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• W2116573044 abstract "Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophilePyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5′ to 3′ direction) requiring ATP, and nuclease activity, which prefers free 5′-ends of ssDNA as substrate. These activities depend on MgCl2 concentrations. Dna2Pho requires a higher concentration of MgCl2 for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5′-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiaewas reported to be stimulated by RNA segments in the 5′-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem.38022–38031). Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophilePyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5′ to 3′ direction) requiring ATP, and nuclease activity, which prefers free 5′-ends of ssDNA as substrate. These activities depend on MgCl2 concentrations. Dna2Pho requires a higher concentration of MgCl2 for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5′-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiaewas reported to be stimulated by RNA segments in the 5′-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem.38022–38031). single-stranded double-stranded Dna2 from P. horikoshii open reading frame nucleotide(s) peptidyl-prolyl cis-trans-isomerase protein DNA replication is a fundamental process that assures the maintenance of integrity of the genome. The various steps in DNA synthesis are basically similar among all organisms in eukarya, bacteria, and archaea, although replication components differ. In eukaryotic cells, the final processing of Okazaki fragments on the lagging strand requires that the ribonucleotide portions as primers are completely removed by the concerted action of three nucleases, Fen-1, RNaseH, and Dna2. Next, the corresponding sections of the molecule are filled by DNA polymerases. The resulting nicks on the lagging strand are then sealed by DNA ligase (1MacNeill S.A. Curr. Biol. 2001; 11: R842-R844Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 2Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 3Goulian M. Richards S.H. Heard C.J. Bigsby B.M. J. Biol. Chem. 1990; 265: 18461-18471Abstract Full Text PDF PubMed Google Scholar, 4Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19727-19733Abstract Full Text PDF Google Scholar, 5Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (660) Google Scholar, 6Lieber M.R. Bioessays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar). Dna2 fromSacharomyces cerevisiae encodes a 172-kDa protein that has single-stranded (ss)1DNA-dependent ATPase, DNA helicase, and ssDNA-specific endonuclease activity (7Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (150) Google Scholar, 8Bae 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). The genetic and physical interactions of Dna2 with Rad27 (a yeast homologue of mammalian Fen-1) suggest that Dna2 plays a role in Okazaki fragment metabolism (9Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). The endonuclease activity associated with Dna2 preferentially cleaves ssDNA with free ends, and the helicase activity unwinds duplex DNA in the 5′ to 3′ direction. The cleavage reaction was stimulated by the presence of an RNA segment at the 5′-end of flap DNA. The 5′-end region of the Okazaki fragment is efficiently processed by Dna2 when it is displaced from the template by the DNA polymerase δ extending the upstream primer. These enzymatic properties of Dna2 provide a biochemical basis for a role in Okazaki fragment maturation (10Bae S.H. Seo Y.S. J. Biol. Chem. 2000; 275: 38022-38031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). Archaea, the third domain of life, resemble bacteria in morphology and genomic organization (i.e. lack of a nucleus and a single circular genome). However, archaea and eukarya likely have a common ancestor that is separated from bacteria (12Woese C.R. Fox G.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5088-5090Crossref PubMed Scopus (2413) Google Scholar, 13Woese C.R. Kandler O. Wheelis M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4576-4579Crossref PubMed Scopus (4466) Google Scholar). Archaeal genome sequence analyses reveal that the cellular components for genetic processes such as DNA replication, transcription, and translation share many common features with eukarya, whereas those for metabolic processes exhibit similarities to bacteria (14Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Yoshizawa T. Nakamura Y. Robb F.T. Horikoshi K. Masuchi Y. Shizuya H. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (553) Google Scholar, 15Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Yoshizawa T. Nakamura Y. Robb F.T. Horikoshi K. Masuchi Y. Shizuya H. Kikuchi H. DNA Res. 1998; 5: 147-155Crossref PubMed Scopus (132) Google Scholar, 16Bult C.J. White O. Olsen G.J. Zhou R. Fleischmann R.D. Sutton G.G. Blake J.A. Fitzgerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Weidmann J.F. Fuhrmann J.L. Nguyen D. Utterback T.R. Kelley J.M. Peterson J.D. Sadow P.W. Hanna M.C. Cotton M.D. Roberts K.M. Hurst M.A. Kain B.P. Borodovsky M. Klenk H.P. Fraser C.M. Smith H.O. Woese C.R. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2281) Google Scholar, 17Klenk H.P. Clayton R.A. Tomb J. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. Richardson D.L. Kerlavage A.R. Graham D.E. Krypides N.C. Fleischmann R.D. Quakenbusch J. Lee N.H. Sutton G.G. Gill S. Kirkness E.F. Dougherty B.A. McKenney K. Adams M.D. Loftus B. Peterson S. Reich C.I. McNeil L.K. Badger J.H. Glodek A. Zhou L. Overbeek R. Gocayne J.D. Weidmann J.F. McDonald L. Utterback T. Cotton M.D. Spriggs T. Artiach P. Kaine B.P. Sykes S.M. Sadow P.W. D'Andrea K.P. Bowman C. Fujii S.A. Mason T.M. Olsen G.J. Fraser C.M. Smith H.O. Woese C.R. Venter J.C. Nature. 1997; 390: 364-370Crossref PubMed Scopus (1198) Google Scholar, 18Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H.M. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicare R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrovski S. Church G.M. Daniels C.J. Mao J.I. Rice P. Nolling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1036) Google Scholar). Although the molecular mechanism of DNA replication in archaea seems to be a simplified version of the eukaryotic one, knowledge of archaeal DNA replication is still rudimentary (19Böhlke K. Pisani F.M. Rossi M. Antranikian G. Extremophiles. 2002; 6: 1-14Crossref PubMed Scopus (25) Google Scholar, 20MacNeill S.A. Mol. Microbiol. 2001; 40: 520-529Crossref PubMed Scopus (20) Google Scholar, 21Cann I.K.O. Ishino Y. Genetics. 1999; 152: 1249-1267Crossref PubMed Google Scholar). The gene encoding the Dna2 homologue protein (PH0109) inPyrococcus horikoshii (Dna2Pho) exists in an operon containing three other ORFs that are important in DNA replication. Although the gene encoding one ORF (PH0108) found upstream of the Dna2Pho gene is unknown, two other downstream ORFs (PH0112 and PH0113) are homologous to subunits of eukaryal replication factor C that load PCNA onto the template DNA. Moreover, the Dna2Pho gene is located only 9.5 kilobase pairs away from an operon adjacent to the replication origin, which consists of the genes encoding the Rad51 homologue (PH0119), small and large subunits of DNA polymerase D (PH0123 and PH0121), and origin recognition complex 1 (PH0124) (Fig. 1) (22Shen Y. Musti K. Hiramoto M. Kikuchi H. Kawarabayashi Y. Matsui I. J. Biol. Chem. 2001; 29: 27376-27383Abstract Full Text Full Text PDF Scopus (45) Google Scholar, 23Kelman Z. Trends Biochem. Sci. 2000; 25: 521-523Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 24Myllykallio H. Lopez P. López-Garrcı́a P. Heilig R. Saurin W. Zivanovic Y. Philippe H. Forterre P. Science. 2000; 288: 2212-2215Crossref PubMed Scopus (191) Google Scholar, 25Hayashi I. Morikawa K. Ishino Y. Nucleic Acids Res. 1999; 27: 4695-4702Crossref PubMed Scopus (33) Google Scholar). This clustering of genetically essential genes suggests that Dna2Pho possibly plays an important role in DNA replication similar to Dna2 in Eukarya. In the present study, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophileP. horikoshii (Dna2Pho). Ultracompetent Escherichia coli XL2-Blue MRF′ cells and E. coli strain BL21-CodonPlus (DE3)-RIL competent cells were obtained from Stratagene (La Jolla, CA). The pET-21b vector was purchased from Novagen (Madison, WI). Vent DNA polymerase and KOD polymerase were purchased from New England Biolabs (Beverly, MA) and Toyobo (Osaka, Japan), respectively. Restriction enzymes were bought from Takara Shuzo (Kyoto, Japan) andPromega (Madison, WI) and used as recommended by the manufacturers. The ligation kit was purchased from Takara Shuzo and used according to the manufacturer's directions. T4 polynucleotide kinase was obtained fromPromega, while protease inhibitor mixture tablets (EDTA-free) were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Chromosomal DNA of P. horikoshii OT3 was prepared from a sarcosyl lysate of the cells as described previously (26Imanaka T. Tanaka T. Tsunekawa H. Aiba S. J. Bacteriol. 1981; 147: 776-786Crossref PubMed Google Scholar) with slight modification. The digestion of DNA with restriction enzymes and analysis of DNA fragments by agarose gel electrophoresis were performed under standard conditions (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation was carried out by the calcium chloride procedure (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Miniscale preparation of E. coli plasmid DNA was performed by the alkaline lysis method (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) or with a QIAprep spin miniprep kit (Qiagen, Hilden, Germany). A QIAquick gel extraction kit (Qiagen) was used to recover DNA fragments from agarose gel. The gene for Dna2Pho from P. horikoshii was amplified by polymerase chain reaction (PCR) with the primers P1 and P2 (Table I, Fig. 1). The amplified fragment was digested with NdeI and SalI and inserted into an expression vector pET-21b digested with NdeI andXhoI. The constructed plasmid was designated pET-Dna2PhoS(21b). Since the Dna2 homologue (Dna2Pab) fromPyrococcus abyssi was longer by 124 amino acids at the NH2-terminal than Dna2Pho, and another initiation codon TTG was found at the position of Dna2Pho corresponding to the initiation codon (GTG) of Dna2Pab, the ORF coding Dna2Pho was elongated by 122 amino acids. The elongated NH2-terminal region was amplified by PCR against the chromosomal DNA of P. horikoshii as template using the primers P3 and P4 (Table I, Fig.1). The amplified fragment was digested with NdeI and inserted into pET-Dna2PhoS(21b) digested with NdeI andSmaI. The newly constructed plasmid was designated pET-Dna2PhoL(21b).Table IPrimers used in cloning and construction of the expression vectorsPrimerSequences and restriction sites in the primer1-aThe restriction sites are underlined.P15′-TTTTAAACCTGAACAATATCCATATGTATCAAGCTCCACTCATAGGAAAGATAGGG-3′NdeIP25′-CTCGAGTGCGGCCGCAAGCTTGTCGACCTCTCCAGCCCACCTAAACAC-3′SalIP35′-GTAGAGGTGGAAACATATGGAATTTGGGGAGTTACATCCCAGCG-3′NdeIP45′-GGGGCCAGGGAAAATTATCCTTAGTGACAATGGCTAGC-3′NheIP55′-GCTTGGTGATCCATATGACGAGGAAGCTG-3′NdeIP65′-AAAAGGATCCGGTACCTCTACTCGAGCCCAC-3′BamHI KpnIP75′-TTTTGGTACCCCTCTAGAAATAATTTTG-3′KpnI1-a The restriction sites are underlined. Open table in a new tab To construct the co-expression vector for PH0108 and Dna2Pho, the PH0108 gene was amplified with primers P5 and P6 (Table I, Fig. 1). The amplified fragment was digested with NdeI andBamHI and inserted into pET-21b digested withNdeI and BamHI. The new plasmid was designated pET-PH0108(21b). To add a KpnI site upstream of the ribosome binding site (RBS) in pET-Dna2PhoL(21b), PCR was performed using primers P4 and P7 (Table I, Fig. 1). After digestion withKpnI, the fragment was subcloned into pUC19 digested withKpnI and SmaI. ThePvuII-NheI fragment was inserted into pET-Dna2PhoL(21b) digested with NdeI (TheNdeI-digested end was blunted by the Klenow fragment) andNheI. The resulting plasmid was then digested withKpnI and Bpu1102I and inserted into pET-PH0108(21b) digested with KpnI and Bpu1102I. The final co-expression vector was named pET-PH108/Dna2PhoL(21b). The sequences of the genes were verified using an ABI PRISM kit and model 310 capillary DNA sequencer (Applied Biosystems, Foster City, CA). The newly constructed vectors were transformed into host E. coliBL21-CodonPlus (DE3)-RIL. The transformed cells were grown in 2× yeast tryptone (2YT) medium (16 g of trypton, 10 g of yeast extract, and 5 g of NaCl in 1 liter of deionized water) containing ampicillin (50 μg/ml) and chloramphenicol (34 μg/ml) at 37 °C. The overnight culture was inoculated (1% inoculation) into fresh 2YT medium containing ampicillin (100 μg/ml), and incubation was continued at 37 °C until the optical density at 660 nm reached 0.4. The inducer isopropyl-β-d-thiogalactopyranoside was added (final concentration, 1 mm), and cultivation was continued for another 4 h at 30 °C. The cells were harvested by centrifugation (7,000 × g at 4 °C for 10 min) and resuspended in buffer A (20 mm Tris-HCl (pH 8.0) and 20 mm NaCl) containing the protease inhibitor, DNase RQ1 (final concentration, ∼1 unit/ml), and MgCl2 (final concentration, ∼6 mm). The cells were disrupted by sonication, and the resultant solution was incubated at 37 °C for 10 min. Cleared supernatants were obtained by centrifugation (24,000 × g, at 4 °C for 20 min) and then applied to the matrix of a nickel column (Novagen) equilibrated with buffer B (20 mm Tris-HCl (pH 8.0) and 500 mm NaCl) and washed with buffer B containing 10 mm imidazole. The enzymes were eluted with buffer B containing 500 mmimidazole. The eluted fraction was heated at 75 °C for 10 min and centrifuged (24,000 × g, at 4 °C for 20 min) to remove denatured proteins. The supernatant was supplemented with 5m NaCl to a final concentration of 2.3 m NaCl and applied to a HiTrap phenyl-Sepharose HP column (Amersham Biosciences, Buckinghamshire, UK) equilibrated with buffer C (20 mm Tris-HCl (pH 8.0) and 2 m NaCl). The column was washed with the same buffer, and protein was eluted with a 2–1m NaCl gradient. The eluted fraction was dialyzed against buffer D (20 mm Tris-HCl (pH 8.0) and 100 mmNaCl) and stored at 4 °C. Protein concentrations were determined by Coomassie protein assay reagent (Pierce). The purified samples were analyzed by 0.1% sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained by Coomassie Brilliant Blue R-250 (Nacalai Tesque, Kyoto, Japan). NH2-terminal peptide sequencing was examined as follows. After electrophoresis, separated proteins were electrotransferred to a polyvinylidene difluoride membrane (0.2 μm) (Bio-Rad) using a semidry apparatus (Bio-Rad). The NH2-terminal amino acid sequence was determined with an Applied Biosystems model 492 protein sequencer (Foster City, CA). The oligomers used in this study (TableII) were labeled at the 5′-end with T4 polynucleotide kinase and [γ-32P]ATP. The labeled oligonucleotide (5 pmol) was first heated with 2 μg of M13mp18 ssDNA or 98-mer oligonucleotide (5 pmol) in annealing buffer (20 mm Tris-HCl (pH 7.5), 10 mm MgCl2, and 50 mm NaCl) at 100 °C for 5 min, then kept at 67 °C for 1 h, and subsequently allowed to stand at 37 °C for 30 min (28Seki M. Enomoto T. Yanagisawa J. Hanaoka F. Ui M. Biochemistry. 1988; 27: 1766-1771Crossref PubMed Scopus (34) Google Scholar). The annealing mixture was slowly cooled to room temperature. For the preparation of 3′-labeled substrate, the partial duplex substrate was prepared as described above and then labeled using the Klenow enzyme and [α-32P]dATP.32P-Labeled oligonucleotide substrate was purified using a PCR purification kit (Qiagen). M13 DNA substrate was purified using a MicroSpin S-400 column (Amersham Biosciences) and PCR purification kit (Qiagen). To determine the direction of translocation, the 5′-labeled or 3′-labeled substrate was digested with SmaI at 30 °C for 6 h and purified using the PCR purification kit (Qiagen).Table IIOligonucleotides used in this studyOligonucleotideSequence15′-GCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC GTA ATC ATG GTC-3′ (63)2-aThe underline indicates theSmaI cleavage site used to prepare a linear M13 ssDNA. The numbers in parentheses indicate the length of each oligonucleotide.25′-GGT GCC GGA AAC CAG GCA AAG CGC CAT TCG-3′ (30)35′-GAA TAC AAG CTT GGG CTG CAG GTC GAC TCT AGA GGA TCC CCG GGC GAG CTC GAA TTC GGG TCT CCC TAT AGT GAG TCG TAT TAA TTT CGA TAA GCC AG-3′ (98)45′-CTG GCT TAT CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC CCG AAT TCG AGC TCG CCC GGG GAT CCT CTA GAG TCG ACC TGC AGC CCA AGC TTG TAT TC-3′ (98)55′-AGA GTC GAC CTG CAG CCC AAG CTT GTA TTC-3′ (30)65′-CTG GCT TAT CGA AAT TAA TAC GAC TCA CTA-3′ (30)75′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAG AGT CGA CCT GCA GCC CAA GCT TGT ATT C-3′ (70)85′-UUU UUU UUU UUU TTT TTT TTT TTT TTT TTT TTT TTT TTT TAG AGT CGA CCT GCA GCC CAA GCT TGT ATT C-3′ (70)2-bThe bold type indicates ribonucleotides.2-a The underline indicates theSmaI cleavage site used to prepare a linear M13 ssDNA. The numbers in parentheses indicate the length of each oligonucleotide.2-b The bold type indicates ribonucleotides. Open table in a new tab ATPase activity was measured in reaction mixtures (20 μl) containing 50 mm HEPES (pH 7.5), 1 mm dithiothreitol, 0.01% bovine serum albumin, 0.2 nmol of [γ-32P]ATP (15 Ci/mmol), 1 mmMgCl2, the indicated amount of polynucleotide, and the purified Dna2Pho helicase (30 ng). After incubation at 50 °C for 30 min, the reaction was terminated by adding 2 μl of 100 mmNa2EDTA. An aliquot (2 μl) was spotted onto a polyethyleneimine-cellulose thin-layer plate. The reaction products (ATP, inorganic phosphate (Pi)) were separated by chromatography in a 1 m formic acid, 0.5 m LiCl solution (29Chong J.P.J. Hayashi M.K. Simon M.N. Xu R.M. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1530-1535Crossref PubMed Scopus (260) Google Scholar). The extent of ATP hydrolysis was quantified with the GS-525 molecular imager system (Bio-Rad). The standard reaction mixture (20 μl) contained 50 mm HEPES (pH 7.5), 1 mmdithiothreitol, 0.01% bovine serum albumin, 2 mm ATP, 1 mm MgCl2, 32P-labeled helicase substrate, and the purified Dna2Pho helicase. After incubation at 50 °C for 1 h, the reaction was terminated by adding 4 μl of a solution containing 50 mm Na2EDTA, 0.5% SDS, 25% glycerol, and 0.025% bromphenol blue. The sample (10 μl) was loaded onto a 15% polyacrylamide gel in TBE buffer (89 mmTris borate (pH 8.2) and 2 mm EDTA) and electrophoresed at 10 mA. Gels were dried, and the helicase or nuclease products were analyzed and quantified with the GS-525 molecular imager system. The helicase activity was calculated with the formula X =P/(P + S), in which P is the value of the displaced oligonucleotides, and S is the value of nondisplaced substrates. The helicase activity was normalized with the positive and negative controls by the following formula: DNA helicase activity (%) = 100 × [(X sample −X n)/(X p −X n)], in which the X nvalue was determined by negative control assays at 50 °C without enzyme, and X p was obtained as a positive control with the reaction mixture boiled for 5 min without enzyme (30Seki M. Enomoto T. Hanaoka F. Yamada M. Biochemistry. 1987; 26: 2924-2928Crossref PubMed Scopus (33) Google Scholar). Purified Dna2Pho (130 ng) was incubated with32P-labeled substrate at 50 °C in a 20-μl reaction mixture the same as in the helicase reaction except that the concentration of MgCl2 was 10 mm. For denaturing gel analysis, after mixing with 10 μl of sequencing gel loading buffer (95% formamide, 10 mm EDTA (pH 8.0), and 0.1% (w/v) bromphenol blue), samples were boiled for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7 m urea). The gel was dried, and the radioactivity was visualized using the GS-525 molecular imager system (31Matsui E. Kawasaki S. Ishida H. Ishikawa K. Kosugi Y. Kikuchi H. Kawarabayashi Y. Matsui I. J. Biol. Chem. 1999; 274: 18297-18309Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). According to the genomic sequence of P. horikoshii, the PH0109 gene encodes a protein of 1188 amino acids with a predicted molecular mass of 137 kDa. The PH0109 gene was first cloned into pET-21b to yield a construct encoding a fusion protein tagged at the COOH terminus with VEHHHHHH (Fig. 1). The newly constructed plasmid was designated pET-Dna2PhoS(21b). Dna2Pho was expressed in the E. coli strain BL21-CodonPlus(DE3)-RIL. However, the majority of the protein was in insoluble fractions. The Dna2 homologue (Dna2Pab) fromP. abyssi is longer by 124 amino acids at the NH2-terminal region than Dna2Pho. Another initiation codon (TTG), corresponding to the initiation codon (GTG) of Dna2Pab, is located 366 nucleotides upstream from the initiation codon (GTG) of Dna2Pho. Therefore, the ORF coding Dna2Pho was elongated by 122 amino acids (Fig. 2). To obtain a soluble recombinant protein, the elongated Dna2Pho was inserted into pET-21b to yield a construct encoding a fusion protein tagged at the COOH terminus with VEHHHHHH (predicted mass, 153 kDa, designated pET-Dna2PhoL(21b)) (Fig. 1). Although Dna2Pho protein was successfully expressed in the soluble fraction using pET-Dna2PhoL(21b), only a small amount was purified (data not shown). The gene encoding one ORF (PH0108) is found upstream of the Dna2Pho gene. Since the subunits of DNA polymerase (DP1Pho and DP2Pho) from P. horikoshii were successfully overexpressed in soluble fractions using a co-expression vector as previously described (22Shen Y. Musti K. Hiramoto M. Kikuchi H. Kawarabayashi Y. Matsui I. J. Biol. Chem. 2001; 29: 27376-27383Abstract Full Text Full Text PDF Scopus (45) Google Scholar), to obtain large amounts of purified Dna2Pho, a co-expression vector designated pET-PH0108/Dna2Pho(21b) was constructed, although the function of PH0108 protein was unknown. In this vector, the PH0108 and Dna2Pho genes were connected in tandem into two ORFs. The two genes are transcribed under the control of a single transcription promoter and terminator (T7 promoter and T7 terminator). PH0108 and Dna2Pho are expressed as native and His-tagged forms of proteins, respectively (Fig. 1). Both of them were successfully expressed in soluble fractions. Dna2Pho protein was purified in larger amounts using the co-expression system than with pET-Dna2PhoL(21b) as described under “Experimental Procedures.” Fig.3 shows the SDS-PAGE of the purified sample. A major protein band of His-tagged Dna2Pho around 150 kDa and a minor protein band around 22 kDa were observed, whereas a band for PH0108 was not detected. The amino-terminal sequence of the 22 kDa protein was determined as MKVAKDLVVSL, which completely agreed with the amino-terminal sequence of FKBP-type peptidyl-prolylcis-trans-isomerase protein (FKBP-PPIase) (21 kDa) fromE. coli (32Wuelfing C. Lomardero J. Plueckthun A. J. Biol. Chem. 1994; 269: 2895-2901Abstract Full Text PDF PubMed Google Scholar), suggesting an intimate interaction between Dna2Pho and a chaperon-like protein, FKBP-PPIase. Since we could not separate Dna2Pho from FKBP-PPIase with any further purification steps, this protein sample was used for the experiments in this report. We also constructed a co-expression vector that produced PH0108 His-tagged at the COOH terminus and the native form of Dna2Pho. SDS-PAGE analysis showed that there was no band of Dna2Pho protein after nickel column chromatography (data not shown). These results may indicate that Dna2Pho does not interact with PH0108 or that the interaction between them is weak. Therefore, pET-PH0108/Dna2Pho(21b) was used to obtain a large amount of the purified Dna2Pho protein. It is not known what effect addition of the His tag may have had on the activities of Dna2Pho, although it is clear that the tagged protein has retained the DNA helicase and the nuclease activities.Figure 3SDS-PAGE of the purified Dna2 homologue fromP. horikoshii (Dna2Pho). Mdenotes molecular mass standards (150, 100, 75, 50, 35, and 25 kDa) purchased from Novegen. The numbers shown on theleft of the figure indicate the sizes of the markers. The purified sample (0.5 μg of protein) was loaded onto the gel. Thearrow indicates the band of FKBP-type peptidyl-prolylcis-trans-isomerase co-purified with Dna2Pho.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The alignment of sequences from Dna2Pho elongated by 122 amino acids, Dna2Pab, Dna2Sce, Dna2Spo, and Dna2Xla revealed many conserved residues over the entire length of the protein. Helicase and RecB-like nuclease motifs are found in Dna2 homologues as shown in Fig. 2 (33Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1026) Google Scholar, 34Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4730Crossref PubMed Scopus (827) Google Scholar, 35Aravind L. Walker D.R. Koonin E.V. Nucleic Acids Res. 1999; 27: 1223-1242Crossref PubMed Scopus (484) Google Scholar). The Dna2 P504S protein ofS. cerevisiae gives a temperature-sensitivein vivo phenotype. This mutation affects ATPase, helicase, and nuclease activities. The amino acid residue corresponding to Pro504 of the Dna2Sce is not observed in Dna2Pho based on the alignment of amino acid sequences (data not shown), whereas Asp142, Glu155, and Tyr173 are found in the RecB-like motif of Dna2Pho equivalent to the catalytic residues of Dna2, Asp657, Glu675, and Tyr693, respectively (36Budd 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, 37Budd 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). The cysteine cluster conserved among eukaryotic Dna2 proteins is also observed in archaeal Dna2 homologues. However, the function of this cluster is still unknown (35Aravind L. Walker D.R. Koonin E.V. Nucleic Acids Res. 1999; 27: 1223-1242Crossref PubMed Scopus (484) Google Scholar). Dna2Pho possesses seven typical helicase motifs (I, Ia, II, III, IV, V, and VI) (33Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1026) Google Scholar, 34Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4730Crossref PubMed Scopus (827) Google Scholar). The sequence (GTGKT) in the walker A box, the NTP binding motif within motif I as shown in Fig. 2, is completely conserved. Although many residues are conserved within RecB-like nuclease and helicase motifs, no conserved residues were identified in other regions. Dna2Pho possesses a RecB-like nuclease motif and helicase motifs. However, the location of these motifs in Dna2Pho is different from that in Dna2Sce (Fig. 2,upper panel). Since theinset in Fig. 4 shows that Dna2Pho hydrolyzed ATP in a dose-dependent manner, Dna2Pho (30 ng) was added to the reaction mixture for the ATPase assay. Pur" @default.
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• W2116573044 title "Helicase and Nuclease Activities of Hyperthermophile Pyrococcus horikoshii Dna2 Inhibited by Substrates with RNA Segments at 5′-End" @default.
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