The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m709749200
ISSN1083-351X
AutoresVenkateswarlu Popuri, Csanád Z. Bachrati, Laura Muzzolini, Georgina Mosedale, Silvia Costantini, Elisa Giacomini, Ian D. Hickson, Alessandro Vindigni,
Tópico(s)Microtubule and mitosis dynamics
ResumoRecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3′-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1–56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells. RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3′-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1–56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells. RecQ helicases are a ubiquitous family of DNA strand-separating enzymes that defend the genome against instability. They derive their name from the prototypical member of the family discovered in Escherichia coli (1Nakayama K. Irino N. Nakayama H. Mol. Gen. Genet. 1985; 200: 266-271Crossref PubMed Scopus (123) Google Scholar). Since this discovery, many other RecQ helicases have been found in various organisms ranging from prokaryotes to mammals (2Cobb J.A. Bjergbaek L. Nucleic Acids Res. 2006; 34: 4106-4114Crossref PubMed Scopus (50) Google Scholar, 3Hartung F. Puchta H. J. Plant Physiol. 2006; 163: 287-296Crossref PubMed Scopus (58) Google Scholar, 4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar, 5Wu L. Hickson I.D. Annu. Rev. Genet. 2006; 40: 279-306Crossref PubMed Scopus (143) Google Scholar). Five members of the RecQ family have been found in human cells: BLM, RECQ1 (also known as RECQL or RECQL1), RECQ4, RECQ5, and WRN (4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar, 6Opresko P.L. Cheng W.H. Bohr V.A. J. Biol. Chem. 2004; 279: 18099-18102Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Mutations in the genes encoding RECQ4, BLM, and WRN are responsible for distinct genetic disorders, named Rothmund-Thomson syndrome, Bloom's syndrome, and Werner's syndrome, respectively (7Ellis 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 (1204) Google Scholar, 8Kitao S. Lindor N.M. Shiratori M. Furuichi Y. Shimamoto A. Genomics. 1999; 61: 268-276Crossref PubMed Scopus (143) Google Scholar, 9Yu 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 (1479) Google Scholar). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that these three enzymes are involved in different DNA metabolic pathways. For example, Werner's syndrome patients show typical premature aging features, such as premature graying and thinning of hair, osteoporosis and cataracts, whereas Bloom's syndrome is unique among cancer predisposition syndromes in that Bloom's syndrome patients are predisposed to the development of most types of cancers. Rothmund-Thomson syndrome individuals are characterized by skin and skeletal abnormalities as well as an increase in cancer incidence, predominantly osteosarcoma. Mutations in the RECQ1 and RECQ5 genes may be responsible for additional cancer predisposition disorders that are distinct from Rothmund-Thomson syndrome, Bloom's syndrome, and Werner's syndrome, but this remains to be proven. In this regard, interesting candidates are patients with a phenotype similar to that of Rothmund-Thomson syndrome individuals who do not carry any mutations in the RECQ4 gene. Moreover, recent studies have linked a single nucleotide polymorphism present in the RECQ1 gene to a reduced survival in pancreatic cancer patients (10Li D. Frazier M. Evans D.B. Hess K.R. Crane C.H. Jiao L. Abbruzzese J.L. J. Clin. Oncol. 2006; 24: 1720-1728Crossref PubMed Scopus (124) Google Scholar, 11Li D. Liu H. Jiao L. Chang D.Z. Beinart G. Wolff R.A. Evans D.B. Hassan M.M. Abbruzzese J.L. Cancer Res. 2006; 66: 3323-3330Crossref PubMed Scopus (72) Google Scholar).RecQ helicases are ATP- and Mg2+-dependent enzymes that unwind DNA with a 3′ to 5′ polarity. Some RecQ helicases are also able to promote the annealing of complementary DNA duplexes in an ATP-independent fashion (12Cheok C.F. Wu L. Garcia P.L. Janscak P. Hickson I.D. Nucleic Acids Res. 2005; 33: 3932-3941Crossref PubMed Scopus (129) Google Scholar, 13Garcia P.L. Liu Y. Jiricny J. West S.C. Janscak P. EMBO J. 2004; 23: 2882-2891Crossref PubMed Scopus (169) Google Scholar, 14Machwe A. Xiao L. Groden J. Matson S.W. Orren D.K. J. Biol. Chem. 2005; 280: 23397-23407Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Macris M.A. Krejci L. Bussen W. Shimamoto A. Sung P. DNA Repair (Amst.). 2006; 5: 172-180Crossref PubMed Scopus (128) Google Scholar, 16Sharma S. Sommers J.A. Choudhary S. Faulkner J.K. Cui S. Andreoli L. Muzzolini L. Vindigni A. Brosh Jr., R.M. J. Biol. Chem. 2005; 280: 28072-28084Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Additionally, WRN possesses a 3′ to 5′ exonuclease activity that distinguishes it from the other human RecQ enzymes (17Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (374) Google Scholar, 18Shen J.C. Gray M.D. Oshima J. Kamath-Loeb A.S. Fry M. Loeb L.A. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Suzuki N. Shiratori M. Goto M. Furuichi Y. Nucleic Acids Res. 1999; 27: 2361-2368Crossref PubMed Scopus (90) Google Scholar). A property common to all RecQ helicases is their ability to unwind DNA structures other than conventional B-form DNA duplexes. In particular, several RecQ enzymes, including BLM and WRN, preferentially unwind G-quadruplex DNA and synthetic X-junctions that model the Holliday junction recombination intermediate (20Constantinou A. Tarsounas M. Karow J.K. Brosh R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Crossref PubMed Scopus (336) Google Scholar, 21Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 22Karow 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 (419) Google Scholar, 23Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar, 24Sun H. Bennett R.J. Maizels N. Nucleic Acids Res. 1999; 27: 1978-1984Crossref PubMed Scopus (189) Google Scholar, 25Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 26Wu X. Maizels N. Nucleic Acids Res. 2001; 29: 1765-1771Crossref PubMed Scopus (86) Google Scholar). Although some differences in substrate specificity between the human, lower eukaryotic, and prokaryotic RecQ enzymes have been described, little is known about the differences that may distinguish the activity of the five human enzymes (27Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (311) Google Scholar). A comparative analysis of the substrate specificity of human RecQ helicases can provide valuable insights into the molecular basis of the different cellular functions of these enzymes.A previous study showed that BLM and WRN have similar activity toward a panel of model DNA substrates of different structure and length, suggesting that, at least for these two enzymes, differences in helicase substrate specificity are not a fundamental distinguishing feature for defining their specific role in cellular DNA metabolism (23Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar). In this study, we compared the substrate specificity of RECQ1 and BLM using a number of substrates of different structure and length, including substrates that have not been analyzed previously for either RECQ1 or BLM. Our findings highlight several differences between the enzymatic properties of RECQ1 and BLM and suggest that the role of RECQ1 in the maintenance of genome stability is distinct from that of BLM. The possible functions of these RecQ helicases in human cells are discussed.EXPERIMENTAL PROCEDURESProteins—Recombinant His6-tagged RECQ1 and BLM were expressed and purified following previously described procedures (28Cui S. Arosio D. Doherty K.M. Brosh Jr., R.M. Falaschi A. Vindigni A. Nucleic Acids Res. 2004; 32: 2158-2170Crossref PubMed Scopus (94) Google Scholar, 29Karow 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). The RECQ1-(57–649) cDNA was PCR-amplified with a forward primer containing an NheI site at the 5′-end and a reverse primer containing a XhoI site at the 5′-end. RECQ1-(57–649) cDNA was then cloned into NheI and XhoI sites of a pET-28a(+) vector (Novagen), and the insert was excised using XbaI and XhoI in order to add the His6 tag at the N terminus. The cDNA with the additional sequence for the His6 tag was subcloned into the pFastBac1 vector using XbaI and XhoI. To obtain mutant RECQ1-(1–579), a termination codon after residue 579 was created by QuikChange XL site-directed mutagenesis kit (Stratagene). For the experiments with the untagged RECQ1, the His6 tag sequence was removed by digestion with thrombin (1:500 ratio) for 3 h at 4 °C in a buffer of 20 mm Tris-HCl, pH 7.4, 150 mm KCl, 5 mm β-mercaptoethanol. The sample was then incubated with the TALON metal affinity resin (Clontech) for 2 h at 4 °C. The flow-through containing the untagged RECQ1 was collected and concentrated using a Vivaspin filter (Vivascience). The experiments with untagged BLM were performed with a truncated variant of the protein, BLM-(642–1290), expressed and purified from E. coli following previously described procedures (30Janscak P. Garcia P.L. Hamburger F. Makuta Y. Shiraishi K. Imai Y. Ikeda H. Bickle T.A. J. Mol. Biol. 2003; 330: 29-42Crossref PubMed Scopus (116) Google Scholar).DNA Substrates—All of the oligonucleotides used in this study are listed in Table 1. For each substrate, a single oligonucleotide was 5′-end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. The kinase reaction was performed in PNK buffer (70 mm Tris-HCl, pH 7.6, 10 mm MgCl2, 5 mm dithiothreitol) at 37 °C for 1 h. [γ32P]ATP-labeled oligonucleotides were then annealed to a 1.4-fold excess of the unlabeled complementary strands in annealing buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl) by heating at 95 °C for 6 min and then cooling slowly to room temperature. The purification of the forked duplex substrates was performed using Micro Bio-Spin columns (Bio-Rad) or ProbeQuant G-50 Micro columns (Amersham Biosciences). The G4 and G2′ DNA substrates carrying the consensus repeat from the murine immunoglobulin Sγ2b switch region or the Oxytricha telomeric repeat sequence were prepared as described previously (24Sun H. Bennett R.J. Maizels N. Nucleic Acids Res. 1999; 27: 1978-1984Crossref PubMed Scopus (189) Google Scholar, 25Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 31Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (129) Google Scholar). The Holliday junction substrates were purified using Sepharose-4B columns (Amersham Biosciences). The plasmid-based D-loop substrates were generated by RecA-mediated strand invasion of oligonucleotides DL3, DLm, and DL5 into pUC18 and were purified as described previously (32Bachrati C.Z. Borts R.H. Hickson I.D. Nucleic Acids Res. 2006; 34: 2269-2279Crossref PubMed Scopus (173) Google Scholar, 33Bachrati C.Z. Hickson I.D. Methods Enzymol. 2006; 409: 86-100Crossref PubMed Scopus (36) Google Scholar). For the RNA-DNA heteroduplex, the radiolabeled rDL30m RNA oligonucleotide was annealed to pUCbottom in a 1:1.2 molar ratio, as described (33Bachrati C.Z. Hickson I.D. Methods Enzymol. 2006; 409: 86-100Crossref PubMed Scopus (36) Google Scholar) and was used without purification. For the oligonucleotide-based R-loop, the RNA-DNA heteroduplex was further annealed to pUCtop in a 1:1.2 molar ratio and used without purification. The plasmid-based fork regression substrate was prepared as described previously by Ralf et al. (34Ralf C. Hickson I.D. Wu L. J. Biol. Chem. 2006; 281: 22839-22846Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Briefly, the pG68 and pG46 plasmids that contain an array of BbvCI restriction endonuclease recognition sites were nicked by N.BbvC IA and N.BbvC IB, respectively. The two plasmids were annealed to create a paranemic joint, which was converted to a plectonemic joint by DNA topoisomerase I treatment to create the RF substrate molecule. The RF1 molecule was radiolabeled on the 3′ end using [α-32P]TTP and Klenow enzyme.TABLE 1Sequence of oligonucleotides used for the substrate preparationNumberNameSequence (5′-3′)1Fork 30 (U)GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT2Fork 30 (D)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC3Fork 8 (U)GAACGAACACATCGGGTACGTTTTTTTT4Fork 8 (D)TTTTTTTTCGTACCCGATGTGTTCGTTC5G4-TPTGGACCAGACCTAGCAGCTATGGGGGAGCTGGGGAAGGTGGGAATGTGA6G4-TP20TGGACCAGACCTAGCAGCTATGGGGGAGCTGGGGAAGGTGGGAATGTGATTTTTTTTTTTTT7OX-1TACTGTCGTACTTGATATTTTGGGGTTTTGGGGAATGTGA8X12-1GACGCTGCCGAATTCTGGCTTGCTAGGACATCTTTGCCCACGTTGACCCG9X12-2CGGGTCAACGTGGGCAAAGATGTCCTAGCAATGTAATCGTCTATGACGTC10X12-3GACGTCATAGACGATTACATTGCTAGGACATGCTGTCTAGAGACTATCGC11X12-4GCGATAGTCTCTAGACAGCATGTCCTAGCAAGCCAGAATTCGGCAGCGTC12X0-1TGGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAATGTAATCGTCAAGCTTTATGCCGTT13X0-2GACGCTGCCGAATTCTACCAGTGCCAGCGACGGACATCTTTGCCCACCTGCAGGTTCACCC14X0-3CAACGGCATAAAGCTTGACGATTACATTGCTACATGGAGCTGTCTAGAGGATCCGACTATCGA15X0-4ATCGATAGTCGGATCCTCTAGACAGCTCCATGATCACTGGCACTGGTAGAATTCGGCAGCGT16rDL30mGAGAGUGCACCAUAUGCGGUGUGAAAUACC17pUCbottomCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGAC18pUCtopGTCGGGGCTGGCTTAACTATGCGGCATCAGACGTAGCTTGTGATTCTAGGTGGACTCGAGGTGCCCAGCAGGTATGCGAAGGATGAAGGAGAAAATACCGCATCAGGCGCCATTCG19DL3AATTCTCATTTTACTTACCGGACGCTATTAGCAGTGGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGT20DLmGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGT21DL5GCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTTAGAAGCGTTATTCTCTGGGCGAATAGAGTGCTATC Open table in a new tab DNA Helicase Assays—Helicase assays were performed in 20 μl of a reaction mixture containing buffer A (20 mm Tris-HCl, pH 7.5, 8 mm dithiothreitol, 5 mm MgCl2, 10 mm KCl, 10% glycerol, 80 μg/ml bovine serum albumin), 5 mm ATP, and 32P-labeled helicase substrate (0.5 nm). These buffer and substrate concentrations were used in all unwinding assays, except where stated. For the G-quadruplex substrates, reactions were performed in buffer B (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 50 mm NaCl, 100 μg/ml bovine serum albumin) using 5 mm ATP and 30 nm 32P-labeled helicase substrate, as described previously (25Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). The experiments with the plasmid-based D-loops, the RNA-DNA hybrids, and the R-loops were carried out in a 10-μl reaction volume in buffer C (33 mm Tris acetate (pH 7.8), 1 mm MgCl2, 66 mm sodium acetate, 0.1 mg/ml bovine serum albumin, 1 mm dithiothreitol, 1 mm ATP). When RNA-based molecules were used as substrates, RNase inhibitor (New England Biolabs) was added to the reaction to a final concentration of 2 units/μl. Recombinant RecQ helicase protein (RECQ1 or BLM) was added to a concentration indicated in the figures, and the mixture was incubated at 37 °C for the times specified in the figure legends. The reaction was terminated by the addition of 20 μl of 0.4 m EDTA pH 8.0, 10% glycerol (quench solution). Reaction products were resolved using 10% native PAGE, and the extent of unwinding was quantified as described previously (28Cui S. Arosio D. Doherty K.M. Brosh Jr., R.M. Falaschi A. Vindigni A. Nucleic Acids Res. 2004; 32: 2158-2170Crossref PubMed Scopus (94) Google Scholar). Fork regression assays were carried out as described previously (34Ralf C. Hickson I.D. Wu L. J. Biol. Chem. 2006; 281: 22839-22846Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar).ATPase and Electrophoretic Mobility Shift Assays—The ATPase and DNA binding assays were performed using procedures described previously (28Cui S. Arosio D. Doherty K.M. Brosh Jr., R.M. Falaschi A. Vindigni A. Nucleic Acids Res. 2004; 32: 2158-2170Crossref PubMed Scopus (94) Google Scholar). The rate of ATP hydrolysis was measured using thin layer chromatography assays. Reactions included different DNA probes at the concentrations indicated in the figure legends and 20 nm RECQ1 or BLM in buffer A. The electrophoretic mobility shift assays were performed in buffer B with various protein concentrations using 30 nm G-quadruplex DNA.RESULTS AND DISCUSSIONThe unusual ability of BLM and WRN to unwind cruciform and G-quadruplex DNA structures provides useful insights into the possible biological function(s) of these enzymes (23Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar). The present study extends these observations, providing the first comparative analysis of the substrate specificity of RECQ1 and BLM (and by extension WRN). A series of oligonucleotide- and plasmid-based DNA substrates with different structures and lengths was generated to compare the substrate specificities of RECQ1 and BLM (Table 2). Initial experiments using forked duplex substrates with an ssDNA 3The abbreviations used are: ssDNA, single-stranded DNA; nt, nucleotide(s); hRPA, human replication protein A; HJ, Holliday junction(s). tail of 30 or 8 nt confirmed that both enzymes were catalytically active and were able to unwind fork-like structures with an ssDNA tail of ≥8 nt (supplemental Fig. 1) (data not shown). The similar specific activity of RECQ1 and BLM toward the forked duplex substrate was used to standardize each preparation of enzyme for the unwinding experiments using other DNA substrates. Moreover, all experiments were repeated using at least two independent preparations of each enzyme and under identical reaction conditions to minimize any possible interexperimental variation. An untagged version of RECQ1 was also used to exclude any possible contribution of the His6 tag in determining the substrate specificity of RECQ1 (supplemental Fig. 2). The results were consistent with our previous finding that His6-tagged and untagged RECQ1 have identical ATPase, unwinding, and strand annealing activities (35Muzzolini L. Beuron F. Patwardhan A. Popuri V. Cui S. Niccolini B. Rappas M. Freemont P.S. Vindigni A. PLoS Biol. 2007; 5: e20Crossref PubMed Scopus (57) Google Scholar). Regarding BLM, previous studies have already demonstrated that a bacterially expressed (not His6-tagged) truncated variant of BLM, BLM-(642–1290), has the same substrate specificity as the full-length His6-tagged BLM isolated from yeast cells toward various linear duplexes, fork substrates, and Holliday junction structures (30Janscak P. Garcia P.L. Hamburger F. Makuta Y. Shiraishi K. Imai Y. Ikeda H. Bickle T.A. J. Mol. Biol. 2003; 330: 29-42Crossref PubMed Scopus (116) Google Scholar). This result was also confirmed with some of the substrates used in this work (supplemental Fig. 2).TABLE 2Substrate specificity of RECQ1 and BLM Open table in a new tab G-quadruplex DNA; a Putative Role of RecQ Helicases in the Removal of G4 DNA Structures from Ribosomal Gene Clusters, Immunoglobulin Heavy Chain Switch Regions, or Telomeric Repeats—Although G-DNA structures have not been unequivocably observed in vivo, the ease of formation of G-DNA in vitro suggests that G4 DNA structures probably exist at least transiently in cells (31Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (129) Google Scholar). In particular, G-rich regions are abundant in ribosomal DNA gene clusters, in the immunoglobulin heavy chain switch regions, and within telomeric repeats. Although the complementary strand of a duplex would normally protect the G-rich strand from interstrand G-G pairing, cellular processes that promote DNA duplex unwinding, such as replication, transcription, or recombination, generate transient single-stranded DNA stretches that would allow G-quadruplex formation. Previous studies indicated that BLM and WRN could efficiently unwind G-quadruplex DNA substrates (21Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 23Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar, 25Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 26Wu X. Maizels N. Nucleic Acids Res. 2001; 29: 1765-1771Crossref PubMed Scopus (86) Google Scholar, 36Huber M.D. Lee D.C. Maizels N. Nucleic Acids Res. 2002; 30: 3954-3961Crossref PubMed Scopus (171) Google Scholar). Our experiments using different concentrations of BLM confirm this finding (Fig. 1A). Surprisingly, given that BLM, WRN, Sgs1, and E. coli RecQ can all unwind G-quadruplex substrates (23Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar, 24Sun H. Bennett R.J. Maizels N. Nucleic Acids Res. 1999; 27: 1978-1984Crossref PubMed Scopus (189) Google Scholar, 25Sun H. Karow J.K. Hickson I.D. Maizels N. J. Biol. Chem. 1998; 273: 27587-27592Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 34Ralf C. Hickson I.D. Wu L. J. Biol. Chem. 2006; 281: 22839-22846Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 36Huber M.D. Lee D.C. Maizels N. Nucleic Acids Res. 2002; 30: 3954-3961Crossref PubMed Scopus (171) Google Scholar), RECQ1 is not able to unwind this G4 structure even at RECQ1 concentrations of up to 500 nm or in the presence of a saturating concentration of the single-stranded binding protein, human replication protein A (hRPA) (Fig. 1, A and B). The same set of experiments was repeated using a G4 DNA substrate with a 3′-tail of 20 nt, with very similar negative results, indicating that the length of the tail does not affect the ability of RECQ1 to unwind this kind of DNA structure (Fig. 1, C and D). Again, BLM is able to disrupt this structure (Fig. 1, C and D). We also prepared aG2′ DNA substrate, containing the Oxytricha telomeric repeat sequence and a tail of 7 nt at the 3′-end, to test if RECQ1 could unwind this kind of G-quadruplex formed by two antiparallel hairpin dimers (24Sun H. Bennett R.J. Maizels N. Nucleic Acids Res. 1999; 27: 1978-1984Crossref PubMed Scopus (189) Google Scholar). Our data indicate that RECQ1 is unable to unwind G2′ DNA substrates (data not shown). In contrast, BLM can also efficiently unwind this substrate even at a protein concentration 200-fold lower than the highest concentration of RECQ1 used in these unwinding experiments (data not shown).FIGURE 1Analysis of the unwinding activity of RECQ1 and BLM using G-quadruplex substrates. A, unwinding experiments using various concentrations of RECQ1 (0–500 nm) or BLM (0–10 nm) and a G4 DNA substrate with a 3′-tail of 7 nt (30 nm). All of the reactions were stopped after 20 min. B, unwinding experiments in the presence of increasing concentrations of hRPA, as indicated, and 50 nm RECQ1. Lanes C and Δ represent controls without enzyme and with heat-denatured substrate, respectively. All of the reactions were stopped after 20 min. C, unwinding experiments using various concentrations of RECQ1 (0–200 nm) or BLM (0–20 nm) and a G4 DNA substrate with a3′ tail of 20 nt (30 nm). Lanes C and Δ represent controls without enzyme and with heat-denatured substrate, respectively. All of the reactions were stopped after 20 min. D, quantification of the data from C. The data points represent the mean of three independent experiments with an S.D. value that was always ≤10%. •, RECQ1; ○, BLM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate the reason for the different unwinding activity of these two human RecQ helicases toward G4 DNA, we compared their ability to catalyze ATP hydrolysis in the presence of G-quadruplexes as cofactors. The calculated kinetic constant (kcat) values for the ATP hydrolysis reactions catalyzed by BLM using G4 or ssDNA probes as DNA co-factors are 260 ± 10 and 330 ± 10 min–1, respectively (Fig. 2A). The equivalent values for RECQ1 are 25 ± 2 and 91 ± 2 min–1, respectively. These results suggest that the inability of RECQ1 to unwind this particular kind of DNA structure might be, at least partially, related to its poor ability to hydrolyze ATP in the presence of G-quadruplexes. Our gel mobility shift experiments showed, however, that RECQ1 efficiently binds G4 substrates with either a 7- or 20-nt 3′-tail with an affinity similar to that measured for the BLM protein (Fig. 2B). These results suggest that binding of RECQ1 to G4 DNA may not trigger the same conformational change necessary for the stimulation of the ATPase activity that is likely to take place with the BLM helicase.FIGURE 2ATPase and DNA binding assays using G4 DNA. A and B, thin layer chromatography assays performed using G4 DNA or oligo(dT)40 (32 μm) and 20 nm RECQ1 (A) or BLM (B) in the presence of increasing concentrations of ATP (5, 50, 100, 200, 400, and 800μm). C and D, DNA binding assays at increasing protein concentrations using the G4 DNA probe and RECQ1 (C; 0.25, 0.5, 1, 2, 5, 10, 20, and 50 nm) or BLM (D; 0.25, 0.5, 1, 2, 5, 10, 20, and 50 nm). The data points represent the mean of three independent experiments with an S.D. that was always ≤10%. The insets show representative examples of the electrophoretic mobility shift assay using increasing protein concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, these findings point to a clear difference in substrate specificity between RECQ1 and all other members of the RecQ helicase family tested thus far,
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