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• W1993640924 abstract "The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. TheXrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) inEscher- ichia coli, and purified the Xrcc2·Rad51D complex to homogeneity. The Xrcc2·Rad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3· Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2·Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2·Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3·Rad51C. The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. TheXrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) inEscher- ichia coli, and purified the Xrcc2·Rad51D complex to homogeneity. The Xrcc2·Rad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3· Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2·Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2·Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3·Rad51C. Chromosomal DNA is vulnerable to attacks from the environment and sustains multiple types of damage, including the double strand break (DSB), 1The abbreviations used are: DSBdouble strand breakHRRhomologous recombinational repairssDNAsingle-stranded DNAdsDNAdouble-stranded DNANi-NTAnickel-nitrilotriacetic acidBSAbovine serum albuminwhich is a lethal DNA lesion for cells if it is not repaired. Homologous recombinational repair (HRR) is one of the major pathways for the repair of DSBs. When cells are defective in HRR, unrepaired DSBs accumulate in chromosomes (1.Sonoda E. Sasaki M.S. Buerstedde J.-M. Bezzubova O. Shinohara A. Ogawa H. Takata M. Yamaguchi-Iwai Y. Takeda S. EMBO J. 1998; 17: 598-608Crossref PubMed Scopus (705) Google Scholar). double strand break homologous recombinational repair single-stranded DNA double-stranded DNA nickel-nitrilotriacetic acid bovine serum albumin In Escherichia coli, the RecA protein catalyzes homologous pairing, which is a key step in HRR (2.Shibata 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, 3.McEntee K. Weinstock G.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2615-2619Crossref PubMed Scopus (241) Google Scholar). In homologous pairing, RecA binds the single-stranded DNA (ssDNA) produced at DSB sites and forms nucleoprotein filaments. Then, the nucleoprotein filaments bind double-stranded DNA (dsDNA) and form a three-component complex that includes ssDNA, dsDNA, and RecA. In the three-component complex, the homology between ssDNA and dsDNA is searched, and joint molecules, in which the ssDNA invades into the homologous region of the dsDNA, are formed as products of homologous pairing. In eukaryotes, the Rad51 protein has been identified as a homologue of RecA (4.Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar). The Rad51 protein, which is conserved from yeast to human (5.Shinohara A. Ogawa H. Matsuda Y. Ushio N. Ikeo K. Ogawa T. Nat. Genet. 1993; 4: 239-243Crossref PubMed Scopus (30) Google Scholar), forms a nucleoprotein filament that is strikingly similar to that formed by RecA (6.Ogawa T. Yu X. Shinohara A. Egelman E. Science. 1993; 259: 1896-1899Crossref PubMed Scopus (561) Google Scholar), suggesting their functional similarity in HRR. Actually, the Saccharomyces cerevisiae and human Rad51 proteins (ScRad51 and HsRad51, respectively) catalyze homologous pairing (7.Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 8.Baumann P. Benson F.E. West S.C. Cell. 1996; 87: 757-766Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 9.Gupta 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). We have found that the human Xrcc3 and Rad51C/Rad51L2 proteins (Xrcc3 and Rad51C, respectively) form a complex and catalyze homologous pairing (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar) in addition to HsRad51. Both Xrcc3 and Rad51C are members of the Rad51 paralogs (Xrcc2 (11.Cartwright R. Tambini C.E. Simpson P.J. Thacker J. Nucleic Acids Res. 1998; 26: 3084-3089Crossref PubMed Scopus (128) Google Scholar, 12.Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar), Xrcc3 (12.Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 13.Tebbs R.S. Zhao Y. Tucker J.D. Scheerer J.B. Siciliano M.J. Hwang M. Liu N. Legerski R.J. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6354-6358Crossref PubMed Scopus (239) Google Scholar), Rad51B/hREC2/Rad51L1 (14.Albala J.S. Thelen M.P. Prange C. Fan W. Christensen M. Thompson L.H. Lennon G.G. Genomics. 1997; 46: 476-479Crossref PubMed Scopus (106) Google Scholar, 15.Rice C.M. Smith S.T. Bullrich F. Havre P. Kmiec E.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7417-7422Crossref PubMed Scopus (85) Google Scholar, 16.Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (74) Google Scholar), Rad51C/Rad51L2 (17.Dosanjh M.K. Collins D.W. Wufang F. Lennon G.G. Albala J.S. Shen Z. Schild D. Nucleic Acids Res. 1998; 26: 1179-1184Crossref PubMed Scopus (145) Google Scholar), and Rad51D/Rad51L3 (16.Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (74) Google Scholar, 18.Kawabata M. Saeki K. Biochem. Biophys. Res. Commun. 1999; 257: 156-162Crossref PubMed Scopus (20) Google Scholar, 19.Pittman D.L. Weinberg L.R. Schimenti J.C. Genomics. 1998; 49: 103-111Crossref PubMed Scopus (113) Google Scholar), etc.) and share 20–30% amino acid identity with HsRad51. The Xrcc2 and Xrcc3 genes were first identified as human genes that complement the DNA damage-sensitive hamster cell lines, irs1 and irs1SF, respectively (13.Tebbs R.S. Zhao Y. Tucker J.D. Scheerer J.B. Siciliano M.J. Hwang M. Liu N. Legerski R.J. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6354-6358Crossref PubMed Scopus (239) Google Scholar, 20.Jones N.J. Cox R. Thacker J. Mutat. Res. 1987; 183: 279-286Crossref PubMed Scopus (184) Google Scholar, 21.Fuller L.F. Painter R.B. Mutat. Res. 1988; 193: 109-121Crossref PubMed Scopus (163) Google Scholar, 22.Jones N.J. Zhao Y. Siciliano M.J. Thompson L.H. Genomics. 1995; 26: 619-622Crossref PubMed Scopus (26) Google Scholar, 23.Thacker J. Tambini C.E. Simpson P.J. Tsui L.C. Scherer S.W. Hum. Mol. Genet. 1995; 4: 113-120Crossref PubMed Scopus (47) Google Scholar), and both genes were confirmed to be involved in HRR in vivo (24.Johnson R.D. Liu N. Jasin M. Nature. 1999; 401: 397-399Crossref PubMed Google Scholar, 25.Pierce A.J. Johnson R.D. Thompson L.H. Jasin M. Genes Dev. 1999; 13: 2633-2638Crossref PubMed Scopus (1058) Google Scholar). Cells lacking Xrcc2 or Xrcc3 show extreme sensitivity to DNA cross-linking reagents like cisplatin and ionizing radiation and have significantly increased missegregation of chromosomes (12.Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 13.Tebbs R.S. Zhao Y. Tucker J.D. Scheerer J.B. Siciliano M.J. Hwang M. Liu N. Legerski R.J. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6354-6358Crossref PubMed Scopus (239) Google Scholar, 26.Griffin C.S. Simpson P.J. Wilson C.R. Thacker J. Nat. Cell Biol. 2000; 2: 757-761Crossref PubMed Scopus (212) Google Scholar). Knock-out experiments in the chicken DT40 cell lines showed that theRad51B/Rad51L1, Rad51C/Rad51L2, andRad51D/Rad51L3 genes, as well as the Xrcc2 andXrcc3 genes, are involved in the HRR pathway (27.Takata M. Sasaki M.S. Sonoda E. Fukushima T. Morrison C. Albala J.S. Swagemakers S.M.A. Kanaar R. Thompson L.H. Takeda S. Mol. Cell. Biol. 2000; 20: 6476-6482Crossref PubMed Scopus (225) Google Scholar, 28.Takata M. Sakaki M.S. Tachiiri S. Fukushima T. Sonoda E. Schild D. Thompson L.H. Takeda S. Mol. Cell. Biol. 2001; 21: 2858-2866Crossref PubMed Scopus (462) Google Scholar). The Xrcc2 protein also has homology to the S. cerevisiae Rad55 protein (29.Thacker J. Trends Genet. 1999; 15: 166-168Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), which interacts with the S. cerevisiae Rad57 protein (30.Hays S.L. Firmenich A.A. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6925-6929Crossref PubMed Scopus (286) Google Scholar, 31.Johnson R.D. Symington L.S. Mol. Cell. Biol. 1995; 15: 4843-4850Crossref PubMed Scopus (211) Google Scholar, 32.Sung P. Genes Dev. 1997; 11: 1111-1121Crossref PubMed Scopus (463) Google Scholar). Interestingly, the Xrcc2 knock-out mice exhibited embryonic lethality associated especially with a high frequency of apoptotic death of neurons in the developing brain (33.Deans B. Griffin C.S. Maconochie M. Thacker J. EMBO J. 2000; 19: 6675-6685Crossref PubMed Scopus (197) Google Scholar). Xrcc2 interacts with the human Rad51D/Rad51L3 protein (Rad51D), another Rad51 paralog (34.Braybrooke J.P. Spink K.G. Thacker J. Hickson I.D. J. Biol. Chem. 2000; 275: 29100-29106Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 35.Schild D. Lio Y. Collins D.W. Tsomondo T. Chen D.J. J. Biol. Chem. 2000; 275: 16443-16889Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The Rad51D knock-out mice also showed embryonic lethality (36.Pittman D.L. Schimenti J.C. Genesis. 2000; 26: 167-173Crossref PubMed Scopus (127) Google Scholar), indicating that both Xrcc2 and Rad51D are essential for embryonic development. In the present study, we purified the Xrcc2·Rad51D complex and characterized it biochemically. The purified Xrcc2·Rad51D complex catalyzed homologous pairing between ssDNA and dsDNA, similar to that of the Xrcc3·Rad51C complex. The Xrcc2·Rad51D complex formed a multimeric ring structure in the absence of DNA and formed nucleoprotein filaments in the presence of ssDNA. These biochemical characteristics of Xrcc2·Rad51D are consistent with the idea that these proteins act in an early step of the HRR pathway. The Xrcc2 and Rad51D genes were cloned from human brain cDNA (purchased from CLONTECH) by PCR. The overexpression plasmid for Xrcc2 and Rad51D was introduced into E. coli JM109(DE3) cells with the plasmid containing the genes for E. coli tRNA Arg-3 and tRNA Arg-4, which recognize the CGG and AGA/AGG codons, respectively. The cells were grown in 10 liters of LB medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol at 30 °C for 10 h, and then 200 μm isopropyl β-thiogalactopyranoside was added to induce protein expression. Xrcc2 and Rad51D were produced for 12 h at 18 °C in the presence of isopropyl β-thiogalactopyranoside, and the cells were harvested. Both Xrcc2 and Rad51D contain a His6 tag at the N terminus. The cells producing Xrcc2 and Rad51D were resuspended in 20 ml of buffer containing 20 mm sodium phosphate (pH 8.5) and 0.5m NaCl and were disrupted by sonication. The samples were centrifuged for 20 min at 30,000 × g, and the supernatants were incubated with 4 ml of Ni-NTA-agarose (Qiagen) for 1 h at 4 °C. Then, the Xrcc2 and Rad51D-bound resin was packed into a column and washed with 300 ml of buffer containing 20 mm Tris-HCl (pH 8.5), 30 mm imidazole, and 0.5m NaCl. Subsequently, the resin was washed with 150 ml of buffer containing 20 mm Tris-HCl (pH 8.5) and 30 mm imidazole. Xrcc2 and Rad51D were eluted by a gradient of imidazole from 30 to 400 mm in 20 mm Tris-HCl (pH 8.5). The samples eluted from the Ni-NTA column were applied to a Mono-Q column (Amersham Biosciences) previously equilibrated with buffer containing 20 mm Tris-HCl (pH 8.5), 2 mm dithiothreitol, and 10% glycerol. The proteins were eluted by a gradient of NaCl from 0 to 1200 mm and were dialyzed against a buffer containing 20 mm Tris-HCl (pH 8.1), 2 mm dithiothreitol, and 10% glycerol. Xrcc2 and Rad51D were further purified by gel filtration chromatography on Superdex 200HR resin (Amersham Biosciences). The purified proteins were stored in 20 mm Tris-HCl (pH 8.1), 5 mmdithiothreitol, and 10% glycerol. The protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad) using bovine serum albumin (BSA) as the standard protein. High pressure liquid chromatography-purified oligonucleotides (120 mer) for the D-loop formation assay were purchased from Roche Molecular Biochemicals. The nucleotide sequences are as follows: 5′-ATTTC TTCAT TTCAT GCTAG ACAGA AGAAT TCTCA GTAAC TTCTT TGTGC TGTGT GTATT CAACT CACAG AGTGG AACGT CCCTT TGCAC AGAGC AGATT TGAAA CACTC TTTTT GTAGT-3′ and were derived from a clone of human α-satellite DNA. The 5′ ends of the oligonucleotides were labeled with T4 polynucleotide kinase (New England Biolabs) in the presence of [γ-32P]ATP. DNA concentrations are expressed in moles of nucleotides. To avoid irreversible denaturation of the dsDNA, we prepared plasmid DNA without any treatment that would potentially cause denaturation, such as alkaline treatment. The plasmid DNA (pGsat4; 3216 base pairs) containing the human α-satellite sequence was introduced into theE. coli DH5α strain, and the cells were cultured for 12 to 16 h at 37 °C. The cells were harvested, mildly disrupted with 0.5 mg/ml lysozyme and 0.1% sarkosyl, and centrifuged at 28,000 rpm for 1 h. The supernatant containing the pGsat4 plasmid DNA was extracted with phenol/chloroform three times, and the pGsat4 DNA in the aqueous phase was precipitated by ethanol. The pellet was dissolved in 1 ml of TE buffer (10 mm Tris·HCl (pH 8.0), 1 mm EDTA) and treated with 0.15 mg/ml RNase A at 37 °C for 30 min. The pGsat4 DNA was purified by a 5–20% sucrose gradient centrifugation at 24,000 rpm for 18 h. DNA concentrations are expressed in moles of nucleotides. TheE. coli cells (JM109) containing pGsat4 DNA were cultured in LB medium until an A660 of 0.3 was achieved and were infected with the helper phage R408 at a multiplicity of infection of ∼20. After infection, the cells were continuously cultured for 6 h with vigorous agitation. Then, the supernatant was harvested and treated with DNase I (10 unit/ml) and RNase A (10 μg/ml) for 15 min at 37 °C. The phage containing pGsat4 ssDNA was precipitated by adding 0.25 volume of phage precipitation solution containing 3.75m ammonium acetate (pH 7.5) and 20% polyethylene glycol 8000. The resulting pellet was resuspended with TE buffer, and the pGsat4 ssDNA was extracted by chloroform:isoamyl alcohol (24:1) followed by phenol:chloroform saturated with TE buffer. After several phenol:chloroform extractions, the aqueous phase was extracted by chloroform, and the pGsat4 ssDNA was precipitated with 2 volumes of ethanol in the presence of 3.25 m ammonium acetate. The resulting pellet was rinsed with 70% ethanol and dried under vacuum. The pGsat4 ssDNA was dissolved in H2O; its concentration is expressed in moles of nucleotides. Single-stranded pGsat4 DNA (30 μm) was mixed with Xrcc2·Rad51D in 10 μl of standard reaction buffer containing 20 mm Tris-HCl (pH 8.1), 2 mm ATP, 1 mm dithiothreitol, 100 μg/ml BSA, 2 mm MgCl2, and 3% glycerol. The reaction mixtures were incubated at 37 °C for 10 min and then analyzed by 0.8% agarose gel electrophoresis in 0.5× TBE buffer. The bands were visualized by ethidium bromide staining. Superhelical pGsat4 DNA (18 μm, 3216 base pairs) was mixed with Xrcc2·Rad51D in 10 μl of standard reaction buffer containing 20 mmTris-HCl (pH 8.1), 2 mm ATP, 20 mm creatine phosphate, 1 mm dithiothreitol, 100 μg/ml BSA, 12 units/ml creatine phosphokinase, 2 mm MgCl2, and 6% glycerol. The reaction mixtures were incubated at 37 °C for 10 min, and the samples were electrophoresed on an 0.8% agarose gel for 4 h at 3 V/cm in 0.5× TBE buffer. The bands were visualized by ethidium bromide staining. A32P-labeled single-stranded 120 mer oligonucleotide (1.8 μm) was mixed with Xrcc2·Rad51D (2 μm) in 10 μl of standard reaction buffer containing 50 mmTris-HCl (pH 8.0), 2 mm ATP, 20 mm creatine phosphate, 1 mm dithiothreitol, 100 μg/ml BSA, 12 units/ml creatine phosphokinase, and 2 mmMgCl2. The mixtures were incubated at 37 °C for 5 min, and the reaction was started by the addition of pGsat4 form I DNA (20 μm). After an incubation at 37 °C for the indicated time, the reaction was terminated by the addition of 0.5% sodium dodecyl sulfate (SDS), and the proteins were removed from the DNA by treatment with proteinase K (700 μg/ml) followed by an incubation at 37 °C for 15 min. The products of homologous pairing (D-loops) were separated from the unreacted DNA substrates by 0.8% agarose gel electrophoresis in 0.5× TBE buffer. The amount of labeled ssDNA fragments incorporated into the D-loops was quantified with a Fuji BAS2500 image analyzer. The reactions were conducted with a 4.8-fold excess amount of ssDNA (molecule). In the experiments with RecA, a 32P-labeled single-stranded 120 mer oligonucleotide (1.8 μm) was mixed with RecA (2 μm) in 10 μl of standard reaction buffer containing 50 mm Tris-HCl (pH 8.0), 20 mm creatine phosphate, 1 mm dithiothreitol, 100 μg/ml BSA, 12 units/ml creatine phosphokinase, and 10 mm MgCl2 in the presence or absence of 2 mm ATP. The mixtures were incubated at 37 °C for 5 min, and the reaction was started by the addition of pGsat4 form I DNA (20 μm). After an incubation at 37 °C for 10 min, the reaction was terminated, and the products were separated from the unreacted DNA substrates by 0.8% agarose gel electrophoresis in 0.5× TBE buffer. The amount of labeled ssDNA fragments incorporated into the D-loops was quantified with a Fuji BAS2500 image analyzer. Xrcc2·Rad51D or HsRad51 (1–2 μm) was incubated with or without circular ssDNA (φX174; 10 μm) or nicked circular dsDNA (φX174; 10 μm) in buffer, containing 20 mm Tris-HCl (pH 8.1), 1 mm dithiothreitol, and 2 mmMgCl2, in the presence or absence of 2 mm ATP. The mixtures were incubated at 37 °C for 10 min and were adsorbed onto a freshly glow-discharged carbon-coated grid. The proteins or the protein-DNA complexes were visualized by negative staining with 2% uranyl acetate. Several splicing variants of Rad51D have been reported in human cells (18.Kawabata M. Saeki K. Biochem. Biophys. Res. Commun. 1999; 257: 156-162Crossref PubMed Scopus (20) Google Scholar). To identify the Rad51D transcript that interacts with Xrcc2, we performed two-hybrid screening with the human brain cDNA library. Two Rad51D clones that positively interact with Xrcc2 were obtained. Both Rad51D clones encode a protein consisting of 328 amino acid residues, which is identical to HsR51H3 (Rad51D/Rad51L3) (16.Cartwright R. Dunn A.M. Simpson P.J. Tambini C.E. Thacker J. Nucleic Acids Res. 1998; 26: 1653-1659Crossref PubMed Scopus (74) Google Scholar). We coexpressed Xrcc2 and Rad51D in the E. coli recA− JM109(DE3) strain as N-terminal His6-tagged (20 amino acid residues) proteins. It has been reported that chicken DT40 cells lacking either the Xrcc2 or Rad51D gene were extremely sensitive to cisplatinum, which induced intra- and interstrand DNA cross-inks (28.Takata M. Sakaki M.S. Tachiiri S. Fukushima T. Sonoda E. Schild D. Thompson L.H. Takeda S. Mol. Cell. Biol. 2001; 21: 2858-2866Crossref PubMed Scopus (462) Google Scholar). As shown in Fig. 1, Band C, the His6-tagged Xrcc2 and Rad51D proteins fully complemented the cisplatinum-sensitive phenotypes in theXrcc2−/− and Rad51D−/− chicken DT40 cells, respectively (Fig. 1, B and C), indicating that the His6 tag did not affect the functions of these proteins. After coexpression, both Xrcc2 and Rad51D were detected in the soluble fraction and were coeluted from the Ni-NTA-agarose (Qiagen). The Xrcc2 and Rad51D proteins were further copurified by Mono-Q (Amersham Biosciences) column chromatography and Superdex 200HR gel filtration chromatography (Amersham Biosciences) (Fig. 1A, lane 2). These results indicate that Xrcc2 and Rad51D form a complex. Endonuclease and exonuclease activities were not detected in the Xrcc2·Rad51D fractions with superhelical and linearized dsDNAs as substrates (data not shown). We have previously reported that the Xrcc3·Rad51C complex catalyzes homologous pairing between a single-stranded oligonucleotide and a superhelical dsDNA by the D-loop formation assay, which is a standard assay for homologous pairing by RecA (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar). As shown in Fig. 2, the Xrcc2·Rad51D complex also catalyzes homologous pairing. The superhelical dsDNA used in this assay was prepared by a method without alkali treatment to avoid irreversible denaturation of the double helix of the dsDNA (37.Shibata T. Cunningham R.P. Radding C.M. J. Biol. Chem. 1981; 256: 7557-7564Abstract Full Text PDF PubMed Google Scholar). The homologous pairing by Xrcc2·Rad51D was an ATP-independent reaction (Fig. 2A, lane 7), like those catalyzed by Xrcc3·Rad51C (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar), the human Rad52 protein (38.Kurumizaka H. Aihara H. Kagawa W. Shibata T. Yokoyama S. J. Mol. Biol. 1999; 291: 537-548Crossref PubMed Scopus (62) Google Scholar, 39.Kagawa W. Kurumizaka H. Ikawa S. Yokoyama S. Shibata T. J. Biol. Chem. 2001; 276: 35201-35208Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and the E. coli RecT protein (40.Noirot P. Kolodner R.D. J. Biol. Chem. 1998; 273: 12274-12280Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and was detected only with a homologous combination of ssDNA and dsDNA but not with a heterologous combination or with BSA instead of Xrcc2·Rad51D (Fig. 2, A,lane 6, and B, lane 1). The migration distance of the D-loop formed by Xrcc2·Rad51D was exactly the same as that of the control D-loop formed by RecA (Fig. 2B,lanes 3 and 4). These data clearly show that the products formed by Xrcc2·Rad51D were authentic D-loops. A low MgCl2 condition (2 mm) was preferred in the homologous pairing promoted by Xrcc2·Rad51D as compared with a high MgCl2 condition (10 mm) (Fig. 2B,lanes 4 and 5), and the activity observed with the low MgCl2 condition was about 20% of that observed with RecA at its optimum MgCl2 condition (10 mm) (Fig. 2C). In the Superdex 200HR gel filtration chromatography, Xrcc2·Rad51D was eluted near the void volume, and no proteins were detected in the fractions corresponding to the molecular size (71 kDa) of the Xrcc2·Rad51D heterodimers (Fig. 3). This result indicates that Xrcc2·Rad51D exists as a multimer rather than a heterodimer. Therefore, we analyzed the structure of the Xrcc2·Rad51D multimer by electron microscopy. As shown in Fig. 4, Xrcc2·Rad51D formed multimeric ring structures (Fig. 4, A–E). The ring structure formed by Xrcc2·Rad51D is similar to that of HsRad52 in its size and shape (39.Kagawa W. Kurumizaka H. Ikawa S. Yokoyama S. Shibata T. J. Biol. Chem. 2001; 276: 35201-35208Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar,41.Stasiak 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).Figure 4Electron microscopic images of Xrcc2·Rad51D.A–E, images of Xrcc2·Rad51D in the absence of DNA. All images were photographed by negative staining with uranyl acetate. The white and black bars denote 50 and 10 nm, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We tested the DNA binding activity of Xrcc2·Rad51D. Xrcc2·Rad51D bound both ssDNA and dsDNA (Fig. 5, A and B). Although significant amounts of ssDNA and dsDNA were still bound to Xrcc2·Rad51D in the absence of ATP, agarose gel electrophoresis revealed that the migration distances of the complexes between Xrcc2·Rad51D and DNA were drastically changed by the addition of ATP (Fig. 5, A, lane 5, and B, lane 5). These results suggest that less Xrcc2·Rad51D bound to ssDNA in the absence of ATP. Both Xrcc2 and Rad51D have an ATP-binding motif. It has been reported that disruptions of the ATP-binding motif of Xrcc2 did not affect the complementation of the phenotypes of the Xrcc2-deficient cell line, irs1 (42.O'Regan P. Wilson C. Townsend S. Thacker J. J. Biol. Chem. 2001; 276: 22148-22153Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The ATP may be important in regulating the DNA binding of Rad51D or may bind different sites from the ATP-binding motifs of Xrcc2 and Rad51D. Further studies are required to elucidate the roles of ATP binding by Xrcc2·Rad51D. In the absence of DNA, Xrcc2·Rad51D formed a multimeric ring structure (Fig. 4), and no filamentous structure was observed. However, an electron microscopic analysis showed that Xrcc2·Rad51D formed filamentous structures in the presence of ssDNA (Fig. 6A). To compare the Xrcc2·Rad51D filament with the HsRad51 filament, we visualized the HsRad51·ssDNA complex under the same conditions (Fig. 6B) used for the Xrcc2·Rad51D filament observation. As shown in Fig. 6B, HsRad51 formed short nucleoprotein filaments with ssDNA, but the length of the filaments were shorter than that of the Xrcc2·Rad51D filament, which fully covered circular ssDNA (Fig. 6A). The ssDNA circle (5386 bases) covered with Xrcc2·Rad51D was significantly smaller than the same length of the dsDNA circle (5386 base pairs) covered by HsRad51 (Fig. 6, Aand C). Thus, the DNA bound to Xrcc2·Rad51D may be packaged differently from that bound to HsRad51, possibly wrapping around the Xrcc2·Rad51D ring as in the case of HsRad52 (43.Parsons C.A. Baumann P. Van Dyck E. West S.C. EMBO J. 2000; 19: 4175-4181Crossref PubMed Scopus (72) Google Scholar). Although both HsRad51 and Xrcc2·Rad51D formed filamentous complexes in the presence of ssDNA, the appearance of those filaments was different. HsRad51 formed a nucleoprotein filament, which was clearly helical in appearance (Fig. 6, B and C). This helical filament is strikingly similar to that of RecA (44.Flory J. Tsang S.S. Muniyappa K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7026-7030Crossref PubMed Scopus (108) Google Scholar). In contrast, the filaments formed by Xrcc2·Rad51D did not have a RecA-like helical appearance (Fig. 6A) and interestingly, did have significant similarities with those formed by Xrcc3·Rad51C (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar) or HsRad52 (39.Kagawa W. Kurumizaka H. Ikawa S. Yokoyama S. Shibata T. J. Biol. Chem. 2001; 276: 35201-35208Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). It has been reported that Xrcc2 is required for embryonic neurogenesis (33.Deans B. Griffin C.S. Maconochie M. Thacker J. EMBO J. 2000; 19: 6675-6685Crossref PubMed Scopus (197) Google Scholar). Both Xrcc2 and Xrcc3are expressed in the brain (12.Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar), where the expression ofRad51 is very low (5.Shinohara A. Ogawa H. Matsuda Y. Ushio N. Ikeo K. Ogawa T. Nat. Genet. 1993; 4: 239-243Crossref PubMed Scopus (30) Google Scholar). Our present and previous two-hybrid analyses have shown that Xrcc2 and Xrcc3 interact with Rad51D and Rad51C, respectively, in the human brain cDNA library (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar). These facts suggest that Xrcc2·Rad51D and Xrcc3·Rad51C may have a specific function in the brain. In nondividing neuronal cells, the homologous chromosomes, not the sister chromatids, must be the templates for homologous pairing, because the cells are arrested before DNA synthesis (the G0 phase). Xrcc2·Rad51D and Xrcc3·Rad51C may have a preference for homologous pairing between homologous chromosomes rather than sister chromatids. Both Xrcc2 and Xrcc3 are required independently for HRR in mammalian cells, because no other proteins can complement their mutant phenotypes. These facts suggest that Xrcc2· Rad51D and Xrcc3·Rad51C are functionally similar but have different roles in the HRR pathway. Schild et al. (35.Schild D. Lio Y. Collins D.W. Tsomondo T. Chen D.J. J. Biol. Chem. 2000; 275: 16443-16889Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) detected an indirect interaction between Xrcc2 and Rad51C by the three-hybrid system in the presence of Rad51D, suggesting a weak interaction between Rad51C and Xrcc2·Rad51D. This interaction may function to form a multiprotein complex containing all four proteins onto the DSB site. Recently, it was reported that Xrcc2·Rad51D interacts with Rad51B and Rad51C, forming a complex distinct from that of Xrcc3·Rad51C (45.Masson J.-Y. Tarsounas M.C. Stasiak A.Z. Stasiak A. Shah R. McIlwraith M.J. Benson F.E. West S.C. Genes Dev. 2001; 15: 3296-3307Crossref PubMed Scopus (299) Google Scholar,46.Miller K.A. Yoshikawa D.M. McConnell I.R. Clark R. Schild D. Albala J.S. J. Biol. Chem. 2002; 277: 8406-8411Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Xrcc2·Rad51D may be a part of the complex containing Xrcc2, Rad51B, Rad51C, and Rad51D. Further analyses are required to elucidate the biological significance of these homologous pairing activities in the human system. The Xrcc2, Xrcc3, Rad51C, andRad51D genes are also expressed in dividing cells, where theRad51 expression is observed (12.Liu N. Lamerdin J.E. Tebbs R.S. Schild D. Tucker J. Shen M.R. Brookman K.W. Siciliano M.J. Walter C.A. Fan W. Narayana L.S. Zhou Z. Adamson A.W. Sorensen K.J. Chen D.J. Jones N.J. Thompson L.H. Mol. Cell. 1998; 1: 783-793Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 17.Dosanjh M.K. Collins D.W. Wufang F. Lennon G.G. Albala J.S. Shen Z. Schild D. Nucleic Acids Res. 1998; 26: 1179-1184Crossref PubMed Scopus (145) Google Scholar, 19.Pittman D.L. Weinberg L.R. Schimenti J.C. Genomics. 1998; 49: 103-111Crossref PubMed Scopus (113) Google Scholar). Interestingly, the mutant cell lines lacking one of the Xrcc2,Xrcc3, Rad51C, and Rad51D genes significantly attenuated the Rad51 assembly on chromosomes after exposure to γ-rays (28.Takata M. Sakaki M.S. Tachiiri S. Fukushima T. Sonoda E. Schild D. Thompson L.H. Takeda S. Mol. Cell. Biol. 2001; 21: 2858-2866Crossref PubMed Scopus (462) Google Scholar, 47.Bishop D.K. Ear U. Bhattacharyya A. Calderone C. Beckett M. Weichselbaum R.R. Shinohara A. J. Biol. Chem. 1998; 273: 21482-21488Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). These results suggest that Xrcc2·Rad51D and Xrcc3·Rad51C are assembled on the damaged sites of chromosomes before Rad51 and that they initiate homologous pairing in cooperation with Rad51 in dividing cells. In addition to the similarity between Xrcc2·Rad51D and Xrcc3·Rad51C, we also found significant similarity between Xrcc2·Rad51D and HsRad52, an important protein in the HRR pathway. HsRad52 is known to form a multimeric ring structure similar to that of Xrcc2·Rad51D (41.Stasiak 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). Furthermore, we found that HsRad52 also formed nucleoprotein filaments with ssDNA and catalyzed homologous pairing as did Xrcc2·Rad51D and Xrcc3·Rad51C (10.Kurumizaka H. Ikawa S. Nakada M. Eda K. Kagawa W. Takata M. Takeda S. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5538-5543Crossref PubMed Scopus (111) Google Scholar, 39.Kagawa W. Kurumizaka H. Ikawa S. Yokoyama S. Shibata T. J. Biol. Chem. 2001; 276: 35201-35208Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). These results indicate that the characteristics of HsRad52 are similar to those of the Rad51 paralogs, although HsRad52 lacks obvious sequence homology with any of the Rad51 paralogs. The multiple homologous pairing activities may be necessary to support the HRR pathway in human chromosomes, which are more complex than those of bacteria and unicellular organisms such as yeast. We are grateful to Keiko Eda for technical assistance and to Drs. Naoshi Domae and Koji Takio for protein sequencing. We thank Drs. Shunichi Takeda (Kyoto University) and Minoru Takata (Kawasaki Medical School) for the in vivo analysis with chicken DT40 cells." @default.
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• W1993640924 title "Homologous Pairing and Ring and Filament Structure Formation Activities of the Human Xrcc2·Rad51D Complex" @default.
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