Werner Syndrome Protein Contains Three Structure-specific DNA Binding Domains
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m308338200
ISSN1083-351X
AutoresCayetano von Kobbe, Nicolas H. Thomä, Bryan K. Czyzewski, Nikola P. Pavletich, Vilhelm A. Bohr,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoWerner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3′-5′-exonuclease, an ATPase, and a 3′-5′-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5′-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one N-terminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways. Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3′-5′-exonuclease, an ATPase, and a 3′-5′-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5′-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one N-terminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways. Werner syndrome (WS) 1The abbreviations used are: WSWerner syndromeWRNWerner syndrome proteinRQCRecQconserved domainHRDChelicase, RNase Dconserved domainssDNAsingle-stranded DNAdsDNAdouble-stranded DNABLMBloomGSTglutathione S-transferaseaaamino acidsHJHolliday junctionATPγSadenosine 5′-O-(thiotriphosphate). is a rare autosomal recessive disorder characterized by the early onset of aging symptoms, such as graying of the hair, cataracts, osteoporosis, atherosclerosis, type II diabetes mellitus, and high incidence of malignant neoplasm (1Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar). The gene mutated in WS (WRN), encodes a nuclear 1432-amino acid protein (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 (1496) Google Scholar). WRN belongs to the RecQ helicase family of proteins, which is a conserved group of proteins implicated in several aspects of DNA metabolism (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 (1496) Google Scholar, 3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (160) Google Scholar). Three of the RecQ helicases, WRN, Bloom (BLM), and Rothmund Thomsons, are associated with heritable human diseases (3Karow J.K. Wu L. Hickson I.D. Curr. Opin. Genet. Dev. 2000; 10: 32-38Crossref PubMed Scopus (160) Google Scholar). WRN is a multifunctional protein with three DNA-dependent catalytic activities: 3′-5′-exonuclease (4Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar, 5Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), ATPase, and 3′-5′-helicase (6Gray 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) (Fig. 1A). WRN is the only member of the human RecQ family known to include an exonuclease domain.Fig. 1A, functional domains of WRN and recombinant WRN fragments used in DNA binding studies. Solid lines are fragments that cover the entire WRN sequence. Dashed lines are C-terminal WRN fragments. Numbers indicate WRN amino acid coordinate. The abbreviations used are as follows: NTS, nucleolar targeting sequence. B, WRN fragments were purified and analyzed by SDS-PAGE and visualized by Coomassie Blue staining. Approximately 1 μg of each recombinant fragment was loaded, except in lanes 7 and 10, where ∼3 and ∼6 μg of each recombinant were loaded, respectively. Numbers indicate WRN amino acid coordinate. The asterisk indicates mobility of full-length GST-WRN helicase domain (aa 500-946). C, strategy to identify WRN DNA binding regions. D, limited proteolysis of WRN RQC and HRDC domains. Purified WRN RQC-HRDC (aa 950-1360) protein (lane 1) was treated with different concentrations of subtilisin (lanes 2-8) as described under "Materials and Methods." The resulting fragments were separated by SDS-PAGE and stained with Coomassie Blue. GST alone is a leftover of the purification, and it serves as an internal control of protease resistant folded protein. Asterisks indicate intermediate products of the subtilisin digestion. Black arrows indicate the two protease-resistant fragments. The N-terminal sequence revealed that the 17- and 14-kDa fragments start at the amino acid 958 and either 1128 or 1148, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Werner syndrome Werner syndrome protein RecQconserved domain helicase, RNase Dconserved domain single-stranded DNA double-stranded DNA Bloom glutathione S-transferase amino acids Holliday junction adenosine 5′-O-(thiotriphosphate). Cells from WS patients display replication defects, genomic instability, and altered telomere dynamics, suggesting an important role for WRN in DNA metabolic pathways (for review, see Ref. 7Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar). This is consistent with the observation that many proteins that interact with WRN are involved in recombination, replication, transcription, and telomere structure or repair (8Brosh 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, 9Brosh 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, 10Spillare E.A. Robles A.I. Wang X.W. Shen J.C. Yu C.E. Schellenberg G.D. Harris C.C. Genes Dev. 1999; 13: 1355-1360Crossref PubMed Scopus (153) Google Scholar, 11Szekely A.M. Chen Y.H. Zhang C. Oshima J. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11365-11370Crossref PubMed Scopus (102) Google Scholar, 12Lebel M. Spillare E.A. Harris C.C. Leder P. J. Biol. Chem. 1999; 274: 37795-37799Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 13Cooper M.P. Machwe A. Orren D.K. Brosh R.M. Ramsden D. Bohr V.A. Genes Dev. 2000; 14: 907-912PubMed Google Scholar, 14von Kobbe C. Karmakar P. Dawut L. Opresko P. Zeng X. Brosh Jr., R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 22035-22044Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). In vitro, WRN binds and is catalytically active toward non-canonical DNA structures such as recombination intermediates (16Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar), replication forks (17Opresko 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), repair intermediates (13Cooper M.P. Machwe A. Orren D.K. Brosh R.M. Ramsden D. Bohr V.A. Genes Dev. 2000; 14: 907-912PubMed Google Scholar, 18Harrigan J.A. Opresko P.L. von Kobbe C. Kedar P.S. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 2003; 278: 22686-22695Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and telomeric ends (19Orren D.K. Theodore S. Machwe A. Biochemistry. 2002; 41: 13483-13488Crossref PubMed Scopus (85) Google Scholar). For example, WRN interacts with Holliday junctions, forked duplexes, 5′-overhang duplexes, and D-loops. Typically, WS cells carry a C-terminal truncated WRN that lacks a nuclear localization signal (20Matsumoto T. Shimamoto A. Goto M. Furuichi Y. Nat. Genet. 1997; 16: 335-336Crossref PubMed Scopus (161) Google Scholar). The C-terminal region of WRN also plays a central role in WRN protein-protein interactions (for review, see Ref. 7Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar) and nucleolar targeting (30von Kobbe C. Bohr V.A. J. Cell Sci. 2002; 115: 3901-3907Crossref PubMed Scopus (68) Google Scholar). The conserved RQC domain (RecQconserved) includes the nucleolar targeting sequence (nuclear localization signal-dependent) and participates in WRN protein-protein interactions (9Brosh 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, 30von Kobbe C. Bohr V.A. J. Cell Sci. 2002; 115: 3901-3907Crossref PubMed Scopus (68) Google Scholar). The C-terminal region of WRN also contains the conserved HRDC (helicase, RNase DConserved) domain (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 (1496) Google Scholar), whose function is still unclear, but which may play a role in DNA binding in Saccharomyces cerevisiae Sgs1 and human RecQ homologues (21Liu Z. Macias M.J. Bottomley M.J. Stier G. Linge J.P. Nilges M. Bork P. Sattler M. Struct. Fold. Des. 1999; 7: 1557-1566Abstract Full Text Full Text PDF Scopus (120) Google Scholar). While the enzymatic activities of WRN have been studied extensively (7Opresko P.L. Cheng W.H. von Kobbe C. Harrigan J.A. Bohr V.A. Carcinogenesis. 2003; 24: 791-802Crossref PubMed Scopus (160) Google Scholar), very little is known about the DNA binding properties of WRN. A more complete understanding of WRN DNA binding properties is essential to define the mechanism by which WRN recognizes, binds, and processes DNA substrates. Earlier biochemical studies on full-length WRN suggest that WRN binds DNA with low efficiency (22Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Crossref PubMed Scopus (81) Google Scholar, 23Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). This study examines the DNA binding properties of a series of truncated recombinant WRN proteins and addresses whether distinct functional domains of WRN display differential DNA binding properties that might be required in different stages of helicase and exonuclease reactions. These WRN variants were challenged with several WRN DNA substrates including long forked duplex, Holliday junction, 5′-overhang duplex, ssDNA, and blunt-ended duplex (Table I). The results demonstrate that WRN has three distinct structure-specific DNA binding domains that co-localize with the exonuclease, RQC, and HRDC regions of WRN, respectively. The importance of this finding and its implications for WRN cellular functions are discussed.Table IBiological pathways in which the substrates used in this study could be formed Open table in a new tab Recombinant Proteins—The cloning and expression of most of the GST-WRN fragments was described previously (9Brosh 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, lysates from Escherichia coli BL21(DE3) pLysS expressing recombinant proteins were obtained by sonication of bacterial pellets in lysis buffer (2× phosphate-buffered saline and a mixture of protease inhibitors, Amersham Biosciences). After sonication, Triton X-100 was added to a final concentration of 0.1%. Lysates were then cleared by centrifugation at 35 K for 60 min at 4 °C in a Beckman Ti60 rotor. Glutathione-Sepharose beads were added to the lysates, and the mixture was rotated for 1 h 30 min at 4 °C. Beads were washed five times with lysis buffer plus Triton X-100 (0.1%), and the recombinant proteins were eluted with glutathione elution buffer (100 mm Tris-HCl, pH 8, 150 mm NaCl, 5% glycerol, 0.1% Triton X-100, and 10 mm glutathione). The purified GST-WRN fragments were then analyzed by SDS-PAGE and visualized by Coomassie staining. Recombinant, hexahistidine-tagged WRN and the N-terminal domain of WRN (N-fragment, aa 1-368) were purified as described previously (24Machwe A. Ganunis R. Bohr V.A. Orren D.K. Nucleic Acids Res. 2000; 28: 2762-2770Crossref PubMed Scopus (58) Google Scholar). GST-WRN RQC (aa 950-1360) was cloned into the NotI-KpnI restriction sites of pGEX-TEV vector and purified basically as described above. DNA Substrates—The telomeric short and long forked, blunt-ended duplex, 5′-overhang duplex, ssDNA, and Holliday junction (HJ) substrates were described elsewhere (15Opresko P.L. von Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 16Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (491) Google Scholar, 25Karmakar P. Piotrowski J. Brosh Jr., R.M. Sommers J.A. Miller S.P. Cheng W.H. Snowden C.M. Ramsden D.A. Bohr V.A. J. Biol. Chem. 2002; 277: 18291-18302Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Electrophoretic Mobility Shift Assay—Binding reactions (20 μl) were conducted in standard binding buffer (40 mm Tris-HCl, pH 7.0, 1 mm EDTA, 20 mm NaCl, 8% glycerol, and 20 μg/ml bovine serum albumin). Protein and DNA substrate concentrations are indicated in the figure legends. Reactions were incubated for 30 min on ice and then directly loaded on 5% nondenaturating polyacrylamide gels (37.5:1). Electrophoresis was carried out at a constant voltage of 12.8 V/cm at 4 °C in 1 × TAE (40 mm Tris acetate, 1 mm EDTA, pH 8.0), for 2 h 20 min. Products were visualized using a PhosphoImager and ImageQuant software (Molecular Dynamics). Helicase Reactions—Reactions were performed in binding buffer plus 4 mm MgCl2 and 2 mm ATP, at 37 °C for 15 min. The DNA substrate (short forked duplex) and protein concentrations are indicated in the figure legend to Fig. 2B. Reactions were terminated by the addition of 3× stop dye (50 mm EDTA, 40% glycerol, 0.9% SDS, 0.1% bromphenol blue, and 0.1% xylene cyanol). Products were analyzed on 12% native polyacrylamide gels as described for electrophoretic mobility shift assay.Fig. 2DNA binding properties of WRN N-and C-terminal domains.A, gel mobility shift assay; full-length WRN (0.1, 0.2, and 1 pmol, lanes 2-4), recombinant WRN fragments (1 pmol, lanes 7-10), or GST (1 pmol, lane 6) were incubated with 30 fmol of radiolabeled long forked duplex DNA substrate for 30 min on ice. The protein-DNA complexes were then analyzed as described under "Materials and Methods." Dashed lines indicate the origin of electrophoresis. B, helicase assay; GST (2 pmol, lane 2) or GST-WRN helicase domain (1 and 2 pmol, lanes 3 and 4, respectively) were incubated with 10 fmol of radiolabeled short forked duplex for 15 min at 37 °C. the filled triangle (lane 5) is control reaction in which substrate was denatured by boiling. C, gel mobility shift assay; GST (1 pmol, lanes 6 and 12) or GST-WRN helicase domain (1 pmol, lanes 2-5 and 8-11) were incubated with 30 fmol long (lanes 1-6) or short forked substrate (lanes 7-12) for 30 min. The reactions were carried out on ice (lanes 1-4 and 7-10) or at 37 °C (lanes 5, 6, 11, and 12) in the presence or absence of ATP, ATPγS, and/or Mg2+ as indicated in the figure. D, top, functional domains of WRN and recombinant C-terminal fragments (dashed lines). Numbers indicate WRN amino acid coordinate. Bottom, gel mobility shift assay; recombinant WRN fragments (1 pmol, lanes 2-6) or GST (1 pmol, lane 2) were incubated with 30 fmol of radiolabeled long forked duplex for 30 min on ice. Dashed lines indicate the origin of electrophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Limited Proteolysis of the WRN C Terminus and Identification of the C-terminal Domain Structure—A WRN protein fragment comprising amino acids 950-1360 was subjected to partial subtilisin proteolysis. In a typical experiment, 30 μg of protein was digested with increasing amounts of subtilisin (Fluka) ranging from 0.065 to 2% (w/v), for 10 min on ice. The reaction was stopped by addition of phenylmethylsulfonyl fluoride to a final concentration of 10 mm. Samples were subsequently analyzed by SDS-PAGE and transferred to polyvinylidene difluoride membrane for N-terminal sequencing. The DNA binding activity of WRN was localized to specific protein regions by performing DNA binding assays with a series of recombinant truncated WRN variants and various DNA substrates (Figs. 1, A and B). DNA binding was assayed on ice in the absence of ATP and divalent cations so that DNA substrates would not be degraded or altered by WRN catalytic activity. The long forked duplex DNA substrate has ssDNA and dsDNA regions and is a good substrate for WRN enzymatic activities (17Opresko 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). The long forked DNA substrate was used for initial assessment of WRN DNA binding capacity, because it can be recognized as ssDNA, dsDNA, a dsDNA break, or a forked structure (Fig. 1C). The forked DNA substrate is also recognized by replication protein A, a ssDNA binding protein, and Ku 70/80, a dsDNA break binding protein complex (data not shown). As shown in Fig. 2A (lanes 1-4), full-length recombinant WRN binds to the long forked substrate when the binding assay is carried out at a high enzyme concentration (lane 4). WRN binds to DNA more strongly in the presence of ATP than in its absence (22Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Crossref PubMed Scopus (81) Google Scholar), which may explain the relatively weak binding of WRN to the long forked DNA substrate under the standard binding conditions used in these experiments. On the primary amino acid sequence level, WRN can be subdivided into three characterized structural domains, the exonuclease, the RecQ like helicase, and the HRDC domain (Fig. 1A). We examined whether these domains expressed by themselves would constitute autonomously folded domains. The integrity of the catalytic exonuclease (aa 1-368) and helicase (aa 500-946) expression constructs were verified by virtue of their enzymatic activity. The exonuclease construct (aa 1-368) posses 3′-5′-exonuclease activity identical to that of the full-length protein (24Machwe A. Ganunis R. Bohr V.A. Orren D.K. Nucleic Acids Res. 2000; 28: 2762-2770Crossref PubMed Scopus (58) Google Scholar). The helicase domain expression construct (aa 500-946) was initially designed based on sequence alignment with other RecQ helicases and was subsequently shown to be catalytically active (Ref. 18Harrigan J.A. Opresko P.L. von Kobbe C. Kedar P.S. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 2003; 278: 22686-22695Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar and this study; Fig. 2B). In the absence of catalytic activities, the domain organization of the C terminus was assessed on the basis of limited proteolysis experiments (Fig. 1D). The rationale behind this approach is that unfolded proteins are generally less resistant to proteases and disappear without distinct intermediates. Folded proteins are more resistant, and proteolysis often reveals distinct intermediates corresponding to subdomains. Limited subtilisin digest of a purified C-terminal WRN fragment (aa 950-1360), which included the RQC and HRDC domains, produced two protease-resistant fragments (Fig. 1D). N-terminal sequencing showed that the 17-kDa fragment started at residue 958 (corresponding to the RQC domain), while the 14-kDa fragment started at either residue 1128 or 1148 (corresponding to the HRDC domain). This limited proteolysis pattern indicated that the C-terminal region of WRN contains two folded domains that encompass the RQC and HRDC domains, respectively. Based on the observed molecular weight in the SDS-PAGE, we estimated the C terminus of the structural domain corresponding to the RQC domain to be around residue 1092. A recombinant RQC fragment with similar boundaries is resistant to limited proteolysis by subtilisin (data not shown), providing further evidence that the RQC fragment is folded. In the studies of the HRDC domain, we also included the linker region connecting RQC and HRDC domains. This linker is predicted to be unstructured by secondary structure prediction programs (data not shown). Thus, the HRDC construct used starts at amino acid 1072 and ends at 1236. Having established these boundaries, we designed and expressed various constructs containing either RQC (aa 949-1092), HRDC (aa 1072-1236), RQC and HRDC (aa 949-1236), HRDC containing C-terminal domain (aa 1072-1432), and the C-terminal half of WRN starting at 949 up to the very C terminus (aa 949-1432) (Fig. 1, A and B). Four WRN fragments were initially used to localize WRN DNA binding domains. Binding activity was detected in the N-terminal exonuclease domain (aa 1-368; Fig. 2A, lane 7) and a C-terminal fragment (aa 949-1432; Fig. 2A, lane 10), but GST (Fig. 2A, lane 6), the acidic region (aa 239-499; Fig. 2A, lane 8), and the helicase domain (aa 500-946; Fig. 2A, lane 9) did not bind the long forked DNA substrate specifically. However, the GST-WRN helicase domain unwinds DNA in the presence of ATP and Mg2+ (18Harrigan J.A. Opresko P.L. von Kobbe C. Kedar P.S. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 2003; 278: 22686-22695Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), suggesting that the interaction between this WRN fragment and the DNA substrate may have been too weak to detect by gel mobility shift assay in the absence of enzyme cofactors. This idea was tested by comparing binding activity in the presence and absence of ATP, ATPγS, and Mg2+ at 37 °C and on ice. As shown in Fig. 2B, the helicase domain efficiently unwound the short forked DNA substrate in binding buffer plus ATP and Mg2+ (lanes 3 and 4), but GST did not (lane 2). In binding reactions on ice, plus Mg2+ and ATP (Fig. 2C), the WRN helicase domain formed a weak protein-DNA complex with short and long forked DNA substrates (lanes 3, 4, 9, and 10). Specific binding did not occur in reactions at 37 °C, although DNA was detected at the origin of electrophoresis (lanes 5 and 11). This supershifted DNA might be a high molecular weight DNA-helicase domain complex that only forms under these conditions. Under the same conditions, GST does not form a similar high molecular weight complex (lanes 6 and 12). These results indicate that WRN includes three distinct DNA binding regions: the N-terminal region, the C-terminal region, and the helicase domain. The DNA binding activity of the N- and C-terminal domains appears to be fairly robust, but the helicase domain binds DNA weakly and requires ATP/Mg2+ for binding. The WRN C-terminal DNA binding domain was localized more precisely with four purified GST-WRN fragments that lie within the aa 949-1432 region. The long forked DNA was used as the DNA binding substrate (Fig. 2D). Three of the C-terminal subfragments, aa 949-1236, 949-1092, and 1072-1432 formed strong protein-DNA complexes (lanes 3, 4, and 6), but the aa 1072-1236 fragment and GST did not bind the DNA substrate (lanes 2 and 5). These results suggest that the WRN C-terminal region has two DNA binding subdomains, one associated with the RQC domain (aa 949-1092) and one associated with the HRDC containing C-terminal domain (aa 1072-1432). Binding curves were carried out with a constant DNA concentration (long forked substrate) and variable concentrations of the WRN N-terminal, RQC, or HRDC regions of WRN (Fig. 3). The N-terminal region (aa 1-368) and the HRDC-containing domain (aa 1072-1432) only formed stable DNA complexes at high protein concentrations; at a ratio of 1 pmol of protein to 30 fmol of DNA, the N-terminal region bound 40% of the DNA substrate and the HRDC-containing domain bound 85% (Fig. 3, lanes 2 and 13). The RQC domain (aa 949-1092) bound the forked DNA substrate with much higher affinity, forming complexes with 70-99% of the DNA substrate at 0.1-1.0 pmol protein per assay (Fig. 3, lanes 7-10). In addition, three protein-DNA complexes formed when RQC was present at 1 pmol (50 nm; lane 7). At lower protein concentrations (6.2-25 nm), the protein-DNA complexes were faster migrating (lanes 8, 9, and 10, corresponding to 25, 12.5, and 6.2 nm, respectively). This result suggests that the RQC domain may form a higher order oligomer that assembles in a stepwise manner and that may involve distinct binding sites on the DNA substrate (ssDNA, dsDNA, or forked structures). The structure specificity of the three DNA binding regions of WRN was tested with four DNA substrates known to form in vivo in DNA metabolic pathways that are linked to WRN function (Table I). The substrates used were a 34-bp blunt-ended duplex (sequence identical to the long forked substrate), a 5′-overhang duplex, ssDNA (same sequence as labeled strand of 5′-overhang substrate), and a Holliday junction. The N-terminal region and the HRDC-containing domain of WRN did not bind the blunted-ended DNA substrate (Fig. 4A, lanes 1-4 and 9-12), and the RQC-containing domain only bound this substrate at high enzyme concentration (50 nm, 17% binding efficiency; Fig. 3B, lane 7). This result suggests that the RQC domain has lower affinity for blunt-ended than for forked DNA (50 nm, 99% binding efficiency; Fig. 3, lane 7), in agreement with previous reports (22Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Crossref PubMed Scopus (81) Google Scholar, 23Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). The 5′-overhang duplex is a DNA repair intermediate. The N-terminal region bound to this DNA substrate poorly (Fig. 4B, lane 3, 7% binding), whereas the HRDC-containing domain showed 27% binding to the DNA substrate at a high concentration (Fig. 4B, lane 7) and formed a faint smeared species during electrophoresis. In contrast, the RQC domain bound efficiently to this substrate, binding 71% of the DNA substrate at 50 nm and 10% of the DNA substrate at 12.5 nm (Fig. 4B, lanes 5 and 6). The 5′-overhang is a good substrate for the exonuclease activity of full-length WRN and also for the exonuclease domain (aa 1-368), despite the fact that this domain does not form a gel-stable protein-DNA complex under the conditions tested. A similar observation was noted above for the WRN helicase domain (Fig. 2C). ssDNA was only a substrate for the RQC domain, which bound up to 60% of the input DNA substrate (Fig. 4B, lanes 9-12). This is in agreement with previous studies showing that WRN binds efficiently to ssDNA (23Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). The result in Fig. 4 strongly suggests that the RQC domain mediates the interaction between WRN and ssDNA. HJs resemble recombination and replication fork arrest intermediates. Specific binding was not detected between the HJ substrate and the WRN N-te
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