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• W2023563387 abstract "Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directlyin vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex. The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast. The results indicate that the NH2-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3. In vitro assays using purified Top3 and NH2-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids. Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH2 terminus can be suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complexin vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction. Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directlyin vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex. The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast. The results indicate that the NH2-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3. In vitro assays using purified Top3 and NH2-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids. Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH2 terminus can be suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complexin vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction. DNA topoisomerase III methyl methanesulfonate hydroxyurea enzyme-linked immunosorbent assay hemagglutinin glutathione S-transferase dithiothreitol polyacrylamide gel electrophoresis immunoprecipitation radioimmunoprecipitation assay phosphate-buffered saline replication protein A fluoroorotic acid The Saccharomyces cerevisiae SGS1 gene encodes a member of the RecQ family of DNA helicases. In addition to the RecQ protein ofEscherichia coli, this family includes the human BLM, WRN, RECQL4, and RECQ5 proteins as well as Rqh1 fromSchizosaccharomyces pombe (1Ellis N.A. Groden J. Ye T.-Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1211) Google Scholar, 2Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1489) Google Scholar, 3Umezu K. Nakayama H. J. Mol. Biol. 1993; 230: 1145-1150Crossref PubMed Scopus (82) Google Scholar, 4Shimamoto A. Nishikawa K. Kitao S. Furuichi Y. Nucleic Acids Res. 2000; 28: 1647-1655Crossref PubMed Google Scholar, 5Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (574) Google Scholar, 6Murray J.M. Lindsay H.D. Munday C.A. Carr A.M. Mol. Cell. Biol. 1997; 17: 6868-6875Crossref PubMed Scopus (172) Google Scholar, 7Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (329) Google Scholar). These proteins play an important role in DNA metabolism as mutations in the human genes give rise to diseases characterized by genome instability and a predisposition to cancer. Werner's syndrome cells, which result from mutations in WRN (2Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1489) Google Scholar), display a genomic instability termed variegated translocation mosaicism (8Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (212) Google Scholar). Bloom's syndrome cells, which result from mutations in BLM (1Ellis N.A. Groden J. Ye T.-Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1211) Google Scholar), are characterized by increased rates of sister chromatid exchange and sensitivity to DNA-damaging agents (9Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4508-4512Crossref PubMed Scopus (786) Google Scholar). Mutations in RECQL4 are found in a subset of Rothmund-Thomson syndrome cases. These cells are characterized by elevated rates of chromosomal breaks and rearrangements (5Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (574) Google Scholar, 10Miozzo M. Castorina P. Riva P. Dalpra L. Fuhrman Conti A.M. Volpi L. Hoe T.S. Khoo A. Wiegant J. Rosenberg C. Larizza L. Int. J. Cancer. 1998; 77: 504-510Crossref PubMed Scopus (55) Google Scholar). All members of this family contain a COOH-terminal domain with homology to RecQ, and all those that have been tested exhibit a 3′- to 5′-DNA helicase activity (11Umezu K. Nakayama K. Nakayama H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5363-5367Crossref PubMed Scopus (229) Google Scholar, 12Lu J. Mullen J.R. Brill S.J. Kleff S. Romeo A. Sternglanz R. Nature. 1996; 383: 678-679Crossref PubMed Scopus (138) Google Scholar, 13Bennett R.J. Sharp J.A. Wang J.C. J. Biol. Chem. 1998; 273: 9644-9650Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 14Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 15Gray M.D. Shen J.C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (519) Google Scholar). In addition to the helicase domain, the eukaryotic proteins contain a large NH2-terminal domain of about 650 amino acids whose sequence is poorly conserved between members. The NH2-terminal domain is important for activity in yeast (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar), but with the exception of the 3′- to 5′-exonuclease domain of WRN (17Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Bork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (217) Google Scholar, 18Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (377) Google Scholar) the biochemical function of the NH2-terminal domain is unknown. A subset of the eukaryotic RecQ family members has been shown to interact with DNA topoisomerase III (Top3)1 (19Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar, 20Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 21Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (98) Google Scholar, 22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar). Eukaryotic Top3 was first identified as a hyperrecombination mutant in yeast that also displayed a slow growth phenotype (23Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (453) Google Scholar). Top3 has since been identified in several organisms including S. pombe (21Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (98) Google Scholar, 24Maftahi M. Han C.S. Langston L.D. Hope J.C. Zigouras N. Freyer G.A. Nucleic Acids Res. 1999; 27: 4715-4724Crossref PubMed Scopus (69) Google Scholar),Caenorhabditis elegans (25Kim Y.C. Lee J. Koo H.S. Nucleic Acids Res. 2000; 28: 2012-2017Crossref PubMed Scopus (17) Google Scholar), and humans (26Hanai R. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3653-3657Crossref PubMed Scopus (123) Google Scholar, 27Ng S.W. Liu Y. Hasselblatt K.T. Mok S.C. Berkowitz R.S. Nucleic Acids Res. 1999; 27: 993-1000Crossref PubMed Scopus (44) Google Scholar). Like the bacterial enzyme, eukaryotic Top3 is a type I 5′-DNA topoisomerase with weak superhelical relaxing activity and a strict requirement for substrates containing single-stranded DNA or strand-passing activity (28Kim R.A. Wang J.C. J. Biol. Chem. 1992; 267: 17178-17185Abstract Full Text PDF PubMed Google Scholar, 29DiGate R.J. Marians K.J. J. Biol. Chem. 1988; 263: 13366-13373Abstract Full Text PDF PubMed Google Scholar). The biological function of Top3 is unclear, but in addition to its relaxing activity E. colitopoisomerase III is notable for its ability to decatenate gapped single-stranded DNA circles (29DiGate R.J. Marians K.J. J. Biol. Chem. 1988; 263: 13366-13373Abstract Full Text PDF PubMed Google Scholar). The recent demonstration that eukaryotic Top3 and E. coli RecQ helicase functionally interact to catenate fully duplex DNA circles (30Harmon F.G. DiGate R.J. Kowalczykowski S.C. Mol. Cell. 1999; 3: 611-620Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) suggested a role for these enzymes at the termination of DNA replication to decatenate daughter chromosomes (31Wang J.C. J. Biol. Chem. 1991; 266: 6659-6662Abstract Full Text PDF PubMed Google Scholar, 32Rothstein R. Gangloff S. Genome Res. 1995; 5: 421-426Crossref PubMed Scopus (37) Google Scholar). Although it has been suggested that RecQ helicases might function to restart stalled replication forks (7Stewart E. Chapman C.R. Al-Khodairy F. Carr A.M. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (329) Google Scholar,33Courcelle J. Hanawalt P.C. Mol. Gen. Genet. 1999; 262: 543-551Crossref PubMed Scopus (218) Google Scholar, 34Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (421) Google Scholar, 35Chakraverty R.K. Hickson I.D. Bioessays. 1999; 21: 286-294Crossref PubMed Scopus (197) Google Scholar) a role for Top3 in this process is unclear. The SGS1 gene of yeast was identified as a mutation that suppressed the slow growth phenotype of top3 mutants (22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar). Thus, in contrast to top3 strains, top3 sgs1double mutants exhibit a near wild type growth rate as well as suppression of other top3 phenotypes (22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar, 36Gangloff S. de Massy B. Arthur L. Rothstein R. Fabre F. EMBO J. 1999; 18: 1701-1711Crossref PubMed Scopus (107) Google Scholar). Compared with wild type cells the sgs1 single mutant displays increased rates of mitotic recombination, both at the ribosomal DNA locus and throughout the genome (22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar, 37Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar), as well as increased rates of chromosome loss and missegregation (38Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar). Like mutations inBLM, SGS1 mutations result in a hypersensitivity to methyl methanesulfonate (MMS) (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar) and hydroxyurea (HU) (39Yamagata K. Kato J. Shimamoto A. Goto M. Furuichi Y. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8733-8738Crossref PubMed Scopus (268) Google Scholar). SGS1 was cloned in a two-hybrid screen with TOP3, suggesting that Top3 interacted with the first 550 amino acids of Sgs1 (22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar). Because two-hybrid results do not provide evidence for direct binding, we set out to confirm this result biochemically, refine the domain of interaction, and determine whether binding was through direct protein-protein interaction. We identified an Top3·Sgs1 complex by coimmunoprecipitating and cofractionating these proteins from yeast extracts. The results indicate that Sgs1 and Top3 are present in a stable complex and that the NH2-terminal 158 amino acids of Sgs1 are sufficient for complex formation. The proteins do not appear to form a simple heterodimer, however, because the full-length proteins cofractionate at a large native molecular weight. We determined that only the NH2-terminal 158 amino acids of Sgs1 were required to bind Top3 based on an enzyme-linked immunosorbent assay (ELISA) using purified proteins. These biochemical results are consistent with our observation that phenotypes caused by mutations in the first 158 amino acids of Sgs1 can be suppressed by overexpressing Top3, whereas larger deletions cannot. Strain construction, growth, and transformation followed standard protocols (40Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990: 119-133Google Scholar). S. cerevisiae strain NJY620 expresses epitope-tagged versions of Sgs1 and Top3. This strain was constructed by modifying the chromosomalSGS1 gene of wild type strain CHY125 (41Mullen J.R. Kaliraman V. Ibrahim S.S. Brill S.J. Genetics. 2001; 157: 103-118Crossref PubMed Google Scholar) by integratingBglII-linearized plasmid pJM1526, which places three consecutive HA epitopes (YPYDVPDYA) at the COOH terminus of Sgs1. This gene and protein are henceforth called SGS1-HA and Sgs1-HA, respectively. The chromosomal TOP3 gene was modified by integrating SphI-linearized pJM2565, which places a single V5 epitope (GKPIPNPLLGLDSTRTG, Invitrogen) followed by six histidines at the COOH terminus of Top3. This gene is henceforth referred to asTOP3-V5 and its encoded protein as Top3-V5. Strain WFY822 was created by integrating pJM2565 into strain NJY531 (sgs1::loxP) (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). Strain NJY560 was constructed by deleting the SGS1 and SLX4 genes of CHY125 (41Mullen J.R. Kaliraman V. Ibrahim S.S. Brill S.J. Genetics. 2001; 157: 103-118Crossref PubMed Google Scholar) with loxP-KAN-loxP cassettes (42Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2237) Google Scholar) and maintaining the strain with plasmid pJM500 (SGS1/URA3). SGS1 andsgs1–34 were integrated at the LEU2 locus of NJY560 to create strains BSY1228 and BSY1229, respectively.SGS1 mutant phenotypes were assayed as described (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). Plasmid pJM1526, which expresses the epitope-tagged truncation Sgs1645–1447-HA, contains the insert from pSM105-HA (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar) in the vector pRS405 (43Sikorski R.S. Hieter P. Genetics. 1989; 12: 19-27Crossref Google Scholar). Plasmid pJM2565 contains a fragment of theTOP3 gene encoding a COOH-terminal in-frame fusion to the V5-His6 epitope (Invitrogen) in pRS404. To overexpress Top3 in yeast,TOP3 was subcloned downstream of the GAL1promoter in pRS424 to make pJM2566. Plasmids expressing Sgs1-HA truncations were described (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar), except for pKR1554 and pKR1555, which express epitope-tagged proteins Sgs11–158-HA and Sgs11–322-HA, respectively. To create these plasmids the first 474 and 966 base pairs of SGS1 were amplified by polymerase chain reaction so as to place an NdeI site in the context of the initiating ATG and an NotI site at the end of the coding region. These fragments were subcloned intoNdeI/NotI- digested pSM100-HA (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). For expression of recombinant yeast proteins in E. coli, TOP3-V5was subcloned into the T7-inducible vector pET11a (44Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar), yielding plasmid pSAS402. Glutathione S-transferase (GST) fusion proteins were expressed by subcloning NdeI/BamHI fragments from pKR1554 and pKR1555 into pET11GTK-WF to create pKR1564 and pKR1565. Plasmid pET11GTK-WF was created by destroying theNdeI site of pET11GTK (45Ruppert J.M. Stillman B. Mol. Cell. Biol. 1993; 13: 3811-3820Crossref PubMed Scopus (49) Google Scholar) and placing an in-frameNdeI downstream of the GST target coding region by polymerase chain reaction. Extract preparation and chromatography were performed at 4 °C. To prepare large scale extracts, yeast cells were grown in 12 liters of yeast extract-peptone-dextrose (YPD) at 30 °C to A600 = 1.5; the medium was supplemented with an additional 2% dextrose and growth continued toA600 = 2.8, which yielded 90 g of cells, wet weight. Cells were washed once with H2O and resuspended in Buffer A (25 mm Tris-HCl (pH 7.5), 1 mmEDTA, 0.01% (v/v) Nonidet P-40, 10% (v/v) glycerol, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm DTT) plus 200 mm NaCl and the following protease inhibitors: 10 μg/ml pepstatin, 5 μg/ml leupeptin, 10 mm benzamidine, 100 μg/ml bacitracin. The cells were broken in a bead-beater (Biospec Products) with 50% volume of glass beads in 30-s bursts (separated by 90-s pauses) for a total of 5 min of breakage. The lysate was centrifuged at 16,000 × g for 10 min and the resulting supernatant cleared at 235,000 × g for 90 min in a Beckman Ti45 rotor. This centrifugation was observed to pellet a significant portion of the chromatin as reported (46Frei C. Gasser S.M. Genes Dev. 2000; 14: 81-96Crossref PubMed Google Scholar). The cleared lysate was precipitated by stirring 350 mg of (NH4)2SO4 /ml of lysate for 60 min followed by centrifugation at 188,000 × g for 15 min. The pellet was resuspended in 84 ml of Buffer A and dialyzed to a conductivity of Buffer A plus 250 mm NaCl. Small scale extracts for immunoprecipitations were prepared as described (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin as a standard. Superose 6 chromatography was performed in Buffer B (25 mm Hepes-HCl (pH 7.5), 1 mm EDTA, 0.01% (v/v) Nonidet P-40, 0.1 mmphenylmethylsulfonyl fluoride, 1 mm DTT) containing 150 mm NaCl at 0.4 ml/min. Fractions were collected, precipitated with trichloroacetic acid, and resolved by 10% SDS-PAGE. Immunoprecipitations (IPs) were performed at 4 °C essentially as described (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). Unless otherwise indicated, all IPs were performed by incubating extract with 1 μl of anti-HA (Roche Molecular Biochemicals, 5 μg/μl) or anti-V5 (Invitrogen, 1 μg/μl) antibodies for 1 h in RIPA buffer (150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS) (47Harlow E. Lane D. Antibodies. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 447-470Google Scholar). 20 μl of protein-A Sepharose beads (Amersham Pharmacia Biotech) was added to each sample, followed by rocking for 1 h. The immune complexes were then washed three times with 1 ml of RIPA buffer. Following SDS-PAGE the gels were transferred to nitrocellulose membranes (48Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44922) Google Scholar) and treated with either anti-V5-horseradish peroxidase or anti-V5 as the primary antibody (1:10,000). Blots were treated with anti-mouse horseradish peroxidase conjugate secondary antibody as required (1:10,000; Life Technologies, Inc.) and developed with chemiluminescence reagents (Life Technologies, Inc.) to detect Top3-V5. Blots were reprobed with anti-HA (1:10,000) as the primary antibody and treated as above to detect Sgs1-HA. For phosphate labeling experiments, yeast cells were grown and labeled with [32P]PO4 as described (49Din S. Brill S.J. Fairman M.P. Stillman B. Genes Dev. 1990; 4: 968-977Crossref PubMed Scopus (246) Google Scholar). Extract preparation and immunoprecipitations were then performed as described above. Plasmids pET11GTK (expressing GST alone), pKR1564 (GST-Sgs11–158-HA), pKR1565 (GST-Sgs11–322-HA), and pSAS402 (Top3-V5) were transformed into E. coli BL21-RIL cells (Life Technologies, Inc.). Cells were grown by shaking in LB medium containing 0.1 mg/ml ampicillin at 37 °C to an A600 of 0.4. To induce the expression of the recombinant protein, cultures were treated with isopropyl-1-thio-d-galactopyranoside at a final concentration of 0.1 mm for 2 h at 37 °C, except for cells expressing Top3-V5, which were induced for 6 h at 20 °C. Induced cells were pelleted and resuspended in Buffer A plus protease inhibitors (above) containing 250 mm NaCl for GST and GST fusions, and 150 mm KCl for Top3. Extractions and chromatography were performed at 4 °C, except where noted. Cell suspensions were incubated with 0.1 mg/ml lysozyme for 30 min and then sonicated three times for 1 min using a Branson sonifier 450 microtip at setting 4, 60% duty cycle. Lysed cells were clarified by centrifugation at 32,500 × g and the supernatant collected as extract. GST and GST-Sgs11–158-HA proteins were purified by batch binding the extract from 1 liter of cells to 1 ml of glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) for 2 h. The resin was washed with 3 column volumes of Buffer A plus 250 mm NaCl, then half-column volume fractions were eluted at room temperature with Buffer A (pH 8.0) plus 150 mm NaCl and 10 mm glutathione. The peak fraction was determined by Bradford assay and SDS-PAGE, then 200 μl was fractionated on a Superdex 75 (Amersham Pharmacia Biotech) gel filtration column in Buffer B plus 150 mm NaCl to achieve greater purity. GST-Sgs11–322-HA extract from 2 liters of cells was diluted in Buffer A to a conductivity of Buffer A plus 50 mm NaCl and bound to an SP-Sepharose (Amersham Pharmacia Biotech) column at 20 mg of extract/ml of resin. SP-Sepharose was washed with 3 column volumes of Buffer A plus 200 mm NaCl, then GST-Sgs11–322-HA was eluted in Buffer A plus 500 mm NaCl. The resulting SP 500 mm pool was diluted in half with Buffer A and affinity purified by glutathione-Sepharose 4B and Superdex 75 chromatography as above. Top3-V5 containing extract from 3 liters of cells was bound to P-11 phosphocellulose (Whatman) at a ratio of 10 mg of extract/ml of resin in Buffer A plus 150 mm KCl. The column was washed with 3 column volumes of Buffer A plus 400 mm KCl, then Top3-V5-containing fractions were eluted from the column in Buffer A plus 600 mm KCl. Top3-V5-containing fractions were precipitated with 400 mg/ml (NH4)2SO4 for 1 h and then pelleted at 32,500 × g. The resulting Top3-V5-containing pellet was resuspended in Buffer N (25 mm Tris-HCl (pH 8.0), 0.01% (v/v) Nonidet P-40, 10% (v/v) glycerol, 0.1 mm phenylmethylsulfonyl fluoride, 250 mm NaCl) plus 20 mm imidazole and batch bound to 1.5 ml of Probond nickel resin (Invitrogen) for 4 h. Resin was poured into a column and washed with 3 column volumes of Buffer N plus 20 mm imidazole and 10 column volumes of Buffer N plus 50 mm imidazole. Top3-V5 protein was then eluted in 6 half-column volume fractions of Buffer N plus 250 mmimidazole. To detect a direct interaction between Top3 protein and the NH2 terminus of Sgs1, 15 pmol of purified GST and GST-Sgs1-HA fragments were first immobilized in DYNEX Imulon 2 HB 0.4-ml wells. Immobilization of GST and GST-Sgs1-HA fragments was carried out in 75 μl of PBS (10.1 mmNa2HPO4, 2.4 mmKH2PO4, 137 mm NaCl, 2.7 mm KCl), pH 7.2, containing 0.1% Tween 20 (PBST) and 1 mm DTT by shaking at 60 rpm for 1 h at room temperature. After immobilization, wells were washed once with 0.4 ml of PBST plus 1 mm DTT, then blocked with 0.4 ml of 5% dried milk (w/v) in PBST plus 1 mm DTT for 1 h at room temperature. After blocking, cells were washed three times with 0.4 ml of PBST plus 1 mm DTT. A titration of 0–40 pmol of Top3 protein was added to each set of coated wells in PBST plus 1 mm DTT in a volume of 75 μl and incubated for 30 min at room temperature. After incubation with Top3-V5 protein the wells were washed three times with 0.4 ml of PBST. To detect the Top3-V5 protein, 100 μl of anti-V5 antibody (diluted 1:5,000 in PBST plus 0.5% dried milk) was added to each well for 1 h at room temperature. Wells were then washed three times with PBST, and 100 μl of anti-mouse horseradish peroxidase conjugate secondary antibody (diluted 1:5,000 in PBST plus 0.75% (w/v) dried milk) was added to each well and incubated for 1 h at room temperature. After the secondary antibody incubation, wells were washed three times with 0.4 ml of PBST, and then 200 μl of 3,3′,5,5′-tetramethylbenzidine liquid substrate system for ELISA (Sigma) was added to each well and incubated for 30 min at room temperature. After incubation, 100 μl of 0.5 nH2SO4 was added to each well and theA450 of each solution read to determine the amount of Top3-V5 protein present. To characterize the interaction between Top3 and Sgs1, we constructed yeast strains whose chromosomal copies of the SGS1 andTOP3 genes were modified to express the COOH-terminally tagged proteins Sgs1-HA and Top3-V5 (see “Experimental Procedures”). These strains allowed us to immunoprecipitate and immunoblot the products of stable single-copy genes expressed under their native promoters. To verify that the epitope-tagged alleles behaved like wild type, we tested their ability to complement varioussgs1 and top3 phenotypes. Two very sensitive measures of SGS1 and TOP3 activity are resistance to the DNA-damaging agent MMS and resistance to the DNA synthesis inhibitor HU (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar). The strains expressing the tagged proteins were serially diluted and replica plated to medium containing MMS or HU. As shown in Fig. 1, the epitope-tagged strains grew as well as wild type on YPD plates and did not show the HU or MMS hypersensitivity characteristic of sgs1 ortop3 strains. For example, top3 mutants grow very slowly on YPD; SGS1 TOP3-V5 cells do not display the slow growth of SGS1 top3 cells and in fact grow at the wild type rate (data not shown). Similarly, sgs1 strains grow somewhat slower than wild type, and SGS1-HA TOP3 cells grow noticeably faster than sgs1 cells. Whereas sgs1and top3 single mutants are hypersensitive to MMS and HU (Fig. 1), the SGS1-HA and TOP3-V5 strains do not display either of these sensitivities; these strains grow like wild type in the presence of these drugs as does the SGS1-HA TOP3-V5 double-tagged strain. Based on these growth phenotypes we conclude that the epitope-tagged alleles SGS1-HA andTOP3-V5 function exactly like wild type. To identify an interaction between Sgs1 and Top3, extracts were prepared from a wild type strain and from strain NJY620 expressing Sgs1-HA and Top3-V5. Following incubation of the extracts with anti-HA or anti-V5 antibodies, the immune complexes were precipitated with protein A beads and analyzed by immunoblot. Using extracts from cells expressing the tagged proteins, we observed that anti-V5 precipitated Top3-V5, as expected, and coprecipitated Sgs1-HA (Fig.2 A, lane 6). Similarly, anti-HA precipitated Sgs1-HA, as expected, and coprecipitated Top3-V5 (Fig. 2 A, lane 4). These signals are specific to the epitope-tagged proteins as extract from the untagged wild type strain showed no bands of corresponding size. We note that under optimal conditions Top3-V5 coprecipitated Sgs1-HA more efficiently than Sgs1-HA coprecipitated Top3-V5 (Fig. 2 A, compare lanes 2 and 4 with 6 and8). The simplest explanation for this effect is that there is an excess of Top3 over Sgs1 protein in the extract. Such a result is consistent with the genetics of this system; lowering the Top3:Sgs1 ratio either by mutating TOP3 (22Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (617) Google Scholar) or by overexpressingSGS1 (16Mullen J.R. Kaliraman V. Brill S.J. Genetics. 2000; 154: 1101-1114Crossref PubMed Google Scholar) results in a profound growth defect. The previous experiment indicates that Sgs1 and Top3 interact in cell extracts but does not address the strength of the interaction or whether these proteins require DNA to interact. We addressed these questions by varying the conditions of the immunoprecipitation from nonstringent (Buffer A plus 150" @default.
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• W2023563387 title "Mapping the DNA Topoisomerase III Binding Domain of the Sgs1 DNA Helicase" @default.
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