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• W2055652654 abstract "Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity. Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity. single-stranded DNA single-stranded circular DNA replication protein-A nucleotide(s) single-stranded DNA-binding protein Okazaki fragment synthesis requires the action of DNA polymerase α-primase, δ, and/or ε with proliferating nuclear antigen and replication factor C (1Baker T.A. Bell S.P. Cell. 1998; 92: 295-305Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 2Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 3Bambara R.A. Murante R.S. Hendericksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Polymerase α, tightly complexed with DNA primase, plays a role in the initiation of DNA synthesis by providing RNA-DNA primers for both leading and lagging strands. Okazaki fragments, after removal of primer RNAs, are ligated together through a process called Okazaki fragment maturation. This process requires the combined action of Fen-1, RNase HI, and DNA ligase I (3Bambara R.A. Murante R.S. Hendericksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 4Goulian M. Richards S.H. Heard C.J. Bigsby B.M. J. Biol. Chem. 1990; 265: 18461-18471Abstract Full Text PDF PubMed Google Scholar, 5Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19723-19733Abstract Full Text PDF PubMed Google Scholar, 6Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (497) Google Scholar, 7Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (663) Google Scholar, 8Lieber M.R. Bioessays. 1997; 19: 233-240Crossref PubMed Scopus (396) Google Scholar). Genetic studies revealed that Dna2 of Saccharomyces cerevisiae andSchizosaccharomyces pombe is likely to be involved in Okazaki fragment maturation by virtue of its genetic and physical association with several enzymes involved in Okazaki fragment maturation (9Kang H.Y. Choi E. Bae S.H. Lee K.H. Gim B.S. Kim H.D. Park C. MacNeill S.A. Seo Y.S. Genetics. 2000; 155: 1055-1067Crossref PubMed Google Scholar, 10Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar, 11Parenteau J. Wellinger R.J. Mol. Cell. Biol. 1999; 19: 4143-4152Crossref PubMed Google Scholar). The essential DNA2 gene of S. cerevisiae encodes a 172-kDa protein with characteristic DNA helicase motifs, and the Dna2 enzyme possesses DNA helicase activity (12Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (150) Google Scholar). Cells harboring temperature-sensitive alleles of DNA2 arrested at either G2/M with a duplicated DNA content at the restrictive temperature (9Kang H.Y. Choi E. Bae S.H. Lee K.H. Gim B.S. Kim H.D. Park C. MacNeill S.A. Seo Y.S. Genetics. 2000; 155: 1055-1067Crossref PubMed Google Scholar, 13Fiorentino D.F. Crabtree G.R. Mol. Biol. Cell. 1997; 8: 2519-2537Crossref PubMed Scopus (52) Google Scholar). The recombinant S. cerevisiae Dna2 protein purified from insect cells intrinsically contained strong single-stranded DNA specific endonuclease activity in addition to helicase activity (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Further characterization of the endonuclease activity of Dna2 revealed that Dna2 is not a structure-specific endonuclease, unlike Rad27, a yeast homolog of mammalian Fen1, but prefers to cleave single-stranded DNA (ssDNA)1 with free ends (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Importantly, the endonuclease activity of Dna2 is stimulated by the presence of a terminal RNA segment at the 5′ end of ssDNA (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Moreover, cleavage of the 5′ end of Okazaki fragments by Dna2 was readily observed in conjunction with DNA polymerases capable of displacement DNA synthesis. This unique property of Dna2 appears to ensure the complete removal of the initiator RNA segment on Okazaki fragments, providing a biochemical role of Dna2 endonuclease activity in Okazaki fragment processing (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Recently, we discovered that replication protein A (RPA) suppressed the temperature-sensitive phenotype of the mutant dna2Δ405N allele that lacked the N-terminal 405-amino acid region of DNA2 (16Bae S.H. Kim J.A. Choi E. Lee K.H. Kang H.Y. Kim H.D. Kim J.H. Bae K.H. Cho Y. Park C. Seo Y.S. Nucleic Acids Res. 2001; 29: 3069-3079Crossref PubMed Scopus (41) Google Scholar, 17Bae S.H. Bae K, H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). Subsequently we showed that RPA promotes the endonuclease, switching between Dna2 and Fen1 in the following manner (17Bae S.H. Bae K, H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). RPA initially binds to the 5′ end region containing RNA primers displaced by polymerase δ and then recruits Dna2, resulting in increase in Dna2-catalyzed cleavage of the primer RNAs. The cleavage reaction causes RPA to dissociate from the shortened flap, permitting Fen1 to access the remaining flap (17Bae S.H. Bae K, H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). These findings suggested a new model for Okazaki fragment processing in which the endonuclease activity of Dna2, in collaboration with Fen1, plays a direct role in Okazaki fragment maturation (17Bae S.H. Bae K, H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). In support of this critical role of the endonuclease activity of Dna2, the endonuclease-deficient Dna2 did not support cell growth (18Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar, 19Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In contrast to the important role of the Dna2 endonuclease activity in Okazaki fragment processing, the properties of the Dna2 helicase activity are not well characterized. A point mutation in the ATP binding motif (K1080E) of DNA2 led to inactivation of its ATPase and helicase activities and rendered mutant cells not viable (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), indicating the critical role of the unwinding activityin vivo. However, mutant cells expressing Dna2K1080T were capable of growth in media when lactate and glycerol were used as the carbon source instead of glucose, suggesting that Dna2 appears to act in lagging strand metabolism in a role that is optimal with, but does not require, full helicase activity (21Formosa T. Nittis T. Genetics. 1999; 151: 1459-1470Crossref PubMed Google Scholar). Thus, the helicase activity of Dna2 may play a supportive role that becomes essential in rapidly growing cells. In this paper, we have characterized the biochemical properties of the Dna2 helicase activity in an attempt to understand the role of its DNA unwinding activity during Okazaki fragment processing. We present evidence that the helicase activity of Dna2 facilitates its intrinsic endonuclease activity by resolving secondary structures present in the primer-RNA-containing flap during Okazaki fragment maturation. All oligonucleotides used for the construction of various DNA and RNA substrates were synthesized commercially and gel-purified before use. The sequences of all oligonucleotide used in this study are listed in Table I. Oligonucleotides used to prepare substrates, the positions of radioisotope labels in the substrates, and the substrate structures were as described in each figure. The oligonucleotides 1, 2, and 4 (Table I) were complementary to ΦX174 single-stranded circular DNA (sscDNA) (New England Biolabs Inc.) at nucleotides 702–731, 702–753, and 702–792, respectively. [γ-32P]ATP (>5000 Ci/mmol), [α-32P]dideoxy-ATP (>5000 Ci/mmol), and [α-32P]dCTP (>6000 Ci/mmol) were purchased fromAmersham Biosciences.Table IOligonucleotides used in this study1-a The numbers in parentheses indicate the length of each oligonucleotide.1-b The bold type indicates ribonucleotides.1-c The regions underlined indicate the inverted repeat sequence involved in the formation of a hairpin structure. Open table in a new tab 1-a The numbers in parentheses indicate the length of each oligonucleotide. 1-b The bold type indicates ribonucleotides. 1-c The regions underlined indicate the inverted repeat sequence involved in the formation of a hairpin structure. The dna2Δ haploid strain harboring pRS316-DNA2 as an episome (YKH12) and the plasmids pRS314-DNA2 and pRS325-RFA1 were as described previously (16Bae S.H. Kim J.A. Choi E. Lee K.H. Kang H.Y. Kim H.D. Kim J.H. Bae K.H. Cho Y. Park C. Seo Y.S. Nucleic Acids Res. 2001; 29: 3069-3079Crossref PubMed Scopus (41) Google Scholar, 17Bae S.H. Bae K, H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). The plasmids pRS315-DNA2D657A and pRS314-DNA2K1080E that contain the indicated mutations are derivatives of pRS314-DNA2. Expression of mutant Dna2 enzymes in these plasmids is driven by its native promoter. T4 polynucleotide kinase, the Klenow fragment of Escherichia coli DNA polymerase I, and terminal transferase were from New England Biolabs. The recombinant Dna2 and a mutant Dna2K1080E protein (devoid of both ATPase and helicase activities) were prepared as described previously (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). A mutant Dna2D657A protein devoid of endonuclease activity was prepared as described previously (18Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar). RPA was purified from a protease-deficient yeast strain BJ2168 (MATa, ura3-52,trp1-Δ63, leu2-Δ,prb1-1122, pep4-3,prc1- 407, gal2) as described (22Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (189) Google Scholar). The DNA substrates used to examine the DNA unwinding activity of Dna2 protein were constructed by hybridizing the oligonucleotides listed in Table Ito ΦX174 sscDNA as described (23Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The various partial duplex substrates used to characterize the helicase and endonuclease activities of Dna2 were prepared as described previously (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) using the synthetic oligonucleotides listed in Table I. Labeling of the 5′-or 3′ ends of the oligonucleotides in the substrates was as described (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). ATP hydrolysis was measured in reaction mixtures (20-μl) that contained 50 mm Tris-HCl (pH 7.8), 0.3 mm MgCl2, 2 mm dithiothreitol, 0.25 mg/ml bovine serum albumin, 250 μm cold ATP, 20 nm[γ-32P]ATP, and the indicated amounts of cofactor DNAs. After incubation with 2 ng (10 fmol) of Dna2 at 37 °C for 10 min, aliquots (2 μl) were spotted onto a polyethyleneimine-cellulose plate (J. T. Baker Inc.), which was then developed in a solution containing 0.5 m LiCl and 1.0 m formic acid. The products were analyzed using a PhosphorImager (Molecular Dynamics). Helicase assays were performed with the endonuclease-deficient mutant enzyme, Dna2D657A, unless otherwise indicated. The standard reaction mixture (40 μl) contained 50 mm Tris-HCl (pH 7.8), 2 mm MgCl2, 2 mm dithiothreitol, 0.25 mg/ml bovine serum albumin, 2 mm ATP, and 15 fmol of helicase substrate. When necessary, the concentrations of ATP and/or Mg2+ were varied as indicated in the individual experiments. The reactions were preincubated at 37 °C for 5 min and initiated by the addition of enzyme. Reactions were stopped with 6× stop solution (8 μl; 60 mm EDTA (pH 8.0), 40% (w/v) sucrose, 0.6% SDS, 0.25% bromphenol blue, and 0.25% xylene cyanol). The reaction products were subjected to electrophoresis for 1.5 h at 150 V through 10% polyacrylamide gel containing 0.1% SDS in 0.5× TBE (45 mm Tris-base, 45 mm boric acid, 1 mm EDTA). The gel was dried on DEAE-cellulose paper and subjected to autoradiography. Labeled DNA products were quantitated with the use of a PhosphorImager. The reaction conditions used to examine Dna2 endonuclease activity were the same as those used for the DNA unwinding reaction except that ATP was omitted. Endonuclease assays were carried out at 37 °C for 5 min with either wild type Dna2 or Dna2K1080E, an ATPase/helicase-deficient mutant enzyme. Reactions were stopped by the addition of 2× stop solution (95% formamide, 20 mm EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). The nucleolytic products were boiled for 1 min and subjected to electrophoresis for 1.5 h at 35 W in 1× TBE through 12 or 20% denaturing polyacrylamide gel containing 7m urea as described previously (14Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). To examine the effects of ATP on the endonuclease activity of Dna2, ATP (2 mm) was included as indicated. In all of the above reactions, Dna2 was diluted to the appropriate concentrations before use with 50 mm Tris-HCl (pH 7.8) containing 2 mm dithiothreitol, 0.5 mg/ml bovine serum albumin, 10% glycerol, 0.5 m NaCl, and 0.02% Nonidet P-40. Nonidet P-40 stabilizes Dna2 especially at low protein levels. Because the potent endonuclease activity associated with Dna2 interfered with the measurement of its helicase activity, all of the experiments described below are carried out with the mutant Dna2D657A, an enzyme devoid of detectable endonuclease activity (18Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar). To ensure that the point mutation in Dna2D657A did not alter its ability to hydrolyze ATP, we compared the requirements for ATP hydrolysis by the mutant Dna2D657A and wild type Dna2 enzymes (TableII). The mutant Dna2 had a slight decrease (∼6%) in ATPase activity as compared with wild type enzyme. This difference, however, appears insignificant and within an error range of measurement since several other mutations that affected the endonuclease activity did not affect ATPase activity noticeably (18Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar). Both wild type and mutant enzymes were inhibited similarly by increasing the concentrations of NaCl; both enzymes were dependent on Mg2+, whereas Mn2+ was less effective than Mg2+, and Ca2+ was inactive. The DNA effectors that supported ATP hydrolysis included M13 sscDNA, poly(dT), poly(dA), and poly(dG) with M13 sscDNA the most effective and poly(dG) the least effective. Both wild type and mutant enzymes did not hydrolyze ATP in the presence of synthetic single-stranded RNAs such as poly(U), poly(A), and poly(G). The synthetic double-stranded DNA ((poly(dI·dC)·(poly(dI·dC)) supported ATP hydrolysis similarly (44–56%, wild type; 48–59%, Dna2D657A). However, this activity is most likely due to the presence of free ssDNA regions that did not form a duplex region (see below). Supercoiled double-stranded plasmid DNA supported ATP hydrolysis but with a lower efficiency than M13 sscDNA. Linearized plasmid DNA did not support ATP hydrolysis with either enzymes (Table II). Therefore, we conclude that Dna2 requires single-stranded DNA for the hydrolysis of ATP. In support of this, the addition of salt (50 mm NaCl), which stabilizes duplex structures, markedly reduced the hydrolysis of ATP in the presence of supercoiled DNA by both enzymes (data not shown). These results suggest that the D657A mutation did not alter the ATPase activity of Dna2 nor, most likely, the helicase activity.Table IIComparison of the requirements for the hydrolysis of ATP by wild type and mutant Dna2 enzymesAdditions or omissionsAmount addedRelative activityWild type Dna2Dna2D657A %Add NaCl0, 25, 50, 100, 200 mm100,2-aThe amount of ATP hydrolyzed by wild type Dna2 with 50 ng of M13 sscDNA (2004 pmol) represented 100%. 82, 78, 41, 494, 69, 64, 26, 4Omit MgCl2<1<1 Add EDTA2 mm<1<1 Add MnCl22 mm2518 Add CaCl22 mm<1<1Omit M13 sscDNA Add poly(dT)50 ng8286 Add poly(dA)50 ng4548 Add poly(dG)50 ng88 Add poly(U)50 ng<1<1 Add poly(A)50 ng<1<1 Add poly(G)50 ng<1<1 Add poly(dl · dC)100, 50 ng56, 4455, 45 Add pUC19 RFI100, 50 ng21, 1723, 17 Add pUC19 RFIII100, 50 ng<1<12-a The amount of ATP hydrolyzed by wild type Dna2 with 50 ng of M13 sscDNA (2004 pmol) represented 100%. Open table in a new tab To optimize the unwinding reaction catalyzed by Dna2D657A, we determined the ratio of Mg2+ to ATP. The helicase activity of Dna2D657A was measured at various concentrations of ATP and Mg2+ (Fig.1 A). Maximal unwinding activity was observed when the concentrations of Mg2+ were equimolar to those of ATP (Fig. 1 A). The optimal concentrations of Mg2+ increased in proportion to increasing concentrations of ATP (Fig. 1 A). Based upon these results, all subsequent unwinding reactions were carried out at 2 mm of ATP and 2 mm MgCl2. We have shown previously that wild type Dna2 translocated in the 5′ to 3′ direction using reaction conditions in which the nuclease activity was suppressed substantially by lowering the ratio of ATP/MgCl2 (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). We reexamined the polarity of Dna2 helicase activity under the above optimized helicase condition (2 mm ATP and 2 mm MgCl2) using Dna2D657A and oligonucleotide substrates containing either 5′- or 3′-oligo(dT) tails (Fig. 1 B). Consistent with our previous results (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), Dna2D657A unwound the substrate with a 5′-ssDNA tail only, indicating that Dna2 translocates in the 5′ to 3′ direction (Fig. 1 B). In previous studies, the influence of a fork structure on the Dna2 helicase activity could not be evaluated due to the presence of endonuclease activity, which preferentially cleaved the ssDNA tail in the forked substrate (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The availability of the Dna2D657A enzyme containing helicase activity only permitted us to examine this question. For this purpose, a substrate containing a 25-nt 3′-ssDNA tail was prepared. Theoretically Dna2 can interact with this fork structure when it translocates in the 5′ to 3′ direction along the template sscDNA as illustrated in Fig.2 A. The fork structure containing a 3′-ssDNA tail, however, was displaced at the same rate by Dna2D657A as was a flush structure lacking a fork structure (Fig. 2). This result demonstrates that Dna2 is neither dependent on nor stimulated by the fork structure per se. Next we examined the effect of substrates containing 5′-ssDNA tails on the helicase activity of Dna2. For this purpose, several ΦX174-based substrates with preformed fork structures were constructed as shown in Fig.3 A. The substrates shown are identical except for the different lengths (0, 10, 25, and 40 nt) of the 5′-ssDNA tail. DNA unwinding activity of Dna2D657A was significantly (2–4-fold) stimulated by the presence of a 5′-ssDNA tail (Fig. 3, A and B). The amounts of unwound products increased when the length of the 5′-ssDNA tail was increased (Fig. 3, A and B). Unwinding was stimulated significantly (∼2-fold) by a 10-nt tail, and a 5′ tail longer than 25-mer resulted in a 4-fold stimulation. Further increase of the 5′ tail length did not stimulate the unwinding activity of Dna2D657A. When we used a substrate with a 5-nt tail, the unwinding activity of Dna2D657A was hardly stimulated compared with helicase substrates with longer (>10 nt) 5′ tails (data not shown). These results indicate that the stimulation is most likely due to the ability of Dna2 to utilize the 5′-ssDNA tail as an efficient entry site. As illustrated to the right side of Fig. 3 A, substrates with a 5′ tail can be unwound by two different mechanisms; one mechanism includes unwinding by Dna2 bound to the ΦX174 sscDNA strand, and the other includes unwinding by Dna2 bound to the 5′ tail. Because the amount of ssDNA present as the 5′ tail (10–40 nt) and the amount of ssDNA of ΦX174 (∼5200 nt) markedly differ, the unwinding occurring through the short ssDNA 5′ tail is remarkably efficient (Fig. 3 C). It should be noted that Dna2 hardly unwound the oligonucleotide substrate with partial duplexes at both ends (data not shown), consistent with the notion that Dna2 requires free 5′-ssDNA ends to be loaded effectively before acting as helicase.Figure 3Effects of 5′ tails on duplex unwinding by Dna2. A, unwinding of different substrates. ΦX174 DNA substrates with a 30-bp duplex region and varying lengths of a 5′ tail (0, 10, 25, or 40 nt) were prepared by annealing either the oligonucleotides 1, 5, 8, or 9 to ΦX174 sscDNA, respectively. Theasterisks indicate 32P-labeled ends. The reactions were performed with 80 ng (450 fmol) of Dna2D657A as described in Fig. 2. B denotes boiled substrate controls. The arrows indicate the positions where the labeled oligonucleotides migrated. The direction of unwinding (arrows) by Dna2 (hatched circle) on the two different substrates is shown at the right of the figure.B, quantitation of the displacement reaction. The amounts of substrate unwound in A are presented. The symbolsindicate substrates with flush end (open circle), 10-nt oligo(dT) tail (closed circle), 25-nt oligo(dT) tail (open square), and 40-nt oligo(dT) tail (closed square). C, quantitation of the displacement reaction contributed by the presence of the 5′ tail. The differences between the unwinding of flush and 5-tailed substrates were plotted as a function of incubation time.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Dna2 appears to have a limited ability to unwind long duplex DNA regions, because it hardly unwound a duplex DNA longer than 30 nt (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). This could be due to the associated endonuclease activity that degraded the template ssDNA or to its inherently weak helicase activity of Dna2. To distinguish between these two possibilities, we investigated the unwinding activity of the endonuclease-deficient Dna2 using DNA substrates containing increasing duplex regions. The DNA substrates were prepared by annealing oligonucleotides of 30, 52, and 91 nt to ΦX174 sscDNA as described under “Experimental Procedures.” As shown in Fig.4 A, Dna2 was able to unwind all three substrates, but the rate of unwinding became significantly inefficient as the length of duplex DNA increased. During the 10-min incubation period, Dna2D657A displaced the 30-bp duplex DNA more efficiently (2-fold) than the 52-bp duplex DNA (Fig. 4, Aand B). When the duplex DNA was 91 bp in length, unwinding efficiency decreased dramatically (15-fold less than that observed with the 30-bp duplex). The addition of E. coli SSB inhibited Dna2-catalyzed unwinding (Fig. 4 C, lanes 1–5), and the addition of RPA did not affect unwinding efficiency of the 91-bp duplex substrate (Fig. 4 C, lanes 6–8). Therefore, the result that longer primers are displaced less efficiently than shorter ones is not due to the rewinding of displaced DNA after the unwinding action of the enzyme. To examine whether Dna2 acts in a processive manner, we carried out substrate-challenge experiments by adding a 10-fold molar excess of the ΦX174 template to the reactions at different time points (0, 2, and 5 min) after initiation of the unwinding reaction. As shown in Fig.4 D, the unwinding activity of Dna2D657A was immediately blocked by the addition of excess template ssDNA at any time point after initiation. This result demonstrates that Dna2 is a weak helicase and unwinds duplex DNA in a highly distributive manner. Because RNA did not support ATPase activity of Dna2 (Table II), whereas a terminal RNA segment on a ssDNA tail stimulated the endonuclease activity (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), we examined whether the 5′-terminal RNA could also stimulate unwinding activity of Dna2 using flap-structured substrates (shown in Fig. 5 A). The terminal 12-mer oligo(U) RNA segment in the chimeric flap (total, 25 nt) neither stimulated nor inhibited helicase activity compared with that obtained with a flap of 25-nt DNA (Fig. 5 A). This contrasts with the finding that the same terminal RNA segment stimulated the endonuclease activity of Dna2 (Fig. 5 B; see also Ref. 15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) when the overall length of the 5′ flap remained constant. It is noteworthy that the 25-nt chimeric flap substrate was unwound more efficiently (3-fold) than the 13-nt DNA flap substrate (Fig.5 A), suggesting that the overall length of the 5′ tail is important in acting as an entry site for Dna2. In the presence of lower concentrations (0.1–0.5 mm) of MgCl2, the stimulatory effects of the terminal RNA on the endonuclease activity of Dna2 was more dramatic (15Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Under this condition, however, the unwinding activity of Dna2D657A was too weak to be reliably measured (Fig. 1 A). To analyze the effect of RPA on the unwinding activity of Dna2, we examined the rate of unwinding in the presence and absence of RPA. The stimulatory effect of RPA was salt-dependent as shown in Fig.6 A. In the presence of low concentrations of NaCl (25 mm), RPA neither stimulated nor inhibited the unwinding activity of Dna2D657A (Fig. 6 A,lanes 2–5). In contrast, RPA stimulated unwinding activity of Dna2D657A in the presence of 125 mm NaCl (Fig.6 A, lanes 8–11). The addition of 125 mm NaCl nearly completely inhibited the unwinding ac" @default.
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