Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end.

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 hyperthermophile Pyrococcus 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 MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) 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. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).

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 (1)(2)(3)(4)(5)(6). Dna2 from Sacharomyces cerevisiae encodes a 172-kDa protein that has single-stranded (ss) 1 DNA-dependent ATPase, DNA helicase, and ssDNA-specific endonuclease activity (7,8). 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 (9). The endonuclease activity associated with Dna2 preferentially cleaves ssDNA with free ends, and the helicase activity unwinds duplex DNA in the 5Ј to 3Ј direction. The cleavage reaction was stimulated by the presence of an RNA segment at the 5Ј-end of flap DNA. The 5Ј-end region of the Okazaki fragment is efficiently processed by Dna2 when it is displaced from the template by the DNA polymerase ␦ extending the upstream primer. These enzymatic properties of Dna2 provide a biochemical basis for a role in Okazaki fragment maturation (10,11).
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 (12,13). 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 (14 -18). 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 (19 -21).
The gene encoding the Dna2 homologue protein (PH0109) in Pyrococcus 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) (22)(23)(24)(25). 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 hyperthermophile P. horikoshii (Dna2Pho).

EXPERIMENTAL PROCEDURES
Chemicals, Plasmids, Enzymes, and DNA Manipulation-Ultracompetent Escherichia coli XL2-Blue MRFЈ 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) and Promega (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 from Promega, while protease in-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Inst. of Applied Biochemistry, The University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan.
hibitor 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 (26) with slight modification. The digestion of DNA with restriction enzymes and analysis of DNA fragments by agarose gel electrophoresis were performed under standard conditions (27). Transformation was carried out by the calcium chloride procedure (27). Miniscale preparation of E. coli plasmid DNA was performed by the alkaline lysis method (27) 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.
Cloning of the Genes and Construction of Expression Vectors-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 and XhoI. The constructed plasmid was designated pET-Dna2PhoS(21b). Since the Dna2 homologue (Dna2Pab) from Pyrococcus abyssi was longer by 124 amino acids at the NH 2 -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 NH 2 -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 and SmaI. The newly constructed plasmid was designated pET-Dna2PhoL(21b).
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 and BamHI and inserted into pET-21b digested with NdeI 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 with KpnI, the fragment was subcloned into pUC19 digested with KpnI and SmaI. The PvuII-NheI fragment was inserted into pET-Dna2PhoL(21b) digested with NdeI (The NdeI-digested end was blunted by the Klenow fragment) and NheI. The resulting plasmid was then digested with KpnI 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).
Expression and Purification of Enzymes-The newly constructed vectors were transformed into host E. coli BL21-CodonPlus (DE3)-RIL. The transformed cells were grown in 2ϫ 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 g/ml) and chloramphenicol (34 g/ml) at 37°C. The overnight culture was inoculated (1% inoculation) into fresh 2YT medium containing ampicillin (100 g/ml), and incubation was continued at 37°C until the optical density at 660 nm reached 0.4. The inducer isopropyl-␤-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 ϫ 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, ϳ1 unit/ml), and MgCl 2 (final concentration, ϳ6 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 ϫ 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 mM imidazole. The eluted fraction was heated at 75°C for 10 min and centrifuged (24,000 ϫ g, at 4°C for 20 min) to remove denatured proteins. The supernatant was supplemented with 5 M 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 2-1 M NaCl gradient. The eluted fraction was dialyzed against buffer D (20 mM Tris-HCl (pH 8.0) and 100 mM NaCl) and stored at 4°C.
Protein Analysis-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). NH 2 -terminal peptide sequencing was examined as follows. After electrophoresis, separated proteins were electrotransferred to a polyvinylidene difluoride membrane (0.2 m) (Bio-Rad) using a semidry apparatus (Bio-Rad). The NH 2 -terminal amino acid sequence was determined with an Applied Biosystems model 492 protein sequencer (Foster City, CA).
Preparation of Radiolabeled Substrates for DNA Helicase and Nuclease Assay-The oligomers used in this study (Table II) were labeled at the 5Ј-end with T4 polynucleotide kinase and [␥-32 P]ATP. The labeled oligonucleotide (5 pmol) was first heated with 2 g of M13mp18 ssDNA or 98-mer oligonucleotide (5 pmol) in annealing buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 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 (28). The annealing mixture was slowly cooled to room temperature. For the preparation of 3Ј-labeled substrate, the partial duplex substrate was prepared as described above and then labeled using the Klenow enzyme and [␣-32 P]dATP. 32 P-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 5Ј-labeled or 3Ј-labeled substrate was digested with SmaI at 30°C for 6 h and purified using the PCR purification kit (Qiagen).
Dna2 Homologue from Hyperthermophilic Archaeon P. horikoshii 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 ϭ P/(P ϩ 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 (%) ϭ 100 ϫ [(X sample Ϫ X n )/(X p Ϫ X n )], in which the X n value 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 (30). Nuclease Assay-Purified Dna2Pho (130 ng) was incubated with 32 P-labeled substrate at 50°C in a 20-l reaction mixture the same as in the helicase reaction except that the concentration of MgCl 2 was 10 mM. For denaturing gel analysis, after mixing with 10 l 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 (31).

RESULTS
Expression and Purification of Dna2Pho-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) from P. abyssi is longer by 124 amino acids at the NH 2 -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 VEH-HHHHH (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 (22), 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  Table I.  -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 -TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAG  Dna2 Homologue from Hyperthermophilic Archaeon P. horikoshii agreed with the amino-terminal sequence of FKBP-type peptidyl-prolyl cis-trans-isomerase protein (FKBP-PPIase) (21 kDa) from E. coli (32), 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 Dna2 Homologue from Hyperthermophilic Archaeon P. horikoshii 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.
Primary Structure of Dna2Pho-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 (33, 34, 35). The Dna2 P504S protein of S. cerevisiae gives a temperature-sensitive in vivo phenotype. This mutation affects ATPase, helicase, and nuclease activities. The amino acid residue corresponding to Pro 504 of the Dna2Sce is not observed in Dna2Pho based on the alignment of amino acid sequences (data not shown), whereas Asp 142 , Glu 155 , and Tyr 173 are found in the RecB-like motif of Dna2Pho equivalent to the catalytic residues of Dna2, Asp 657 , Glu 675 , and Tyr 693 , respectively (36,37).
The cysteine cluster conserved among eukaryotic Dna2 proteins is also observed in archaeal Dna2 homologues. However, the function of this cluster is still unknown (35). Dna2Pho possesses seven typical helicase motifs (I, Ia, II, III, IV, V, and VI) (33,34). 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).
ATPase Activity Stimulated by a Single-stranded DNA-Since the inset 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. Purified Dna2Pho (30 ng) was assayed for ATPase activity in the presence of increasing concentrations of closed circular single-and doublestranded (ds) M13 DNA. Although the optimal growth temperature of P. horikoshii is 95°C, ATPase assays were performed at 50°C because portions of double-stranded DNA are likely to denature at high temperature. The hydrolysis of ATP by the Dna2 from S. cerevisiae is markedly stimulated by ssDNA (8). ATPase activity of Dna2Pho was also stimulated as the amount of ssDNA increased. By the addition of 40 fmol of M13 ssDNA, the ATPase activity was stimulated ϳ10-fold; however, ATPase activity was not stimulated by dsDNA. These results indicate that Dna2Pho has ATPase activity stimulated by singlestranded DNA (Fig. 4).
Nuclease and DNA Helicase Activities of Dna2Pho Need Both ATP and MgCl 2 -To confirm the existence of helicase and nuclease activities, an M13 single-stranded DNA circle hybridized to a 30-nt oligonucleotide was used as a substrate. As shown in Fig. 5, no bands due to the displacement or degradation of substrate were observed on varying the Mg 2ϩ concentration in the absence of ATP. In the presence of ATP, DNA helicase activity was detected at a lower concentration of Mg 2ϩ (1 mM) than of ATP (2 mM). However, bands of less than 30-mers and smear bands due to the degradation of substrate appeared at higher Mg 2ϩ concentrations (5 and 10 mM) than the ATP concentration (2 mM). Neither helicase nor nuclease activity was detected when EDTA was added to the reaction mixture. These activities were not detected when the reactions were performed with the protein sample purified with the cell extracts of transformants harboring pET-21b vector alone by the same purification procedure. The results indicate that Dna2Pho possesses nuclease and ATP-dependent DNA helicase activities and Mg 2ϩ ion is required for both.
An ATP-dependent DNA Helicase That Unwinds DNA in the 5Ј to 3Ј Direction-Two classes of DNA helicase have been reported. DnaB translocates in the 5Ј to 3Ј direction (32); however, minichromosome maintenance protein translocates in the opposite direction (from 3Ј to 5Ј) (29, 38 -40). In this experiment, the DNA unwinding polarity of Dna2Pho was determined by blocking the nuclease activity using high ratios of ATP (2 mM) to Mg 2ϩ (1 mM). The partial duplex DNA sub-  Table II) labeled at the 5Ј-or 3Ј-end was annealed to M13 ssDNA and digested with SmaI to produce linear substrates that were used to measure the polarity of displacement. The arrow indicates the migration of the displaced 32-mer oligonucleotide. The schematic structure of the substrate used is shown at the left of the figure. The asterisks indicate 32 P-labeled ends. The right panel indicates DNA helicase activity from a single experiment of several trials using 5Ј-labeled (open circle) and 3Ј-labeled (closed circle) oligonucleotides. Displaced 5Ј-and 3Ј-labeled oligonucleotides were quantified with the GS-525 molecular imager system. The values were normalized as described under "Experimental Procedures." One-hundred percent is the value for heat-denatured substrate.
Dna2 Homologue from Hyperthermophilic Archaeon P. horikoshii strates, consisting of linear M13 ssDNA containing either 3Јlabeled or 5Ј-labeled 32-nucleotide (nt) fragments at both ends, were prepared by digesting with SmaI as shown in Fig. 6 and under "Experimental Procedures." Since the substrates comprise duplex regions at both ends of a long linear molecule, Dna2Pho must first bind to the internal single-stranded regions of these substrates. If the enzyme subsequently moves from the 3Ј to 5Ј along the single-stranded DNA segment, it would displace the 5Ј-labeled fragment from the substrate. In contrast, the 3Ј-labeled fragment would be displaced, if the enzyme migrates in a 5Ј to 3Ј direction.
As shown in Fig. 6, the 3Ј-labeled fragment was displaced gradually from the substrate as the addition of Dna2Pho to the reaction mixture increased, whereas little displaced 5Ј-labeled fragment was observed. The displaced fragments were quantified and the values were normalized as described under "Experimental Procedures." The value of DNA helicase activity was more than 70% (closed circle) using a 3Ј-labeled substrate, while less than 12% (open circle) using a 5Ј-labeled substrate when Dna2Pho (100 ng) was added to the reaction mixture. When a 30-mer duplex fragment, which was blunt-ended at both ends, was assayed, the value of DNA helicase activity increased gradually as the amount of Dna2Pho in the reaction increased. The helicase activity was ϳ18% when Dna2Pho (120 ng) was added to the reaction mixture (data not shown). These results suggest that the DNA helicase activity using a 5Јlabeled substrate was due to movement of the enzyme in the 5Ј to 3Ј direction from the blunted end; hence, Dna2Pho translo-cates in the 5Ј to 3Ј direction, similarly to the yeast Dna2 (10).
Dna2Pho Has Nuclease Activity That Prefers the Free 5Ј-End to 3Ј-End-To identify the nuclease activity of Dna2Pho, single-stranded, double-stranded, 3Ј-overhang, and 5Ј-overhang DNA were used as substrates at high ratios of Mg 2ϩ (10 mM) to ATP (2 mM). As shown in Fig. 7A, the single-stranded DNA substrate was rapidly degraded to oligonucleotides varying in size with the time course of reaction, whereas the doublestranded DNA (dsDNA) substrate was degraded slowly. The trinucleotide observed using dsDNA substrate might be due to unwinding from the 5Ј-labeled end and subsequently cleavage of the translocating side of dsDNA. These results demonstrate that the nuclease activity of Dna2Pho prefers ssDNA. When the 3Ј-overhang substrate was used for nuclease assay, the nuclease activity was decreased, compared with the activity against the single-stranded DNA (Fig. 7, A and B). On the other hand, the 5Ј-overhang DNA substrate was more efficiently degraded than the 3Ј-overhang DNA substrate, indicating that for nuclease activity, Dna2Pho is likely to prefer ssDNA with free 5Ј-end as substrate (Fig. 7, A and B).  Table II. M denotes molecular size markers prepared by labeling a synthetic mixture (2,3,4,6,40,50, and 63-mers) and commercial size markers ((dGATC) n , where n denotes oligonucleotides 8 -32-mers in length, Amersham Biosciences) at their 5Ј-ends with T4 polynucleotide kinase. The numbers shown on the left of the figures indicate the size of the markers. The reaction mixtures contained 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 0.01% bovine serum albumin, 2 mM ATP, 10 mM MgCl 2 , the 32 P-labeled substrate as described under "Experimental Procedures," and Dna2Pho (130 ng in a 20 l-reaction mixture), at 50°C. Samples were removed (0, 5, 10, 20, 40, 60, and 120 min), quenched, and loaded on a 15% denaturing polyacrylamide gel (7 M urea). The gel was dried, and the radioactivity was visualized using the GS-525 molecular imager system. B, the nuclease activity of Dna2Pho using two partial duplex DNA (5Ј-overhang and 3Ј-overhang) as substrate. Both Nuclease and Helicase Activities Were Inhibited by RNA/DNA Chimeric Substrate-The endonuclease activity of Dna2 from S. cerevisiae is stimulated by substrate with an RNA segment at the 5Ј-end, and the helicase activity enhances the endonuclease activity. Therefore it is considered that Dna2 removes RNA-DNA primers of Okazaki fragments with both unwinding and cleavage activity coupled to each other during Okazaki fragment processing (10,11,41).
To investigate the effect of RNA/DNA chimeric substrate on the activities, a Y-structured substrate containing a 12-nt oligonucleotide (U) segment at its 5Ј-end was prepared as shown in Fig. 8. In the DNA helicase assay, displaced fragments were observed as the Dna2Pho concentration increased. However, no displaced fragments were produced from the substrate with an RNA segment at the 5Ј-end, indicating that Dna2Pho could not unwind the substrate (Fig. 8A).
The nuclease activity was assayed on single-stranded DNA or single-stranded RNA/DNA chimera, respectively. As shown in Fig. 8B, the single-stranded DNA was degraded into pieces varying from 50-mer to the size of a monomer, whereas RNA-DNA oligonucleotide was not digested by the nuclease activity. The Y-structured DNA substrate was also degraded by Dna2Pho just like single-stranded DNA. However, the Y-structured RNA-DNA chimeric substrate was not degraded by the nuclease activity. These results demonstrate that Dna2Pho can not displace and cleave substrates with an RNA segment at the 5Ј-end and that the 5Ј-end moiety of ssDNA is important for the recognition or binding of Dna2Pho to the substrate. DISCUSSION In this report, we have identified a number of biochemical properties of the Dna2Pho from P. horikoshii. The gene for Dna2Pho is encoded in an operon, in which two subunits of replication factor C are also present. Moreover, the Dna2Pho gene is located only 9.5 kilobase pairs away from an operon adjacent to the replication origin, which consists of genes encoding a Rad51 homologue, small and large subunits of DNA polymerase D, and origin recognition complex 1 (14,15,23,24). This clustering of genetically essential genes indicates that Dna2Pho possibly plays an important role in DNA replication.
Based on the genome information of P. horikoshii (14,15), we cloned Dna2Pho from genomic DNA by PCR. Dna2Pho was expressed in E. coli as a protein of 1188 amino acids, but was unstable and difficult to purify. Therefore we expressed a protein with an additional 122 amino acids at the NH 2 terminus. The enlarged Dna2Pho, consisting of 1310 amino acids, was successfully produced in E. coli. Moreover, the enlarged Dna2Pho was coexpressed with PH0108 protein, and purified in larger amounts than Dna2Pho expressed alone in E. coli. It appears likely that PH0108 protein participates in assisting Dna2Pho to fold into a normal structure or stabilize the mRNA transcribed from the Dna2Pho gene. Although further analysis of the functions of PH0108 protein is needed, we focused on the biochemical characteristics of Dna2Pho in this report.
The Dna2Pho protein of 1310 amino acids was compared with Dna2 homologues from several species. RecB-like nuclease and helicase motifs are found in Dna2Pho (33,34,35). The location of the RecB-like motif in Dna2Pho is different from that in Dna2 from S. cerevisiae (Dna2Sce). The NH 2 -terminal domain of Dna2Sce functions as a regulator of both helicase and nuclease activities essential for the optimal function of Dna2Sce in Okazaki fragment processing (42). Although a sequence homologous to the NH 2 -terminal region of Dna2Sce is not observed in Dna2Pho, the amino acid sequences within RecB-like nuclease and helicase motifs are remarkably conserved among Dna2 homologues. Dna2Pho possesses ATPase, DNA helicase, and nuclease activities. The ATPase activity is stimulated by single-stranded DNA. The DNA helicase activity is dependent on ATP and Mg 2ϩ ion. The helicase activity is detectable by blocking the nuclease activity at a higher concentration of ATP (2 mM) than Mg 2ϩ (1 mM). The DNA helicase activity unwinds DNA in the 5Ј to 3Ј direction. The nuclease prefers the free 5Ј-end to the 3Ј-end of single-stranded DNA. These properties of Dna2Pho are similar to those of Dna2Sce; however, a critical difference is the specificity for substrate with an RNA segment in the 5Ј-tail. The biochemical characteristics of Dna2Pho differed from those of Dna2Sce as follows.
1) The nuclease activity of Dna2Pho could not cleave the RNA/ DNA chimeric substrate, whereas that of Dna2Sce is stimulated by substrates with an RNA segment in the 5Ј-end. 2) The helicase activity of Dna2Pho could not displace the Y-structured substrate with an RNA segment at the 5Ј-end.
The eukaryotic primase is known as a DNA polymerase␣primase complex composed of four subunits, p180, p70, p58, and p48. The primase activity requires the p48 and p58 subunits, and especially, p48 is known to be the primase catalytic subunit, which synthesizes the short RNA segment, typically 8 -12 nucleotides in length (43). The primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus, has been characterized by Liu et al. (44). The amino acid sequences of two subunits, Pfup41 and Pfup46, have similarity to p48 and p58 of the eukaryotic DNA primase complex, respectively. The p41-p46 complex can synthesize both DNA (ϳ700 bases in length) and RNA primers (12-40 nucleotides in length), whereas the DNA primase from yeast cannot synthesize DNA primers. We also characterized the DNA primase from P. horikoshii (45). Surprisingly, the complex could synthesize long DNA primers 10 times more effectively than RNA primers and also could synthesize DNA/RNA hybrids. Liu et al. (44) speculated that the primer synthesis was started by RNA using ATP in P. furiosus, because the p41-p46 complex can discriminate ATP from other NTPs. We used a substrate with a poly(U) 12 segment at its 5Ј-end in this report; therefore, further experiments to elucidate specificity against a substrate with a poly(A) segment at the 5Ј-end might be necessary. Furthermore, the isolation and characterization of Okazaki fragments from Pyrococcus cells are very interesting, since it is still unclear whether DNA primers are synthesized in vivo or not. If the DNA primase of Pyrococcus species could synthesize a DNA primer in vivo, the properties of Dna2Pho might be suitable for Okazaki fragment processing in Pyrococcus cells.