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• W2060127560 abstract "RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability. RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability. Cellular processes such as DNA replication, recombination, and repair often involve steps that require unwinding of double-stranded DNA (dsDNA) 1The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; SDSA, synthesis-dependent strand annealing; RPA, replication protein A; HJ, Holliday junction; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; SDSA, synthesis-dependent strand annealing; RPA, replication protein A; HJ, Holliday junction; ATPγS, adenosine 5′-O-(thiotriphosphate). to form transient single-stranded DNA (ssDNA) intermediates. Helicases are a class of enzymes that unwind DNA duplexes with a distinct directional polarity, either 3′ to 5′ or 5′ to 3′ with respect to the strand on which the helicase is presumed to translocate, deriving energy from the hydrolysis of ATP (1Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2002; 35: 431-478Crossref PubMed Scopus (146) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar). An essential “caretaker” role of helicases in DNA metabolic processes is suggested by a number of genetic disorders that have been linked to defects in DNA helicases (3Harrigan J.A. Bohr V.A. Biochimie (Paris). 2003; 85: 1185-1193Crossref PubMed Scopus (40) Google Scholar, 4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar, 5Thompson L.H. Schild D. Mutat. Res. 2002; 509: 49-78Crossref PubMed Scopus (349) Google Scholar).The RecQ family of DNA helicases, named after the Escherichia coli RecQ DNA helicase, is highly conserved from bacteria to humans (4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). RecQ helicases share a centrally located helicase domain of ∼450 residues containing the seven conserved helicase motifs and an additional conserved region of ∼80 amino acids located C-terminal to the helicase domain designated RecQ-Ct (4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar, 6Morozov V. Mushegian A.R. Koonin E.V. Bork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar). The conserved helicase domain couples nucleotide hydrolysis to DNA unwinding, whereas the RecQ-Ct domain is suggested to be involved in protein interaction (7Brosh 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) and DNA binding (8von 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 (108) Google Scholar). In all organisms, RecQ helicases play critical roles in maintaining genomic stability as evidenced by the hyper-recombination phenotype observed in recQ mutants (for review, see Ref. 9Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (310) Google Scholar).There are five human helicases with the defined region of homology in the conserved RecQ helicase motifs: 1) WRN, defective in Werner syndrome; 2) BLM, defective in Bloom syndrome; 3) RTS, defective in Rothmund-Thomson syndrome; 4) RECQ1; and 5) RECQ5 (3Harrigan J.A. Bohr V.A. Biochimie (Paris). 2003; 85: 1185-1193Crossref PubMed Scopus (40) Google Scholar). Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome are all characterized as genomic instability disorders with an elevated cancer incidence and/or premature aging. Human diseases have not yet been genetically linked to mutations in RECQ1 or RECQ5; however, these proteins are likely to have important roles in nucleic acid metabolism based on the biological significance of other RecQ helicases.The biological importance of RECQ1 cannot be clearly evaluated at present because there is little information on the phenotypes of human RECQ1 mutant cells. Recent genetic complementation studies using the chick DT40 cell-based system indicate that RECQ1-/- cells do not display a growth deficiency, sensitivity to DNA damage, or elevated sister chromatid exchange; however, RECQ1-/- BLM-/- cells grow more slowly compared with BLM-/- cells due to an increased population of dead cells, indicating that RECQ1 is involved in cell viability under the BLM-impaired condition (10Wang W. Seki M. Narita Y. Nakagawa T. Yoshimura A. Otsuki M. Kawabe Y. Tada S. Yagi H. Ishii Y. Enomoto T. Mol. Cell. Biol. 2003; 23: 3527-3535Crossref PubMed Scopus (75) Google Scholar). In addition, the double mutant RECQ1-/- BLM-/- cells exhibit elevated mitomycin C-induced sister chromatid exchange compared with the BLM-/- cells, suggesting that RECQ1 may have some redundant functions with the BLM helicase (10Wang W. Seki M. Narita Y. Nakagawa T. Yoshimura A. Otsuki M. Kawabe Y. Tada S. Yagi H. Ishii Y. Enomoto T. Mol. Cell. Biol. 2003; 23: 3527-3535Crossref PubMed Scopus (75) Google Scholar).Recent studies of BLM function in Drosophila suggests a role of the helicase in double-strand break repair by a homologous recombination pathway known as synthesis-dependent strand annealing (SDSA) (11Adams M.D. McVey M. Sekelsky J.J. Science. 2003; 299: 265-267Crossref PubMed Scopus (203) Google Scholar, 12McVey M. Adams M. Staeva-Vieira E. Sekelsky J.J. Genetics. 2004; 167: 699-705Crossref PubMed Scopus (88) Google Scholar). This pathway exploits the cellular recombination machinery to rejoin double-strand breaks through a series of strand invasion, DNA synthesis, and re-annealing steps. Although the precise details of BLM function in SDSA are not well understood, the ability of BLM to unwind D-loop structures (13van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Crossref PubMed Scopus (196) Google Scholar), a key intermediate of the SDSA pathway, is likely to be relevant. Consistent with this, the Drosophila genetic data support a model in which BLM acts down-stream of strand invasion (12McVey M. Adams M. Staeva-Vieira E. Sekelsky J.J. Genetics. 2004; 167: 699-705Crossref PubMed Scopus (88) Google Scholar). Understanding the molecular functions of RECQ1 might provide some insight as to how the enzyme has at least partially redundant or synergistic functions with BLM in a pathway such as SDSA.Recent biophysical and biochemical characterization of the purified recombinant RECQ1 helicase provided evidence that RECQ1 forms dimers in solution (14Cui 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) and is able to unwind short 3′-ssDNA tailed duplex substrates (14Cui 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, 15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Considerably longer substrates have been shown to be unwound in the presence of the human ssDNA-binding protein replication protein A (RPA) (14Cui 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, 15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In this study, we have investigated the unwinding of various DNA structures by RECQ1 helicase. In the course of this work, we determined that RECQ1 has the ability to form stable protein complexes with either ssDNA or dsDNA, suggesting that RECQ1 may utilize its physical interaction with DNA for functions other than duplex DNA unwinding. A novel strand annealing activity of human RECQ5β helicase was reported recently (16Garcia P.L. Liu Y. Jiricny J. West S.C. Janscak P. EMBO J. 2004; 23: 2882-2891Crossref PubMed Scopus (169) Google Scholar). Because RECQ1 is able to efficiently bind ssDNA, we became interested in the possibility that RECQ1 might utilize its ssDNA binding activity to bridge the complementary ssDNA molecules and to promote strand annealing. The results presented here provide evidence that RECQ1 can efficiently catalyze DNA unwinding of model recombination/repair DNA intermediates or promote strand annealing of complementary ssDNA molecules and that these processes are modulated by nucleotide availability, which alters RECQ1 protein conformation. The dual catalytic functions of RECQ1 are likely to be important in a cellular DNA metabolic pathway important for maintenance of genomic stability.MATERIALS AND METHODSProteins—Recombinant His-tagged RECQ1 protein was overexpressed using a baculovirus/Sf9 insect system and purified to near homogeneity as described previously (14Cui 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). A baculovirus encoding mutant recombinant His-tagged RECQ1-K119A protein was constructed as described (17Doherty K.M. Sharma S. Uzdilla L. Wilson T.M. Cui S. Vindigni A. Brosh Jr., R.M. J. Biol. Chem. 2005; 280: 28085-28094Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and the RECQ1-K119A protein was purified using the same procedure as described for wild-type recombinant RECQ1 protein. Purified recombinant human RPA was kindly provided by Dr. Mark Kenny (Albert Einstein Medical Center, Bronx, NY). Purified recombinant RuvA protein was kindly provided by Dr. Michael Cox (University of Wisconsin, Madison, WI). E. coli ssDNA-binding protein was purchased from Promega.DNA Substrates—PAGE-purified oligonucleotides used for preparation of DNA substrates were purchased from Midland Certified Reagent Co. 5′-32P-Labeled duplex DNA substrates were prepared as described previously (18Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Synthetic Holliday junction HJ(X12) was made by annealing four 50-mer oligonucleotides (X12-1, X12-2, X12-3, X12-4) as described previously (19Mohaghegh 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). D-loop substrates were prepared as described previously (13van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Crossref PubMed Scopus (196) Google Scholar).DNA Helicase Assays—Helicase reaction mixtures (20 μl) contained 20 mm Tris-HCl (pH 7.5), 10 mm KCl, 8 mm dithiothreitol, 5 mm MgCl2, 5 mm ATP (unless indicated otherwise), 10% glycerol, 80 μg/ml bovine serum albumin, 0.5 nm DNA substrate, and the indicated RECQ1 concentrations. Reactions were initiated by the addition of RECQ1 unless noted otherwise, and the reaction mixtures were incubated for the indicated times at 37 °C. In kinetic experiments, 200-μl reactions containing forked duplex substrate (0.8 or 3.2 nm, as specified) and either 10 or 20 nm RECQ1 were initiated with 5 mm ATP, and 20-μl aliquots were removed at 0-180 s. Unless indicated otherwise, helicase reactions were terminated by the addition of 20 μl of stop buffer (35 mm EDTA, 0.6% SDS, 25% glycerol, 0.04% bromphenol blue, and 0.04% xylene cyanol) with a 10-fold molar excess of unlabeled competitor oligonucleotide to prevent re-annealing of the unwound ssDNA products. Unlabeled competitor oligonucleotide was not included in the quench for helicase reactions with D-loop or HJ substrates. Reaction products were subsequently incubated with 0.1 mg/ml proteinase K for 15 min at 37 °C, and the products of standard duplex and D-loop substrates were resolved on nondenaturing 12% polyacrylamide gels. Products of the HJ branch migration reactions were resolved on native 10% polyacrylamide gels. Radiolabeled DNA species on polyacrylamide gels were visualized with a PhosphorImager and quantitated using ImageQuant software (Amersham Biosciences). The percent of helicase substrate unwound was calculated by the following formula: percent unwinding = 100 × (P/(S + P)), where P is the product and S is the residual substrate. The values of P and S were corrected after subtracting background values in controls with no enzyme and heat-denatured substrate, respectively. Helicase data represent the mean of at least three independent experiments, with mean ± S.D. indicated by error bars.DNA Binding Assays—Reaction mixtures (20 μl) contained the indicated amounts of RECQ1 and 0.5 nm32P-end-labeled DNA substrates in DNA binding buffer (20 mm Tris-HCl (pH 7.5), 2 mm MgCl2, 20 mm NaCl, 0.1 mg/ml bovine serum albumin, and 1 mm dithiothreitol) containing 1 mm ATPγS or no nucleotide. The reactions were incubated at 24 °C for 15 min after the addition of RECQ1. After incubation, 3 μl of loading dye (74% glycerol, 0.01% xylene cyanol, and bromphenol blue) was added to each reaction, and samples were loaded onto native 5% polyacrylamide gels (19:1 cross-linking ratio) and electrophoresed at 200 V for 2.5 h at 4 °C using 1× Tris borate/EDTA as the running buffer. The resolved radiolabeled species were visualized with a PhosphorImager and analyzed using ImageQuant software.DNA Strand Annealing Assays—The DNA strand annealing activity of RECQ1 was measured using partially or fully complementary synthetic oligonucleotides (each at a concentration of 0.5 nm), one of which was labeled at the 5′-end using [γ-32P]ATP and T4 polynucleotide kinase. Annealing of a 32P-labeled oligonucleotide (0.5 nm) to M13mp18 ssDNA (1 nm) was also measured. Strand annealing reactions (20 μl) were carried out in helicase reaction buffer (20 mm Tris-HCl (pH 7.5), 10 mm KCl, 8 mm dithiothreitol, 5 mm MgCl2) and contained the indicated RECQ1 concentrations. Where specified, RPA (0.19-96 nm), E. coli single-stranded DNA-binding protein (0.75-768 nm), ATP (2 or 5 mm), ATPγS (2 or 5 mm), and ADP (2 mm) were also present. Reactions were initiated by the addition of the unlabeled DNA strand, followed by incubation for 15 min at 37 °C. In kinetic experiments, 200-μl reactions were initiated, and 20-μl aliquots were removed at 0.5, 1, 2, 4, 8, 16, and 32 min. A control reaction mixture was set up in an identical manner to determine the level of spontaneous annealing of oligonucleotides in the absence of RECQ1. Reactions were terminated by the addition of 20 μl of stop buffer. Reaction products were subsequently incubated with 0.1 mg/ml proteinase K for 15 min at 37 °C, and the products were resolved on native 12% polyacrylamide gels. Radiolabeled DNA species on polyacrylamide gels were visualized with a PhosphorImager and quantitated using ImageQuant software.Immunodepletion—One microgram of rabbit anti-RECQ1 polyclonal antibody or normal rabbit IgG (both from Santa Cruz Biotechnology, Inc.) was incubated for 1 h at 4 °C with 40 μl of protein G-agarose beads (Roche Applied Science) that had been equilibrated in helicase reaction buffer (see above). Beads were subsequently washed three times with helicase reaction buffer. Antibody-bound beads were then incubated with 20 nm RECQ1 in helicase reaction buffer (40 μl) for 1 h at 4 °C. Beads were collected by centrifugation, and the supernatant was again incubated with antibody-bound beads for 10 min at 4 °C for a second immunodepletion step. Following centrifugation, supernatants were collected and divided into two equal aliquots, one to be assayed for strand annealing activity as described above and the other to assess the efficiency of RECQ1 depletion by Western blot analysis. In control experiments, protein G-agarose beads lacking any bound antibody were incubated either with or without RECQ1 (20 nm), and the resulting supernatants were tested for strand annealing.Partial Proteolytic Digestion of RECQ1—For the limited proteolysis studies, purified recombinant RECQ1 (2 μg) was incubated with chymotrypsin (0.1 μg; Sigma) in the presence or absence of Mg2+ (3 mm) and ATP or ATPγS (3 mm) in a 20-μl reaction mixture at 37 °C for 45 min. An equal volume of 2× SDS sample buffer (126 mm Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.02% bromphenol blue, and 10% β-mercaptoethanol) was added to the proteolytic digestion mixture, and samples were boiled for 5 min. Proteins were resolved on denaturing SDS-10% polyacrylamide gels and visualized by Coomassie Blue staining.RESULTSAlthough the effect of duplex length on RECQ1 helicase activity has been determined (15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we characterized the unwinding activity of purified recombinant RECQ1 helicase on a variety of DNA substrates that are proposed intermediates of various pathways of cellular DNA metabolism. In addition, we characterized how RECQ1 acts upon unwound ssDNA molecules to convert them to duplex strands by a strand annealing activity.DNA Substrate Specificity of RECQ1 Helicase—To determine the DNA substrate preference for RECQ1 helicase activity, we examined a series of B-form duplex DNA substrates with a variety of combinations of ssDNA or dsDNA tails flanking the 19-bp duplex region. These DNA substrates, characterized by a related nucleotide sequence, were incubated with increasing concentrations of purified recombinant RECQ1 helicase in the presence of ATP, and the reaction products were analyzed on native polyacrylamide gels. The results demonstrate that RECQ1 unwound these substrates to different extents depending on the presence and nature of the 3′- and 5′-tails (Fig. 1). Of the substrates tested, a forked duplex with 5′- and 3′-ssDNA arms of 26 and 25 nucleotides, respectively, was unwound relatively efficiently (Fig. 1, A and C). As expected from the unwinding polarity of RECQ1 (15Cui S. Klima R. Ochem A. Arosio D. Falaschi A. Vindigni A. J. Biol. Chem. 2003; 278: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), a DNA substrate flanked by only a 3′-ssDNA tail was also unwound by RECQ1; however, significantly less unwinding of this substrate was detected compared with the forked duplex with 3′- and 5′-ssDNA arms throughout the RECQ1 protein titration. The greatest difference (2.5-fold) was observed at a RECQ1 concentration of 10 nm (Fig. 1C).The observation that RECQ1 unwound a forked DNA duplex with non-complementary 3′- and 5′-ssDNA tails significantly better than the simple 3′-ssDNA tailed duplex suggested that RECQ1, like WRN and BLM (18Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 19Mohaghegh 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), might unwind other DNA structures with junctions. Forked DNA substrates with either one or both of the arms in the double-stranded state are intermediates of cellular processes, including DNA replication, repair, and recombination. We therefore tested this substrate class, which includes a 3′-flap, a 5′-flap, and a synthetic replication fork with 3′- and 5′-duplex arms. Interestingly, the 3′-flap substrate was unwound the best of all the substrates tested. A significantly greater percent of the 3′-flap substrate (as much as 3.5-fold) was unwound by RECQ1 compared with the forked duplex with 3′- and 5′-ssDNA arms at the lowest RECQ1 protein concentrations tested (0.6, 1.2, and 2.5 nm) (Fig. 1C). The 5′-flap substrate was also unwound by RECQ1, but not nearly to the extent as either the forked duplex with ssDNA arms or the 3′-flap substrate. Despite the absence of a 3′-ssDNA tail, RECQ1 unwound the 5′-flap substrate as efficiently as the 3′-ssDNA tailed duplex, resulting in release of ∼40% of the 5′-flap oligonucleotide at the highest RECQ1 concentration tested (10 nm) (Fig. 1C).We next tested a forked duplex in which both the 5′- and 3′-tails of the fork were double-stranded. This three-way junction structure resembles a stalled replication fork where both the leading and lagging strands have been converted to duplex DNA. As shown in Fig. 1B, the synthetic replication fork was unwound by RECQ1 in a protein concentration-dependent manner. Quantitative analysis demonstrated that nearly the same percent of the synthetic replication fork and 5′-flap substrate was unwound by RECQ1 at a given helicase concentration, suggesting that the enzyme is indifferent to the single- or double-stranded character of the 5′-arm of these substrates. Gel analysis of the unwound products of the RECQ1 helicase reaction with the synthetic replication fork revealed that the substrate was unwound in the direction of the fork, primarily yielding a product with the duplex arm intact (Fig. 1B). These results suggest that RECQ1 recognized and bound the junction and translocated in a 3′ to 5′ direction along the bottom strand toward the direction of the fork, thus generating the partial duplex product. This activity is similar to that observed for the WRN helicase (18Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), but is distinct from that of Drosophila RECQ5β, which was shown to unwind the lagging strand duplex of the synthetic replication fork structure (20Ozsoy A.Z. Ragonese H.M. Matson S.W. Nucleic Acids Res. 2003; 31: 1554-1564Crossref PubMed Scopus (45) Google Scholar). Notable, the results suggest that RECQ1 unwinds conventional duplex DNA substrates that completely lack ssDNA character and that its unwinding properties have both similarities to and differences from the unwinding properties of other RecQ helicases (see “Discussion”).RECQ1 Helicase Activity Is Sensitive to Free Magnesium Cation—The overall duplex DNA unwinding reaction catalyzed by a helicase is an energy-requiring process, and helicases utilize energy from the hydrolysis of ATP or other NTPs, allowing them to translocate and unwind DNA duplexes (1Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2002; 35: 431-478Crossref PubMed Scopus (146) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar). As the ATP·Mg2+ complex is the source of chemical energy for the RECQ1 helicase-catalyzed unwinding, we sought to determine the optimal concentrations of Mg2+ and ATP for RECQ1 helicase activity. Initially, we varied the Mg2+ concentrations at a fixed ATP concentration (5 or 1 mm) to estimate the ratio between ATP and Mg2+ at which the greatest level of RECQ1 unwinding could be detected and then to determine the optimal concentration of the ATP·Mg2+ complex. As shown in Fig. 2A, RECQ1 helicase activity in the presence of 5 mm ATP increased fairly linearly with Mg2+:ATP ratios up to 0.5, but decreased with greater Mg2+:ATP ratios, particularly evident at ratios >1. A steep decrease in RECQ1 helicase activity was observed at a Mg2+:ATP ratio of 1.25, resulting in a 2-fold reduction in the percent of forked duplex substrate unwound. Additional increases in the Mg2+:ATP ratio resulted in a greater reduction in unwinding by RECQ1. At a Mg2+:ATP ratio of 2, only 10% of the substrate molecules were unwound by RECQ1 compared with 80% at a Mg2+:ATP ratio of 0.5. To confirm the effect of free Mg2+ on RECQ1 helicase activity, we carried out the same set of experiments using 1 mm ATP, which also resulted in a steady increase in RECQ1 helicase activity as a function of the Mg2+:ATP ratio up to 0.5. Inhibition of RECQ1 helicase activity at Mg2+:ATP ratios >0.5 was observed; however, the decrease in RECQ1 unwinding did not display as great a dependence on the Mg2+:ATP ratio as observed at 5 mm ATP. These data indicate that RECQ1 helicase is sensitive to the ratio of Mg2+ to ATP, with maximal activity exhibited at Mg2+:ATP ratios of ∼0.5-1.0. Beyond this optimum, the unwinding activity steadily declined with increasing Mg2+:ATP ratios, suggesting that free Mg2+ ion is inhibitory to RECQ1 helicase activity. Sensitivity to free Mg2+ ion has also been reported for the E. coli RecQ helicase (21Harmon F.G. Kowalczykowski S.C. J. Biol. Chem. 2001; 276: 232-243Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar); however, WRN helicase activity, which displays optimal unwinding at an Mg2+:ATP ratio of 1, is not adversely affected by greater Mg2+:ATP ratios up to 4 (22Choudhary S. Sommers J.A. Brosh Jr., R.M. J. Biol. Chem. 2004; 279: 34603-34613Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Fig. 2Dependence of RECQ1 helicase activity on ATP and Mg2+ ion concentrations.A, RECQ1 helicase activity is inhibited by free Mg2+ ion concentration. Helicase reaction mixtures contained 0.5 nm forked DNA substrate, 10 nm RECQ1, and either 5 mm ATP with increasing concentrations of MgCl2 from 0 to 20 mm (○) or 1 mm ATP with increasing concentrations of MgCl2 from 0 to 4 mm (•). Percent unwinding in a 15-min reaction at 37 °C is shown. B, optimal ATP·Mg2+ complex concentration for RECQ1 helicase activity. Helicase reaction mixtures contained 0.5 nm forked DNA substrate, 10 nm RECQ1, and increasing concentrations of ATP·Mg2+ at a ratio of either 1:1 Mg2+: ATP (○) or 0.5:1 Mg2+:ATP (•). Percent unwinding in a 15-min reaction at 37 °C is shown. For A and B, helicase data (percent DNA substrate unwound) represent the mean of at least three independent experiments, with S.D. indicated by error bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next performed experiments to determine the optimal concentration of the ATP·Mg2+ complex for RECQ1 helicase activity. Because RECQ1 is inhibited by free Mg2+, RECQ1 helicase assays were performed in which the concentration of the ATP·Mg2+ complex (maintained at either a 1:1 or 0.5:1 ratio of Mg2+ to ATP) was increased. The percent of forked DNA substrate unwound was plotted as a function of ATP concentration (Fig. 2B). RECQ1 unwinding activity increased linearly with ATP concentration up to ∼4-5 mm ATP, at which a plateau was approached for either fixed Mg2+:ATP ratio (Fig. 2B). A Michaelis-Menten kinetic analysis of the initial hyperbolic regime of the plot shown in Fig. 2B yielded Km and Vmax v" @default.
• W2060127560 created "2016-06-24" @default.
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• W2060127560 creator A5010247067 @default.
• W2060127560 creator A5031550197 @default.
• W2060127560 creator A5036007045 @default.
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• W2060127560 date "2005-07-01" @default.
• W2060127560 modified "2023-09-26" @default.
• W2060127560 title "Biochemical Analysis of the DNA Unwinding and Strand Annealing Activities Catalyzed by Human RECQ1*[boxs]" @default.
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