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Physical and Functional Mapping of the Replication Protein A Interaction Domain of the Werner and Bloom Syndrome Helicases

2005; Elsevier BV; Volume: 280; Issue: 33 Linguagem: Inglês

10.1074/jbc.m500653200

1083-351X

Kevin M. Doherty, Joshua A. Sommers, Matthew D. Gray, Jae Wan Lee, Cayetano von Kobbe, Nicolas H. Thomä, Raichal P. Kureekattil, Mark K. Kenny, Robert Brosh,

Genomics and Chromatin Dynamics

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRNH-R) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRNH-R helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRNH-R had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM642–1290, that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding. The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRNH-R) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRNH-R helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRNH-R had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM642–1290, that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding. Within the last decade, several genetic disorders with premature aging and/or cancer have been identified in which a gene member of the RecQ helicase family is mutated (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (313) Google Scholar, 2Harrigan J.A. Bohr V.A. Biochimie (Paris). 2003; 85: 1185-1193Crossref PubMed Scopus (41) Google Scholar). RecQ helicases share a centrally located domain of ∼450 residues that contains the seven conserved helicase motifs (for review, see Ref. 3Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (580) Google Scholar). The founding member of the RecQ family, Escherichia coli RecQ helicase, has been extensively studied biochemically and has been genetically implicated in DNA recombination. A single yeast RecQ helicase, Sgs1 or Rqh1, is found in the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, respectively, and these helicases are thought to be important in the cellular response to DNA-damaging agents and maintenance of genome stability. RecQ helicases have also been identified in a number of higher eukaryotes, including Xenopus laevis (focus forming activity 1 (FFA-1) 1The abbreviations used are: FFA-1, focus forming activity 1; WRN, Werner syndrome helicase; BLM, Bloom syndrome helicase; WS, Werner syndrome; BS, Bloom syndrome; RQC, helicase domain designated RecQ-Ct; HRD, helicase-related domain; RPA, replication protein A; 3-AT, 3-amino-1,2,4-triazole; GST, glutathione S-transferase; MBP, maltose-binding protein; ELISA, enzyme-linked immunosorbent assay; ssDNA, single-stranded DNA; BSA, bovine serum albumin.), Drosophila melanogaster (DmBLM and DmRecQ5), and Caenorhabditis elegans (WRN-1, Ce-RCQ5, HIM-6, and RECQL/Q1). These helicases have proposed functions in DNA replication or repair; however, the precise details of their roles in cellular pathways of DNA metabolism are still under investigation. There are five human RecQ helicases: 1) WRN, defective in Werner syndrome (WS); 2) BLM, defective in Bloom syndrome (BS); 3) RECQL4, defective in Rothmund-Thomson syndrome and RAPADILINO; 4) RECQ1; and 5) RECQ5. The human RecQ helicase disorders have distinctly different clinical phenotypes; however, WS, BS, and Rothmund-Thomson syndrome are all characterized by genomic instability, an elevated cancer incidence, and/or particular aspects of premature aging. Human diseases have not yet been genetically linked to mutations in RECQ1 or RECQ5. The prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 and RECQ5 helicases are also likely to be important in vivo. In addition to the conserved helicase motifs, the majority of RecQ helicases have a second conserved region of ∼80 amino acids located C-terminal to the helicase domain designated RecQ-Ct (RQC) (4Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends. Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar). Although the conserved helicase domain is responsible for coupling nucleotide hydrolysis to DNA unwinding, recent studies suggest that the RQC motif plays an important role in protein interactions (5Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Crossref PubMed Scopus (68) Google Scholar), nucleolar localization (6von Kobbe C. Bohr V.A. J. Cell Sci. 2002; 115: 3901-3907Crossref PubMed Scopus (68) Google Scholar), and/or DNA binding (7von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The WRN protein is unique among the human RecQ helicases in that it also contains a region of homology to the 3′ to 5′ exonuclease domain of proofreading enzymes (8Yu 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 (1496) Google Scholar). Interestingly, the WRN protein has a direct repeat of a highly acidic 27-amino acid sequence located in the N terminus between the exonuclease and helicase domains (8Yu 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 (1496) Google Scholar). Although the exact functional significance of this repeated sequence is not known, and only one copy of the sequence is found in Xenopus FFA-1, this acidic repeat has been implicated in studies of the human WRN protein to play a role in transcriptional activation (9Balajee A.S. Machwe A. May A. Gray M.D. Oshima J. Martin G.M. Nehlin J.O. Brosh Jr., R.M. Orren D.K. Bohr V.A. Mol. Biol. Cell. 1999; 10: 2655-2668Crossref PubMed Scopus (118) Google Scholar). A C-terminal conserved motif designated helicase-related domain (HRD), found in a number of DNA-metabolizing proteins, is present in many RecQ helicases (4Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends. Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar), and recent evidence suggests that the domain is important in DNA binding (7von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Biochemical analyses of the purified recombinant WRN protein from a number of laboratories, including ours, have characterized the catalytic activities and protein interactions of WRN (for review, see Refs. 5Brosh Jr., R.M. Bohr V.A. Exp. Gerontol. 2002; 37: 491-506Crossref PubMed Scopus (68) Google Scholar and 10Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar). WRN protein has been shown to unwind relatively short DNA duplexes of 20–30 bp in the absence of any auxiliary factors with a 3′ to 5′ polarity (11Gray 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 (523) Google Scholar, 12Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar, 13Suzuki N. Shimamoto A. Imamura O. Kuromitsu J. Kitao S. Goto M. Furuichi Y. Nucleic Acids Res. 1997; 25: 2973-2978Crossref PubMed Scopus (195) Google Scholar). However, WRN is a relatively poor helicase on longer DNA duplexes such as a 34-bp forked duplex (14Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M.M. Bohr V.A. J. Biol. Chem. 2001; 276: 44677-44687Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) or 69-bp M13 partial duplex DNA substrate (15Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), suggesting that the enzyme has limited processivity in the unwinding reaction. WRN helicase can catalyze unwinding of longer DNA duplexes up to 851 bp in a reaction dependent on replication protein A (RPA) (15Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), a single-stranded DNA-binding protein that is implicated in the processes of DNA replication, recombination and repair, and transcription (16Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar). Consistent with the functional interaction between WRN and RPA, the two proteins physically interact (15Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 17Shen J.C. Lao Y. Kamath-Loeb A. Wold M.S. Loeb L.A. Mech. Ageing Dev. 2003; 124: 921-930Crossref PubMed Scopus (50) Google Scholar); however, the mechanism for stimulation of WRN-catalyzed DNA unwinding is not well understood. To address the mechanism for RPA stimulation of WRN helicase activity, we have begun to evaluate the importance of the physical interaction between WRN and RPA for the stimulation of helicase activity. In this work, we have mapped the RPA binding sites on WRN protein using multiple approaches and identified a high affinity RPA interaction site in the N terminus. Biochemical analyses of a purified recombinant WRN protein fragment, WRNH-R, that contains the helicase core domain and RQC motif, but lacks the N-terminal region and C-terminal regions of WRN, retains its ability to unwind short (28 bp) M13 partial duplexes nearly as efficiently as full-length WRN helicase; however, WRNH-R is severely compromised in its ability to unwind longer (50–850 bp) DNA substrates that the full-length WRN protein can efficiently unwind in the presence of RPA. These results demonstrate that WRN helicase binds tightly to RPA via its N-terminal domain and suggest that this physical protein interaction with RPA is likely to be important in the mechanism of RPA-dependent stimulation of WRN helicase activity. In addition to WRN, human BLM (18Brosh Jr., R.M. Li J.L. Kenny M.K. Karow J.K. Cooper M.P. Kureekattil R.P. Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), RECQ1 (19Cui S. Arosio D. Doherty K.M. Brosh Jr., R.M. Falaschi A. Vindigni A. Nucleic Acids Res. 2004; 32: 2158-2170Crossref PubMed Scopus (96) Google Scholar), and RECQ5β (20Garcia P.L. Liu Y. Jiricny J. West S.C. Janscak P. EMBO J. 2004; 23: 2882-2891Crossref PubMed Scopus (171) Google Scholar) helicases also physically and functionally interact with RPA, but the details of how RPA stimulates the unwinding activities of these DNA helicases are also largely unknown. Similar to our findings with WRN, we report here that BLM helicase contains a high affinity N-terminal RPA interaction site. A BLM recombinant fragment lacking this RPA interaction site, but containing the RQC and HRD motifs, was shown to be defective in unwinding long DNA duplex substrates despite the presence of RPA. Our studies indicate that both WRN and BLM helicases contain an N-terminal RPA binding domain that is important for the stimulation of helicase activity by RPA. The important cellular roles of RPA in DNA metabolism are likely to involve specific interactions with human RecQ helicases in pathways that maintain genome stability. Yeast Two-hybrid Reporter Assay—A two-hybrid screen was performed as previously described (9Balajee A.S. Machwe A. May A. Gray M.D. Oshima J. Martin G.M. Nehlin J.O. Brosh Jr., R.M. Orren D.K. Bohr V.A. Mol. Biol. Cell. 1999; 10: 2655-2668Crossref PubMed Scopus (118) Google Scholar). Briefly, L40 S. cerevisiae reporter strain, pBTM116 (LexA DNA-binding domain) and pVP16 (herpes simplex virus VP16 transcriptional activation domain) fusion vectors were kindly provided by Dr. Stanley Hollenberg. Yeast media were purchased from BIO 101 (La Jolla, CA), and 3-amino-1,2,4-triazole (3-AT) was from Sigma. LexA-WRN fusion protein constructs were created using the expression vector pBTM116 (21Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). This plasmid carries the TRP1 gene for selection in yeast and a multiple cloning site inserted after the 202-amino acid open reading frame of the E. coli lexA gene, which encodes an SOS function regulatory protein and is driven by a constitutive yeast ADH promoter in this vector. In-frame fusions between the C terminus of the LexA protein and specified domains of the human WRN protein were created by insertion of PCR-generated WRN cDNA domains into the multiple cloning sites of pBTM116 as previously described (6von Kobbe C. Bohr V.A. J. Cell Sci. 2002; 115: 3901-3907Crossref PubMed Scopus (68) Google Scholar). Briefly, the full-length WRN cDNA fusion, construct WRN+, was created by insertion of the unique BamHI-SspI WRN cDNA fragment into the BamHI and SalI (filled in) sites of WRN domain within pBTM116. To create WRN–1/2, a single copy of the acidic 27-amino acid repeat was removed from construct WRN+ by deletion of the unique AflII-AflII fragment of the WRN cDNA, which occurs within the repeated sequence. To create the full deletion of the repeated sequence, WRN–R, a PCR fragment was generated from WRN cDNA using primers TH1 (5′-GCC AGA TCT TGG AAA CAA CTG CAC-3′) and THΔR (5′-CGG CTT AAG CTC AGT AGA TTT A-3′), which was digested with BamHI and AflII and then substituted for the BamHI-AflII WRN fragment of construct WRN–1/2. This PCR product results in the addition of a novel AflII site located just before the acidic repeat sequence, which becomes fused upon ligation to the distal AflII site within the endogenous WRN message. To create the WRN+, WRN–1/2, and WRN–R fusion protein constructs, regions of the wild-type or deleted repeat domains were amplified directly from the corresponding full-length constructs as previously described (9Balajee A.S. Machwe A. May A. Gray M.D. Oshima J. Martin G.M. Nehlin J.O. Brosh Jr., R.M. Orren D.K. Bohr V.A. Mol. Biol. Cell. 1999; 10: 2655-2668Crossref PubMed Scopus (118) Google Scholar). These PCR products were then inserted into the pBTM116 vector at the chosen restriction sites. VP16-WRN fusion protein constructs were created using the expression vector pVP16 (21Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). This plasmid carries the LEU2 gene for selection in yeast and a multiple cloning site inserted after the herpes simplex virus VP16 transcriptional activation domain. In-frame fusions between the C terminus of the VP16 protein, and specified domains of the human WRN protein were created by insertion of PCR-generated WRN cDNA domains into the multiple cloning sites of pVP16. RPA fusion protein constructs were created by PCR amplification of individual full-length RPA protein subunits (cDNAs were kindly provided by Dr. Marc Wold) using primers containing restriction sites compatible with pBTM116 (for LexA fusions) or pVP16 (for VP16 fusions) multiple cloning sites. For C-terminal truncated versions of the RPA70 subunit, stop codons were introduced within PCR reverse primers directly after the specified amino acid codon or, in the case of the N-terminal truncation, the forward primer initiated at the indicated internal amino acid position within the cDNA sequence. All fusion protein constructs were confirmed by manual sequencing to be in-frame and devoid of mutations. For two-hybrid assays, S. cerevisiae LexA reporter strain L40 (MATa his3Δ200 trp1–901 leu2–3112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-LacZ GAL4 gal80) was co-transformed with the indicated pairs of LexA- and VP16 fusion protein constructs or control vectors. To assess the relative reporter activities of each pair, positive co-transformants were selected on yeast media plates lacking tryptophan and leucine, and multiple colonies of each pairing were restreaked to templates for even growth (2–3 days on the same media). Co-transformant templates were then replica-plated onto yeast media plates lacking tryptophan, leucine, and histidine and containing 0–500 mm 3-AT. Replica plating was performed in reverse order (i.e. from high 3-AT concentration to low) to ensure efficient transfer throughout the series. Relative growth on 3-AT-containing plates was assessed after 10 days of incubation at 30 °C. All reporter assays were repeated in at least three different experiments. Reporter strength was essentially invariable within multiple co-transformants (original colonies isolated) of each construct pair in all 3-AT growth experiments. Proteins—Recombinant hexahistidine-tagged WRN protein was overexpressed using a baculovirus/Sf9 insect cell system and purified as described previously (22Sharma S. Otterlei M. Sommers J.A. Driscoll H.C. Dianov G.L. Kao H.I. Bambara R.A. Brosh Jr., R.M. Mol. Biol. Cell. 2004; 15: 734-750Crossref PubMed Scopus (112) Google Scholar). Glutathione S-Transferase (GST) fusion proteins of WRN were subcloned into the bacterial expression plasmid pGEXCS (Amersham Biosciences) as described elsewhere (23Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). Inframe fusions of GST-2R (WRN residues 406–525) or GST-0R (WRN residues 406–525 with intervening residues 424–475 deleted) were created by inserting the PCR products, generated in the same way as for the two-hybrid system, into the BamHI site within the multiple cloning site of the pGEXCS vector. GST-WRN fragment fusion proteins were expressed and purified as described previously (23Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). A recombinant WRN protein fragment (exact boundaries to be described elsewhere), 2Nicolas H. Thoma, submitted for publication. designated WRNH-R, was overexpressed using a baculovirus insect cell system and purified using immobilized glutathione beads. WRNH-R contains the entire helicase domain (motifs I–VI) and the conserved RQC region but lacks the N-terminal sequence, including the acidic repeats as well as the C-terminal region after the RQC motif (Fig. 5A). Hexahistidine-tagged recombinant BLM protein, was overexpressed in S. cerevisiae and purified as previously described (24Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Expression constructs for maltose-binding protein (MBP) fusion proteins of BLM fragments (25Wu 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 (284) Google Scholar) and the BLM642–1290 protein fragment were kindly provided by Dr. Ian Hickson (Cancer Research UK Laboratories). MBP-BLM fusion proteins were purified as previously described (25Wu 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 (284) Google Scholar). Briefly, MBP-BLM fusion proteins were expressed in BL21(DE3) cells (New England Biolabs) that had been transformed with the pMAL-C2 expression plasmids containing various portions of the BLM cDNA (MBP-BLM1–447 and MBP-BLM966–1417). BL21(DE3) cells transformed with plasmid encoding MBP were used for control experiments. Overnight transformed bacterial cultures were used to inoculate 1 liter of Luria Bertani broth supplemented with 2% glucose and 100 μg/ml ampicillin at a 1:100 dilution, and the cultures were grown at 37 °C to an A600 of ∼0.5. Isopropyl-1-thio-β-d-galactopyranoside was then added to a final concentration of 0.4 mm, and the cultures were allowed to grow for an additional 3 h before being chilled on ice for 30 min. After centrifugation at 10,000 rpm in a Beckman JA10 rotor, the cell pellet was resuspended in 50 ml of column buffer (20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1 mm EDTA, 10 mm β-mercaptoethanol, 1 mm dithiothreitol) supplemented with complete protease inhibitor mixture (Roche Applied Science) at the manufacturer's recommended concentration. Cells were lysed by sonication, and the lysate was clarified by centrifugation at 9,000 × g in a Beckman Ti-70 rotor for 30 min at 4 °C. The crude extract was diluted 1:5 with column buffer and then bound to amylose resin (New England Biolabs) that had been pre-washed with column buffer. The resin was washed with 12 volumes of column buffer before the recombinant protein was eluted from the resin with column buffer supplemented with 10 mm maltose. RPA containing all three subunits (RPA70, RPA32, and RPA14) was purified as previously described (26Kenny M.K. Schlegel U. Furneaux H. Hurwitz J. J. Biol. Chem. 1990; 265: 7693-7700Abstract Full Text PDF PubMed Google Scholar). GST-WRN-Sepharose Pull-down Experiments—GST-WRN pull-down experiments were performed as previously described (23Brosh Jr., R.M. von Kobbe C. Sommers J.A. Karmakar P. Opresko P.L. Piotrowski J. Dianova I. Dianov G.L. Bohr V.A. EMBO J. 2001; 20: 5791-5801Crossref PubMed Scopus (228) Google Scholar). Briefly, GST-WRN fusion proteins were overexpressed in BL21(DE3)pLysS by 1 mm isopropyl-1-thio-β-d-galactopyranoside induction for 8 h at 23 °C. The bacterial cell pellet was lysed by sonication in lysis buffer (phosphate-buffered saline, 10% glycerol, 0.4% Triton X-100). The lysate was clarified by centrifugation at 35,000 rpm (Ti-60 rotor; Beckman) for 1 h at 4 °C. One milliliter of the resulting supernatant was incubated with 100 μl of GS beads (50% v/v) for 1 h at 4 °C. The beads were washed three times with 1 ml of lysis buffer, and split into two aliquots, one for binding experiments and one for determination of background signal in Western blot analysis. For binding experiments, protein-bound beads were incubated for 1 h at 4°C with 1.2 μg of purified recombinant RPA in 250 μl of buffer D (50 mm HEPES, pH 7.1, 100 mm KCl, 10% glycerol). The beads were subsequently washed three times with 500 μl of buffer D and eluted by boiling treatment in 40 μl of Laemmli buffer. Proteins were electrophoresed on 10% polyacrylamide SDS gels and transferred to polyvinylidene difluoride membranes. Control membranes were stained with Amido Black reagent to demonstrate equal loading of protein samples. Membranes were probed with mouse monoclonal anti-RPA antibody (Ab-1, 1:20, Oncogene Research Products), followed by horse anti-mouse IgG-horseradish peroxidase (Vector) and visualized using ECL-Plus (Amersham Pharmacia). ELISA Assays—The indicated purified recombinant WRN or BLM proteins (full-length or fragment) were diluted to a concentration of 18 nm in carbonate buffer (0.016 m Na2CO3, 0.034 m NaHCO3, pH 9.6) and were added to individual wells of a 96-well microtiter plate (50 μl/well), which was then incubated at 4 °C overnight. Bovine serum albumin (BSA) was used in the coating step for control reactions. After the initial binding step, samples were aspirated and the wells were blocked for 2 h at 30 °C with blocking buffer (phosphate-buffered saline, 0.5% Tween 20, and 3% BSA). After washing, RPA was diluted to 144 nm in blocking buffer and was added to the appropriate wells of the ELISA plate (50 μl/well), which was then incubated for 1 h at 30 °C. The samples were aspirated, and the wells were washed five times before addition of anti-RPA (Ab-1) mouse monoclonal antibody (Oncogene Research Products) diluted 1:100 in blocking buffer, followed by incubation at 30 °C for 1 h. Following three washes, horseradish peroxidase-conjugated anti-mouse secondary antibody (1:2500) diluted in blocking buffer was added to the wells, and the samples were incubated for 30 min at 30 °C. After washing five times, RPA bound to the proteins was detected using O-phenylenediamine substrate (Sigma). The reaction was terminated after 5 min with 3 n H2SO4, and absorbance readings were taken at 490 nm. Data analysis for determination of the apparent dissociation constant (Kd) was performed as previously described (18Brosh Jr., R.M. Li J.L. Kenny M.K. Karow J.K. Cooper M.P. Kureekattil R.P. Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; 275: 23500-23508Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Helicase Substrate Preparation—M13mp18 single stranded circular DNA was from New England Biolabs. The oligonucleotides used for the 28-, 50-, 69-, and 100-bp M13 partial duplex helicase substrates were purchased from Midland Certified Reagents Co. (Midland, TX). The 28-, 50-, 69-, 100-, and 849-bp M13mp18 partial duplex substrate were constructed using oligonucleotides complementary to positions 6296–6323, 6272–6322, 6254–6322, and 6039–6138, respectively, in the M13mp18 ssDNA circle. The 849-bp fragment, in which one strand was complementary to positions 1396–2244 of the M13mp18 ssDNA circle was amplified by PCR with the primers 5′-CCTTTAACTCCCTGCAAGCC-3′ and 5′-AAACAAACACTTATAGTTCC-3′. The M13mp18 partial duplex substrates were radioactively labeled and constructed as previously described (15Brosh Jr., R.M. Orren D.K. Nehlin J.O. Ravn P.H. Kenny M.K. Machwe A. Bohr V.A. J. Biol. Chem. 1999; 274: 18341-18350Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The 19-bp forked duplex substrate was radioactively labeled and constructed as previously described (27Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Helicase Assays—Helicase assay reaction mixtures (20 μl) using the M13 partial duplex substrates contained 40 mm Tris (pH 7.4), 4 mm MgCl2, 5 mm dithiothreitol, 2 mm ATP, 0.5 mg/ml tRNA, and the indicated concentration of WRN or BLM protein. For assays using the 19-bp forked duplex substrate, reaction mixtures (20 μl) contained 30 mm HEPES (pH 7.5), 40 mm KCl, 8 mm MgCl2, 100 ng/μl BSA, 5% glycerol, and DNA substrate concentrations indicated in the figure legends. Reaction mixtures were initiated by the addition of WRN or BLM protein. Helicase reactions were incubated at 37 °C and were terminated by the addition of 10 μl of 50 mm EDTA-40% glycerol-0.9% SDS-0.1% bromphenol blue-0.1% xylene cyanol at time points indicated in the figure legends for M13 partial duplex substrates or 37 °C for 15 min using the 19-bp forked duplex substrate. The products of helicase reactions were resolved on nondenaturing 10% or 12% polyacrylamide gels for the reactions with M13 partial duplex (28, 50, 69, and 100 bp) or oligonucleotide based forked duplex substrates, respectively.