Annu. Rev. Biochem. 1992; 61: 603-640Crossref PubMed Scopus (302) Google Scholar, 7Camerini-Otero R.D. Hsieh P. Annu. Rev. Genet. 1995; 29: 509-552Crossref PubMed Google Scholar). In Escherichia coli, two homologous pairing enzymes, the RecA and RecT proteins, have been reported (8McEntee K. Weinstock G.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2615-2619Crossref PubMed Scopus (241) Google Scholar, 9Shibata T. DasGupta C. Cunningham R.P. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1638-1642Crossref PubMed Scopus (279) Google Scholar, 10Hall S.D. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3205-3209Crossref PubMed Scopus (67) Google Scholar). RecA and RecT, which have no amino acid sequence homology, catalyze homologous pairing in separate recombinational repair pathways: the RecBCD and RecF pathways for RecA and the RecE pathway for RecT. (11Ganesan A.K. Seawell P.C. Mol. Gen. Genet. 1975; 141: 189-205Crossref PubMed Scopus (62) Google Scholar, 12Rothman, R. H., Kato, T., and Clark, A. J. (1975) Basic Life Sci. 283–291Google Scholar, 13Takahashi N.K. Kusano K. Yokochi T. Kitamura Y. Yoshikura H. Kobayashi I. J. Bacteriol. 1993; 175: 5176-5185Crossref PubMed Google Scholar). In eukaryotes, two RecA homologues, Rad51 and Dmc1, have been identified, and both proteins promote homologous pairing (14Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 15Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 16Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (239) Google Scholar, 17Li Z. Golub E.I. Gupta R. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11221-11226Crossref PubMed Scopus (128) Google Scholar, 18Masson J.-Y. Davies A.A. Hajibagheri N. Van Dyck E. Benson F.E. Stasiak A., Z. Stasiak A. West S.C. EMBO J. 1999; 18: 6552-6560Crossref PubMed Scopus (118) Google Scholar, 19Masson J.-Y. West S.C. Trends Biochem. Sci. 2001; 26: 131-136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). No proteins with significant sequence homology to RecT have been found in eukaryotes, although recombination pathways that are independent of Rad51 have been identified (20Malkova A. Ivanov E.L. Haber J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7131-7136Crossref PubMed Scopus (344) Google Scholar, 21Kang L.E. Symington L.S. Mol. Cell. Biol. 2000; 20: 9162-9172Crossref PubMed Scopus (46) Google Scholar). An electron microscopic visualization showed that RecT forms a heptameric or octameric ring structure (22Thresher R.J. Makhov A.M. Hall S.D. Kolodner R. Griffith J.D. J. Mol. Biol. 1995; 254: 364-371Crossref PubMed Scopus (41) Google Scholar). Interestingly, the ScRad52 (S accharomyces c erevisiae Rad52) and HsRad52 (H omo s apiens Rad52) proteins also form ring structures similar to that of RecT (23Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (237) Google Scholar, 24Stasiak A.Z. Larquet E. Stasiak A. Muller S. Engel A. Van Dyck E. West S.C. Egelman E.H. Curr. Biol. 2000; 10: 337-340Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). RAD52 genes have been identified in several eukaryotes, including yeast, chicken, mouse, and human (25Adzuma K. Ogawa T. Ogawa H. Mol. Cell. Biol. 1984; 4: 2735-2744Crossref PubMed Scopus (90) Google Scholar, 26Bezzubova O. Schmidt H. Ostermann K. Heyer W.-D. Buerstedde J.-M. Nucleic Acids Res. 1993; 21: 5945-5949Crossref PubMed Scopus (66) Google Scholar, 27Muris D.F.R. Bezzubova O. Buerstedde J.-M. Vreeken K. Balajee A.S. Osgood C.J. Troelstra C. Hoeijmakers J.H.J. Ostermann K. Schmidt H. Natarajan A.T. Eeken J.C.J. Lohman P.H.M. Pastink A. Mutat. Res. DNA Repair. 1994; 315: 295-305Crossref PubMed Scopus (102) Google Scholar). The role of Rad52 in the Rad51-dependent recombination pathway has been extensively studied both in vivo (28Rijkers T. Van Den Ouweland J. Morolli B. Rolink A.G. Baarends W.M. Van Sloun P.P.H. Lohman P.H.M. Pastink A. Mol. Cell. Biol. 1998; 18: 6423-6429Crossref PubMed Scopus (272) Google Scholar, 29Yamaguchi-Iwai Y. Sonoda E. Buerstedde J.-M. Bezzubova O. Morrison C. Takata M. Shinohara A. Takeda S. Mol. Cell. Biol. 1998; 18: 6430-6435Crossref PubMed Scopus (201) Google Scholar) and in vitro. The ScRad52 and HsRad52 proteins both bind single-stranded DNA (ssDNA) and promote annealing of complementary ssDNA molecules (23Shinohara A. Shinohara M. Ohta T. Matsuda S. Ogawa T. Genes Cells. 1998; 3: 145-156Crossref PubMed Scopus (237) Google Scholar,30Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar, 31Reddy G. Golub E.I. Radding C.M. Mutat. Res. 1997; 377: 53-59Crossref PubMed Scopus (85) Google Scholar, 32Benson F.E. Baumann P. West S.C. Nature. 1998; 391: 401-404Crossref PubMed Scopus (334) Google Scholar, 33Sugiyama T. New J.H. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6049-6054Crossref PubMed Scopus (262) Google Scholar, 34Van Dyck E. Hajibagheri N.M.A. Stasiak A. West S.C. J. Mol. Biol. 1998; 284: 1027-1038Crossref PubMed Scopus (93) Google Scholar, 35Parsons C.A. Baumann P. Van Dyck E. West S.C. EMBO J. 2000; 19: 4175-4181Crossref PubMed Scopus (72) Google Scholar), suggesting a role of Rad52 in the single-stranded annealing (36Van Dyck E. Stasiak A.Z. Stasiak A. West S.C. Nature. 1999; 398: 728-731Crossref PubMed Scopus (257) Google Scholar). In addition, ScRad52 and HsRad52 both reportedly enhance the joint molecule formation between circular ssDNA and linear double-stranded DNA (dsDNA) that is promoted by the ScRad51 (S . c erevisiae Rad51) and HsRad51 (H .s apiens Rad51) proteins (32Benson F.E. Baumann P. West S.C. Nature. 1998; 391: 401-404Crossref PubMed Scopus (334) Google Scholar, 37Sung P. J. Biol. Chem. 1997; 272: 28194-28197Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 38New J.H. Sugiyama T. Zaitseva E. Kowalczykowski S.C. Nature. 1998; 391: 407-410Crossref PubMed Scopus (501) Google Scholar, 39Shinohara A. Ogawa T. Nature. 1998; 391: 404-407Crossref PubMed Scopus (408) Google Scholar). Therefore, Rad52 has been proposed to be a mediator protein that binds ssDNA and facilitates the loading of Rad51 onto the recombination site (36Van Dyck E. Stasiak A.Z. Stasiak A. West S.C. Nature. 1999; 398: 728-731Crossref PubMed Scopus (257) Google Scholar, 40McIlwraith M.J. Van Dyck E. Masson J.Y. Stasiak J.Y. Stasiak A. West S.C. J. Mol. Biol. 2000; 304: 151-164Crossref PubMed Scopus (102) Google Scholar, 41Song B.W. Sung P. J. Biol. Chem. 2000; 275: 15895-15904Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In addition to its role in the Rad51-dependent recombination pathway, the role of Rad52 in the Rad51-independent recombination pathways has also been studiedin vivo (42Aguilera A. Curr. Genet. 1995; 27: 298-305Crossref PubMed Scopus (32) Google Scholar, 43Ivanov E.L. Sugawara N. Fishman-Lobell J. Haber J.E. Genetics. 1996; 142: 693-704Crossref PubMed Google Scholar, 44Haber J.E. Nature. 1999; 398: 665-667Crossref PubMed Scopus (82) Google Scholar, 45Bartsch S. Kang L.E. Symington L.S. Mol. Cell. Biol. 2000; 20: 1194-1205Crossref PubMed Scopus (83) Google Scholar). However, its precise molecular mechanism is not well understood. Interestingly, HsRad52 itself promotes homologous pairing between ssDNA fragments and superhelical dsDNA substrates (46Kurumizaka H. Aihara H. Kagawa W. Shibata T. Yokoyama S. J. Mol. Biol. 1999; 291: 537-548Crossref PubMed Scopus (62) Google Scholar). This activity, along with the single-stranded annealing, may provide the framework for the role of Rad52 in such a pathway. In the present study, the homologous pairing activity of HsRad52 has been biochemically characterized. HsRad52 showed maximal homologous pairing activity at a concentration that just saturated the ssDNA. The HsRad52-ssDNA complex formation is a critical step in the homologous pairing reaction, because an incubation of HsRad52 with dsDNA before the addition of ssDNA inhibited the reaction. The conserved N-terminal domain of HsRad52, which was revealed by a limited proteolysis, formed a nucleoprotein filament with ssDNA and catalyzed homologous pairing with an efficiency similar to that of the wild type HsRad52 protein. HsRad52 and HsRad521–237were both purified in a three-step procedure involving Ni-NTA-agarose purification, the removal of the hexahistidine tag, and heparin-Sepharose purification, to facilitate comparison of the activities. MonoS column chromatography was used to concentrate the HsRad52 protein. Both proteins were cloned into the T7 polymerase expression vector pET-15b (Novagen). The proteins were overexpressed in the E. coli strain JM109 (DE3) along with E. colitRNAArg3 and tRNAArg4, which recognize the CGG and AGA/AGG codons, respectively. Expression levels of both proteins were extremely low without the inclusion of the plasmid containing the tRNA genes. For an average purification, HsRad52 and HsRad521–237 were purified from 5- and 1-liter cultures, respectively. All cultures were incubated at 30 °C. At the logarithmic phase of growth (A600 = 0.6), protein expression was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside (final concentration). The cells were harvested after 4–6 h of culture and were lysed by sonication in buffer A (pH 7.8) containing 50 mm Tris-HCl, 0.3 m KCl, 2 mm2-mercaptoethanol, 10 mm imidazole, 10% glycerol, and protease inhibitors (Complete EDTA-free; Roche Molecular Biochemicals) on ice. All manipulations after harvesting were done at 4 °C. The lysates were centrifuged at 27,700 × g for 20 min, and the supernatants were gently mixed by the batch method with 4 ml of Ni-NTA agarose beads (Qiagen) for 1 h. The HsRad52-coupled Ni-NTA-agarose beads were packed into Econo-columns (Bio-Rad) and were washed with 30 column volumes of buffer B (pH 7.8), which contained 50 mm Tris-HCl, 0.3 m KCl, 2 mm2-mercaptoethanol, 50 mm imidazole, and 10% glycerol at a flow rate of about 0.3 ml/min. The HsRad52 proteins were eluted in a 30-column volume linear gradient from 50 to 300 mmimidazole. Peak fractions, which eluted at about 0.15 mimidazole, were collected, and 2 units of thrombin protease (Amersham Pharmacia Biotech)/mg of HsRad52 protein were added to remove the hexahistidine tag. Fractions were immediately dialyzed against buffer C (pH 7.5), which contained 50 mm Tris-HCl, 0.2 mKCl, 0.5 mm EDTA, 2 mm 2-mercaptoethanol, and 10% glycerol, for more than 12 h. After the removal of the hexahistidine tag, the HsRad52 proteins were loaded onto a 4-ml Heparin-Sepharose column (Amersham Pharmacia Biotech). The column was washed with 20 column volumes of buffer C, and the proteins were eluted with a 20-column volume linear gradient from 0.2 to 1 mKCl. HsRad52 and HsRad521–237 both eluted in sharp peaks at about 0.3 m KCl. The peak fractions of HsRad521–237, which had a concentration of 1–2 mg/ml, were dialyzed against buffer D (pH 7.0), which contained 20 mm Hepes-KOH, 0.2 m KCl, 0.5 mmEDTA, 2 mm 2-mercaptoethanol, and 50% glycerol, and were stored at −20 °C. For HsRad52, the heparin-Sepharose peak fractions were dialyzed against buffer E (pH 7.5), which contained 20 mm potassium phosphate, 0.2 m KCl, 0.5 mm EDTA, 2 mm 2-mercaptoethanol, and 10% glycerol, and were applied to a 1-ml MonoS column (Amersham Pharmacia Biotech) for concentration. The MonoS column was washed with buffer E and was eluted in a 20-ml linear gradient from 0.2 to 1 mKCl. The HsRad52 protein sharply eluted at about 0.3 m KCl. Peak fractions were collected, dialyzed against buffer D, and stored at −20 °C. For subsequent experiments, HsRad52 and HsRad521–237 were dialyzed against buffer F (pH 7.0), which contained 20 mm Hepes-KOH, 0.2 m KCl, 0.5 mm EDTA, 2 mm 2-mercaptoethanol, and 10% glycerol. Protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin (Pierce) as the standard. The HsRad51 expression vector was constructed as described (46Kurumizaka H. Aihara H. Kagawa W. Shibata T. Yokoyama S. J. Mol. Biol. 1999; 291: 537-548Crossref PubMed Scopus (62) Google Scholar). Hexahistidine-tagged HsRad51 was overexpressed in E. colistrain JM109 (DE3) along with the tRNA described above. The culture conditions for HsRad51 were identical to those for HsRad52. The HsRad51 protein was purified from a 10-liter LB culture. Harvested cells were lysed by sonication in buffer G (pH 8.0), which contained 50 mm Tris-HCl, 0.5 m NaCl, 5 mm2-mercaptoethanol, 10 mm imidazole, 10% glycerol, and protease inhibitors (Complete EDTA-free; Roche Molecular Biochemicals). Lysates were mixed gently by the batch method with Ni-NTA-agarose beads at 4 °C for 1 h. The HsRad51-coupled Ni-NTA-agarose beads (4 ml) were then packed into an Econo-column (Bio-Rad) and were washed with 30 column volumes of buffer H (pH 8.0), which contained 50 mm Tris-HCl, 0.5 m NaCl, 5 mm2-mercaptoethanol, 60 mm imidazole, and 10% glycerol, at a flow rate of about 0.3 ml/min. Hexahistidine-tagged HsRad51 was eluted in a 30-column volume linear gradient from 60 to 400 mmimidazole. HsRad51, which eluted in a broad peak, was collected, and was treated with 1 unit of thrombin protease (Amersham Pharmacia Biotech)/mg of HsRad51. The HsRad51 protein was immediately dialyzed against buffer I (pH 8.0), which contained 50 mm Tris-HCl, 0.2 m KCl, 0.5 mm EDTA, 2 mm2-mercaptoethanol, and 10% glycerol, at 4 °C. The HsRad51 protein precipitated overnight but redissolved after changing the dialysis buffer the following day. After more than 24 h of dialysis, the HsRad51 protein was collected and filtered to remove residual precipitates. About 20 mg of HsRad51, which was more than 99% pure as judged by SDS-PAGE and Coomassie Brilliant Blue staining, were then applied to a 1-ml MonoQ column (Amersham Pharmacia Biotech) for concentration. The MonoQ column was washed with 20 ml of buffer I and was eluted with a 20-ml linear gradient from 0.2 to 0.6 mKCl. The HsRad51 protein sharply eluted between 0.3–0.4 mKCl, and peak fractions had concentrations of about 2 mg/ml. These fractions were dialyzed against buffer J (pH 8.0), which contained 20 mm Tris-HCl, 0.1 m KCl, 0.5 mmEDTA, 2 mm 2-mercaptoethanol, and 10% glycerol, and were used for subsequent studies. The protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin (Pierce) as the standard. To prevent the dsDNA substrates used in the D loop formation from undergoing irreversible denaturation, alkaline treatment of the cells harboring the plasmid DNA was avoided. Instead, the cells were gently lysed using sarkosyll, as described (47Cunningham R.P. DasGupta C. Shibata T. Radding C.M. Cell. 1980; 20: 223-235Abstract Full Text PDF PubMed Scopus (148) Google Scholar). pGsat4 was created by inserting a 198-base pair fragment of the human α-satellite sequence into the pGEM-T Easy vector (Promega). The resulting 3.2-kilobase plasmid DNA and the ΦX174 form I DNA were both prepared following the method described above. For the ssDNA substrates, the following high pressure liquid chromatography-purified oligonucleotides were purchased from Roche Molecular Biochemicals: SAT-1 (50-mer, 5′-ATT TCA TGC TAG ACA GAA GAA TTC TCA GTA ACT TCT TTG TGC TGT GTG TA-3′) and ΦX-1 (50-mer, 5′-ATT TTG TTC ATG GTA GAG ATT CTC TTG TTG ACA TTT TAA AAG AGC GTG GA-3′). The 5′ ends of the oligonucleotides were labeled with T4 polynucleotide kinase (New England Biolabs) in the presence of [γ-32P]ATP at 37 °C for 90 min. Labeled oligonucleotides were purified with Chromaspin-10 columns (CLONTECH). The TE buffer in the spin columns was exchanged with H2O prior to purification of the oligonucleotides. This allowed accurate absorbance readings of the purified oligonucleotides at 260 nm for concentration determination. HsRad52 (5 μl of 1–3 mg/ml) was mixed with 5 μl of 30 μg/ml proteinase K, and the mixture was incubated at 25 °C for 4–6 h. The proteinase K solution was diluted in 10 mm Tris-HCl (pH 7.5). The reaction mixture was fractionated by 12% SDS-PAGE, and the protein bands were transferred to a polyvinylidene difluoride membrane using a semi-wet blotting apparatus (Bio-Rad). Proteolytic bands were visualized by staining with a 100-fold diluted Coomassie Brilliant Blue staining solution, followed by a brief destaining of the membrane with methanol. Portions of the polyvinylidene difluoride membrane containing the bands were excised for N-terminal amino acid sequencing. For mass determination of the proteolytic fragments, the reaction mixture containing the proteolyzed HsRad52 was treated with 1 μl of 2n HCl to stop further proteolysis by proteinase K. The mixture was diluted 100-fold with H2O and was subjected to mass spectrometry. To maintain the activity of the HsRad52 proteins, the desired concentrations of the HsRad52 proteins were obtained by repeating 2-fold serial dilutions of the protein solution in buffer F. In the HsRad52-catalyzed homologous pairing reactions, the reaction mixture contained final concentrations of 50 mm Hepes-KOH (pH 7.5), 40 mm KCl, 1 mm MgCl2, 0.1 mg/ml bovine serum albumin, 1 mm dithiothreitol, 2% glycerol, 1 μm ssDNA, 0.25–2 μm HsRad52, and 15 μm dsDNA. The initial 9 μl of the reaction mixture contained 2 μl of 5× reaction buffer (250 mm Hepes-KOH (pH 7.5), 5 mmMgCl2, 0.5 mg/ml bovine serum albumin, 5 mmdithiothreitol), 1 μl of 32P-labeled ssDNA (10 μm), appropriate amounts of H2O, and 2 μl of HsRad52, which were mixed on ice. HsRad52 and ssDNA were preincubated for 5 min at 37 °C in this 9-μl reaction mixture. The reaction was initiated by adding 1 μl of 150 μm dsDNA. After a 10-min incubation, the reaction was stopped by adding 1 μl of 5% SDS, followed by immediately adding 1 μl of 6 mg/ml proteinase K (Roche Molecular Biochemicals). The reaction mixtures were further incubated at 37 °C for 15 min. After adding the 6-fold loading dye (15% Ficoll, 0.1% bromphenol blue, 0.1% xylene cyanole), the reaction mixtures were subjected to 1% agarose gel electrophoresis (SeaKem GTG-agarose) in 0.5× TBE buffer. Electrophoresis was run at 3.3 V/cm at room temperature. The recombination products were visualized by autoradiography of the dried gel. Products and reactants were quantified using a Fuji BAS2500 image analyzer. Because the ssDNA is in excess, the yield of products was expressed as a percentage of the pGsat4 dsDNA incorporated into D-loops. The reaction mixture was essentially identical to that used in the assay for the D-loop formation to facilitate comparisons of the results. For the ssDNA binding, one volume of the 32P-labeled SAT-1 ssDNA (10 μm) was mixed with nine volumes of the nonlabeled SAT-1 ssDNA (10 μm) to dilute the 32P label 10-fold. After a 5-min incubation of HsRad52 and SAT-1 ssDNA (1 μm), 1 μl of a 2% glutaraldehyde solution was added to fix the HsRad52-ssDNA complex. The incubation was continued for 20 min. Afterward, the 6-fold loading dye was added, and the reaction mixtures were subjected to 1% agarose gel electrophoresis in 0.5× TBE buffer. Electrophoresis was run at 3.3 V/cm at room temperature. The resulting complexes were visualized by autoradiography of the dried gel. The amounts of free ssDNA were quantified using a Fuji BAS2500 image analyzer. For the dsDNA binding, HsRad52 was incubated with pGsat4 dsDNA (15 μm) for 5 min and subjected to 1% agarose gel electrophoresis without fixation of the complex. The complexes were visualized by ethidium bromide staining (0.5 μg/ml) of the gel. The amounts of free dsDNA were quantified using a MacBAS software. The ability of HsRad52 to bind to Affi-Gel 15-conjugated HsRad51 was observed. An Affi-Gel 15 slurry (250 μl; Bio-Rad) was washed two times with 0.5 ml of H2O, followed by three washes with 0.5 ml of buffer K (pH 8.0), which contained 20 mm Hepes-KOH, 0.1m KCl, 0.5 mm EDTA, 2 mm2-mercaptoethanol, 10% glycerol, and 0.05% Triton X-100. The washed beads were mixed with 0.5 ml of 2.8 mg/ml HsRad51 and were incubated at 4 °C for 5 h. Afterward, the supernatant was removed, and the conjugated beads were washed six times with 0.5 ml of buffer L. The final concentration of HsRad51 conjugated to Affi-Gel 15, determined by the amount of unbound HsRad51, was 3.3 mg/ml. The Affi-Gel 15-HsRad51 matrix was adjusted to a 33% slurry with binding buffer and was stored at 4 °C. For the binding assay, 10 μg of Rad51 (30 μl of the slurry) were mixed with 20 μg of HsRad52 and HsRad521–237. The proteins were mixed at room temperature for 90 min. The unbound HsRad52 proteins were then removed, and the Affi-Gel-HsRad51 beads were washed six times with 0.5 ml of buffer L. SDS-PAGE sample buffer (10 μl of a 2-fold stock) was mixed directly with the washed beads. After heating the mixture at 98 °C for 2 min, the HsRad51 and the bound HsRad52 proteins were fractionated by 15–25% gradient SDS-PAGE. Bands were visualized by Coomassie Brilliant Blue staining. A Superdex 200 HR 10/30 gel filtration column (Amersham Pharmacia Biotech) was used to determine the estimated molecular masses of the HsRad52 proteins. All gel filtrations were done in buffer F. For each injection, 0.1–0.2 ml of 1–2 mg/ml HsRad52 was used. To create a standard molecular mass curve, ferritin, aldolase, albumin, ovalbumin, and ribonuclease A (Amersham Pharmacia Biotech) were used. HsRad52 and HsRad521–237 and their complexes with ΦX174 circular ssDNA were negatively stained on a copper-plated carbon grid with 2% uranyl acetate. The proteins and complexes were observed with a JEOL JEM 2000FX electron microscope. We previously reported that HsRad52 catalyzes homologous pairing independently from HsRad51, as observed in the D-loop formation assay, which is a standard homologous pairing assay for RecA (9Shibata T. DasGupta C. Cunningham R.P. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1638-1642Crossref PubMed Scopus (279) Google Scholar). However, in those previous experiments, we used dsDNA substrates prepared by a method involving alkaline treatment (46Kurumizaka H. Aihara H. Kagawa W. Shibata T. Yokoyama S. J. Mol. Biol. 1999; 291: 537-548Crossref PubMed Scopus (62) Google Scholar), which may irreversibly denature the double helix of the dsDNA. To exclude the possibility of observing an annealing reaction between a denatured dsDNA and its homologous ssDNA, we prepared the dsDNA by a conventional method that does not involve alkaline treatment (47Cunningham R.P. DasGupta C. Shibata T. Radding C.M. Cell. 1980; 20: 223-235Abstract Full Text PDF PubMed Scopus (148) Google Scholar) and successfully reproduced the formation of D-loops by purified HsRad52 (Figs.1 A and2).Figure 2D-loop formed by HsRad52. A, a schematic diagram of the D-loop formation. B, the D-loops formed by HsRad52 (1 μm) and RecA (0.2 μm) migrated the same distance on a 1% agarose gel. The HsRad52 and RecA proteins were reacted in the presence of 10 mmMgCl2, which is required for the optimal activity of the RecA protein. ATP was omitted from the reaction containing HsRad52. In the reaction, a 50-mer ssDNA (1 μm), which was end-labeled with [γ-32P]ATP, was first incubated with HsRad52 or RecA in the reaction mixture containing 1 mmMgCl2. The reaction was initiated by adding pGsat4 dsDNA (final concentration, 15 μm) and MgCl2 (final concentration, 10 mm). The products were not observed without the proteins (lane 1) or without ATP in the presence of RecA (lane 2). The D-loops formed by HsRad52 dissociated when treated with either the NheI, PstI, orScaI restriction enzyme for 30 min at 37 °C (lanes 5–7). Each enzyme cuts the dsDNA at a single, nonhomologous site.C, the HsRad52 protein catalyzed the formation of D-loops in a homology-dependent manner. pGsat4 (3.2 kilobases, 15 μm) and ΦX174 dsDNA (5.4 kilobases, 15 μm) and their homologous ssDNAs (1 μm) were used as DNA substrates. Without HsRad52, D-loop products were not observed with either homologous combination of the DNA substrates (lanes 1 and 2). In the presence of HsRad52 (1 μm), D-loops were observed in homologous combinations that have migration distances corresponding to the length of the dsDNA (lanes 3 and 4). HsRad52 was unable to catalyze any D-loops between heterologous combinations of the DNA substrates (lanes 5 and 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The use of this DNA substrate now allowed us to characterize the homologous pairing activity of HsRad52. In the reaction, a 50-mer oligonucleotide was preincubated with HsRad52, and then the dsDNA was added to initiate the reaction. The migration distance of the reaction product on the agarose gel was identical to that of the D-loop product formed by RecA (Fig. 2 B, lanes 3 and4). When the superhelical tension in the D-loops was released by cutting the dsDNA with restriction enzymes at sites outside the homologous region, the D-loops dissociated by spontaneous branch migration, as in the case of authentic D-loops formed by RecA or a nonenzymatic method (48Holloman W.K. Wiegand R. Hoessli C. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2394-2398Crossref PubMed Scopus (71) Google Scholar, 49Shibata T. Ohtani T. Chang P.K. Ando T. J. Biol. Chem. 1982; 257: 370-376Abstract Full Text PDF PubMed Google Scholar) (Fig. 2 B, lanes 5–7). These two results indicate that the D-loop formed by HsRad52 has the same physical characteristics as that formed by RecA. The HsRad52-catalyzed D-loop formation was a homology-dependent reaction. When ΦX174 dsDNA, which was also prepared by the conventional method, and its homologous ssDNA were used as D-loop substrates, HsRad52 formed D-loops with an efficiency similar to that of the pGsat4 DNA substrates (Fig. 2 C,lanes 3 and 4). In contrast, no D-loops were formed between heterologous combinations of the DNA substrates (Fig.2 C, lanes 5 and 6). The HsRad52 protein converted more than 10% of the dsDNA into D-loops when HsRad52 was preincubated with ssDNA followed by the addition of dsDNA (Fig. 3, A an" @default.
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• W2078932716 title "Homologous Pairing Promoted by the Human Rad52 Protein" @default.
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