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J. Biol. Chem., Vol. 276, Issue 48, 45484-45490, November 30, 2001

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The Archaeal DNA Primase

BIOCHEMICAL CHARACTERIZATION OF THE p41-p46 COMPLEX FROM PYROCOCCUS FURIOSUS^*

Lidong Liu^§, Kayoko Komori, Sonoko Ishino, Arnaud A. Bocquier^¶, Isaac K. O. Cann^**, Daisuke Kohda^¶, and Yoshizumi Ishino

From the  Department of Molecular Biology, and ^¶ Department of Structural Biology, Binomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan

Received for publication, July 9, 2001, and in revised form, September 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase -primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [-³²P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase -primase complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA replication is the fundamental process for the maintenance of life and also for the evolution of extant organisms, by which species transfer genetic information to their offspring. In both the bacterial and eukaryotic DNA replication processes, DNA polymerases are incapable of de novo DNA synthesis, and it is well known that DNA primase works for the de novo synthesis of the short RNA/DNA oligonucleotide on the template DNAs for both the leading and lagging strands. The short oligonucleotide, called the primer, is then extended by DNA polymerases to synthesize the long DNA strand (reviewed in Refs. 1 and 2). In Escherichia coli, the primase activity is derived from the DnaG protein, and it synthesizes a short RNA primer. The DnaG protein interacts with the DNA polymerase III holoenzyme, and this interaction limits the size of the nascent primers to the range of 9-14 nucleotides (3). On the other hand, the eukaryotic primase is known as the DNA polymerase -primase complex (pol -pri),¹ which is composed of four subunits, p180, p70, p58, and p48 (reviewed in Refs. 4 and 5). Among these subunits, the primase activity requires the p48 and p58 subunits, and especially, p48 is known to be the primase catalytic subunit. These two subunits assemble together with the DNA polymerase subunit, p180, and the p70 subunit, whose function is unknown. The current understanding of the molecular mechanism of primer synthesis is as follows: at the replication origin (for the leading strand synthesis) or at the preferred sites (for the lagging strand synthesis), the p48 subunit synthesizes an RNA primer that is typically 8-12 nucleotides long. Then, the pol  subunit elongates the RNA primer to about a 30-mer using dNTPs as substrates to generate the DNA primer, which can be extended by the replicative DNA polymerases (pol  and pol ).

Archaea, the third domain of life (6), is now recognized as biologically and evolutionarily interesting to study. However, the current knowledge of the DNA replication mechanism in Archaea is still rudimentary. Several genes encoding eukaryotic-like DNA replication proteins have been found in archaeal genomes, and numerous biochemical studies have been reported (reviewed in Refs. 7-10). These results have led to the proposal that the mechanism of archaeal DNA replication is basically similar to that of Eukarya. Biochemical characterizations of the individual protein factors as well as genetic analyses of the corresponding genes involved in the initiation and elongation processes are necessary to understand the global mechanism of molecular recognition in archaeal DNA replication. To date, several proteins involved in the elongation step, including DNA polymerases, PCNA (proliferating cell nuclear antigen), RFC (replication factor C), and RPA (replication protein A) have been characterized in vitro, including the homologs from Pyrococcus furiosus (11-19). However, there are no reports about the initiation of archaeal DNA replication or its priming mechanism.

In the archaeal genomes, there is one open reading frame (ORF) that has some sequence similarity to the eukaryotic DNA primase subunit p48 (20). The corresponding genes have been cloned from Methanococcus jannaschii and P. furiosus, and the gene products, Mjpri (21) and Pfup41 (22), respectively, have been characterized. One remarkable property of Pfup41 is that it preferentially uses deoxyribonucleotides as a substrate and has de novo activity to synthesize long DNA strands in vitro (22). The next step of this archaeal primase study is to investigate whether the catalytic primase proteins form a complex with other proteins in the replication machinery, like those of the other two biological domains. There is no ORF with an obvious similarity to the eukaryotic p58 subunit in the archaeal genomes. However, based on our knowledge that in P. furiosus, genes encoding functionary related proteins are often arranged in tandem on the genomic DNA. Therefore, we carefully compared sequences of the ORFs neighboring to Pfup41 with that of eukaryotic p58 and found some similarity in one ORF overlapped with that for Pfup41. These two genes seemed likely to compose an operon.

In this study, we cloned the gene for the p58-like protein from P. furiosus, expressed it in E. coli, and characterized the gene product, named Pfup46. We demonstrated that the Pfup46 protein modulates the activities of Pfup41 for the primase-polymerase. From the biochemical properties of the p41-p46 complex, we propose an archaeal replication model, in which the p41-p46 complex works as the primase-polymerase, like the eukaryotic pol -pri complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequencing of the Genes for Pfup41 and Pfup46-- The two genes were amplified directly from P. furiosus genomic DNA, prepared as described earlier (11), by polymerase chain reaction using PfuDNA polymerase (Stratagene) to maintain the accuracy of amplification. The nucleotide sequences of the genes were confirmed after they were cloned into the TA-cloning vector (Novagen). Preparation of the pPFPR41 plasmid and production of the Pfup41 protein in E. coli were as described previously (22). Due to the difficulty in producing the Pfup41 protein in a high yield, the protein was produced as a hexa-histidine-tagged protein at the N terminus. For the gene encoding the Pfup46 protein, two primers were used, p46-F (5'-CACGACCATGGTAGACCCATTTAGTGAG-3') and p46-R (5-CACGGTCGACTCATTACTGTAGAATTCGCT3'), with NcoI and SalI restriction sites (underlined), respectively. The gene was inserted into the corresponding site of pET21d (Novagen) to obtain the expression plasmid, pPFPR46.

Expression and Purification of Pfup41, Pfup46, and the p41-p46 Complex-- The Pfup41 and Pfup46 proteins were overproduced as follows: E. coli BL21-codonPlus (DE3)-RIL (Novagen) cells harboring pPFPR41 or pPFPR46 were grown at 37 °C in 1 liter of LB medium in the presence of ampicillin and chloramphenicol. When the culture reached an A₆₀₀ of 0.4, the expression of the target genes was induced by adding isopropyl--D-thiogalactopyranoside to 1 mM. After cultivation for a further 5 h, the cells were harvested by centrifugation, with yields of 3.8 and 4.0 g (wet weight), respectively, for Pfup41 and Pfup46. The Pfup41 protein was purified from E. coli/pPFPR41 cells. The cell lysate was prepared by sonication in 70 ml of buffer A (50 mM Tris-HCl, pH 8.0, 2 mM -mercaptoethanol, 0.3 M NaCl) containing 1 mM phenylmethanesulfonyl fluoride. After centrifugation for 20 min at 30,000 × g, the supernatant was incubated at 80 °C for 15 min to remove most of the E. coli proteins, and then the supernatant was loaded onto a chelating column charged with Co²⁺ ions (5 ml, TALON^TM, CLONTECH), connected on an FPLC apparatus (Amersham Pharmacia Biotech). The column was extensively washed with buffer A containing 10 mM imidazole, and the bound proteins were eluted with buffer A containing 100 mM imidazole. The protein fractions were pooled, dialyzed against buffer B (50 mM Tris-HCl, pH 7.0, 0.3 M NaCl), and subjected to cation-exchange chromatography (HiTrap SP, 1 ml, Amersham Pharmacia Biotech) on an AKTA system (Amersham Pharmacia Biotech). The chromatography was developed with a 20-ml linear gradient of 0.3-0.8 M NaCl in 50 mM Tris-HCl, pH 7.0, at a flow rate of 1 ml/min. The active fractions, which eluted at a salt concentration of 0.5-0.7 M NaCl, were pooled, dialyzed against buffer C (10% glycerol, 50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, and 1 mM -mercaptoethanol), and stored at 4 °C. The concentration of purified Pfup41 was determined by using a molar extinction coefficient  of 47,770 M¹ cm¹, which was calculated from the amino acid sequence with the ExPASy-ProParam Tool program. The Pfup46 was purified from E. coli/pPFPR46 cells. The cell lysate was prepared and heat-treated as described above. The supernatant after centrifugation was mixed with polyethyleneimine (Sigma) and NaCl to 0.2% (w/v) and 0.3 M, respectively, and the mixture was stirred for 30 min on ice to precipitate the DNA. The proteins were then precipitated with ammonium sulfate (80% saturation). The precipitate was resuspended in and dialyzed against buffer A. The dialysate was diluted with 50 mM Tris-HCl, pH 8.0, to a final NaCl concentration of 0.1 M and was applied immediately to an anion exchange column (HiTrap Q, 5 ml, Amersham Pharmacia Biotech). The chromatography was developed with a 50-ml linear gradient of 0.1-1.0 M NaCl at a flow rate of 1.0 ml/min. The Pfup46 protein, which eluted at a salt concentration of 0.1 to 0.25 M, was collected and applied to a HiTrap Heparin column (Amersham Pharmacia Biotech). The chromatography was developed with a 20-ml linear 0.05-0.8 M NaCl gradient, and the Pfup46 protein was eluted at a salt concentration of 0.3-0.5 M NaCl. The concentration of Pfup46 protein was determined by using a molar extinction coefficient  of 43,950 M¹ cm¹. For the preparation of the p41-p46 complex, the purified Pfup41 was combined with an excess amount of purified Pfup46 in 50 mM Tris-HCl, pH 7.0, and was subjected to cation-exchange chromatography (HiTrap SP) as described for the Pfup41 purification. The excess, free Pfup46 was found in the flowthrough fraction, and the p41-p46 complex was eluted at a salt concentration of 0.5-0.7 M NaCl.

Gel Mobility Shift Assay-- Three synthetic oligonucleotides, described earlier (18), were used as the substrates. The 49-mer deoxyribonucleotide was labeled at the 5' terminus using [-³²P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (Takara Shuzo) and was used as the single-stranded DNA (ssDNA). A primed DNA (priDNA) and a double-stranded DNA (dsDNA) were prepared by annealing the labeled 49-mer DNA to its unlabeled complementary 17-mer and 49-mer, respectively, with an equal molar ratio. The DNA substrate (50 nM) was incubated for 5 min at 55 °C with various amounts (50-750 nM) of proteins in a total volume of 10 µl, in a buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 20 mM KCl, and 1 mM -mercaptoethanol. The protein/DNA mixtures were loaded onto a 1% agarose gel and were separated by electrophoresis in 0.1 × TAE buffer (4 mM Tris acetate, 0.1 mM EDTA, pH 8.4) at 50 V for 1.5 h at room temperature. The radiolabeled DNA was visualized by autoradiography.

De Novo DNA Synthesis by Pfup41 and p41-p46 Complex-- The reaction mixture (20 µl), containing primase assay buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 1 mM -mercaptoethanol), 1 µg M13 mp18 single-stranded DNA (23), 100 µM each of dGTP, dCTP, and dTTP, and 10 µM dATP containing [-³²P]dATP (Amersham Pharmacia Biotech), was incubated with Pfup41 or p41-p46 (0.35 µM) at 70 °C for 20 min. The reaction products were analyzed by 1% alkaline agarose gel electrophoresis (AGE) in 50 mM sodium hydroxide and 1 mM EDTA, followed by autoradiography. A laser-excited image analyzer (BAS-5000; Fuji Film, Tokyo, Japan) was used to quantify the synthesized strands.

In Vitro Replication of M13 Single-stranded DNA by P. furiosus Proteins-- DNA synthesis reaction was carried out in an assay mixture (20 µl) containing primase assay buffer, 100 µM each of dATP, dGTP, and TTP, 10 µM dCTP containing [-³²P]dCTP (Amersham Pharmacia Biotech), 0.5 µg of M13mp18 single-stranded DNA, and 0.14 µM p41-p46 complex, in addition to 2.5 units of P. furiosus pol I (pol BI) (14) and pol II (pol D) (16), 0.05 µM PfuPCNA (17) and PfuRFC (18), and 0.8 µM PfuRPA (19) at 70 °C for 20 min except for any concrete descriptions. The products were analyzed by 10% polyacrylamide gel electrophoresis (PAGE) in the presence of 8 M urea. To analyze the lengths of DNA fragments, the reaction mixtures were fractionated by 1% alkaline AGE, followed by autoradiography. To compare the product amounts in the presence of different NTP concentrations, the band of the full-length product from each reaction on the autoradiogram was quantified as described above. As a control, a synthetic DNA 30 nucleotides long was annealed to M13 DNA and was used as the template-primer for the DNA synthesis reaction by P. furiosus proteins except p41-p46 as described earlier (17, 18).

Synthesis of RNA Primers and Their Elongation by DNA Polymerases from P. furiosus-- RNA primer synthesis was carried out at 55 °C for 20 min in an assay (20 µl) containing primase assay buffer, 100 µM concentration each of ATP, CTP, and GTP, 10 µM UTP containing [-³²P]UTP (Amersham Pharmacia Biotech), 0.5 µg of M13 mp18 single-stranded DNA, and 0.14 µM p41-p46. For the primer extension reactions, 100 µM concentration each dNTP and 2.5 units of pol I (pol BI) or pol II (pol D) were mixed together with the materials for RNA primer synthesis, and the reactions were carried out at 70 °C for 20 min. The reaction products were analyzed as described above.

Competitions of dNTPs and NTPs-- For the competition of dNTPs and NTPs in the primer synthesis reaction, increasing concentrations of dNTPs were added to the reaction for RNA primer synthesis. The reaction products were analyzed by PAGE on a 10% gel containing 8 M urea, followed by autoradiography. Each product signal was quantified as described above. The concentration ratios of the substrates (dNTPs/NTPs) were no NTPs, 1/1000, 1/100, 1/10, 1/1, and no dNTP. In reverse, increasing concentrations (1, 10, and 100 µM) of ATP or NTPs were added to the reaction for DNA primer synthesis, containing 1 µM concentration of each dNTP ([³²P]dCTP) at 55 °C 30 min. To see the effect of high concentrations of NTPs, dNTP concentration was lowered to 1 µM in this experiment.

Interaction of p41 with p46 in Vivo-- Rabbit polyclonal antibodies were raised against homogeneous Pfup41 and Pfup46, respectively. The antibodies for pol I (pol BI) were as described previously (15). Immunoprecipitation experiments were done as follows at room temperature: 30 µl of protein A-Sepharose (Amersham Pharmacia Biotech) were dispensed into each of four Eppendorf tubes, and the resin was washed three times with PBS (10 mM sodium phosphate, pH 7.5, 150 mM NaCl). The protein A-Sepharose in each tube was then mixed with one of the above antisera (10 µl) and incubated for 1 h on a rotary shaker. Each mixture was washed twice with PBS and once with buffer D (50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol). The contents of each tube were mixed with 400 µl of P. furiosus cell extract (from 40 µg of cells) and were incubated for 30 min on a rotary shaker. Precipitates were washed three times with buffer D, and the immunoprecipitated products were eluted by boiling them in 30 µl of 1 × loading buffer (0.05 M Tris-HCl, pH 6.8, 10% glycerol, 1% 2- mercaptoethanol, 0.04% bromphenol blue, 1% SDS), followed by Western blot analysis. The blots were analyzed with the enhanced chemiluminescence system (Pierce), using peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of the p46 Protein as the Subunit of the P. furiosusPrimase-- In the P. furiosus genome, there is one ORF with an amino acid sequence similar to that of the p48 subunit of the eukaryotic pol -pri complex. We characterized the protein, named Pfup41, as a primase in vitro (22). One remarkable characteristic is that this enzyme has de novo activity to synthesize long DNA strands. To further characterize the function of the primase in P. furiosus cells, we sought to identify additional primase subunits corresponding to those of Eukarya. We found that an ORF overlapping that of Pfup41 has some sequence similarity to that of the p58 subunit of the Pol -pri complex (Fig. 1). It has been shown that there is some remarkable sequence similarity between the eukaryotic p58 subunit and the 8-kDa domain of DNA pol  (20). The conserved residues between p58 and pol  are located in box c in Fig. 1A. These residues are not well aligned when p58 proteins are compared with archaeal proteins. However, there are some localized similarities between p58 proteins and archaeal proteins across the regions, and therefore, we cloned the corresponding gene, expressed it in E. coli, and purified the gene product, named Pfup46. In contrast to the production of Pfup41, which was produced in a histidine-tagged form because of the low production efficiency, Pfup46 was efficiently produced in E. coli, and it was purified to high homogeneity without the need for a tag (Fig. 2, A and B). From 1 liter of each culture, 1.0 and 6.5 mg of His-Pfup41 and Pfup46 were purified, respectively.

View larger version (65K): [in this window] [in a new window]   Fig. 1.   A, five regions with similarities in the archaeal ORFs and the eukaryotic DNA primase p58 subunit. Identical and similar amino acid residues are indicated in black and gray, respectively. The ORF names from each data base were shown. The organisms are: MTH, M. thermoautotrophicus; MJ, M. jannaschii; Ta, Thermoplasma acidophilum; AF, A. fulgidus; VNG, Halobacterium sp. NRC-1; ORF-, S. solfataricus; the yeast (Sce) and human p58 proteins were aligned at the bottom. B, gene organization of the primase complex on the P. furiosus genome. The genes encoding the amino acid sequences similar to those of the p48 and p58 subunits, respectively, of the eukaryotic primase complex were found in tandem with overlapping of their ORF on the P. furiosus genome. The coding regions of p41 and p46 overlap with each other, as shown on the bottom.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Purification of Pfup41 (A), Pfup46 (B), and the p41-p46 complex (C). SDS-PAGE (10%) analysis of the recombinant p41 and p46 proteins from the different stages of the purification. In A and B, lanes: 1, supernatant after sonication; 2, supernatant after heat treatment (80 °C, 15 min); 3, metal affinity fraction in A or HiTrap Q fraction in B; 4, HiTrap SP fraction in A or HiTrap Heparin fraction in B. In C, purified Pfup41 and Pfup46 were mixed and applied to HiTrap SP column. The peak fraction eluted at around 0.6 M NaCl was subjected to 10% SDS-PAGE (lane 2). Lanes 1 and 3 contain purified Pfup41 and Pfup46, respectively. The gels were stained with Coomassie Brilliant Blue.

To investigate whether Pfup41 and Pfup46 form a stable complex, the two purified proteins were mixed in an excess ratio of Pfup46 to Pfup41 and were subjected to cation-exchange chromatography. The Pfup41 and Pfup46 proteins eluted together at a salt concentration of 0.6 M NaCl (the calculated pI of the complex is 9.2), whereas the free Pfup46 (calculated pI, 6.6) passed through the column in the pH 7.0 buffer. The peak fraction contained both Pfup41 and Pfup46 (Fig. 2C, lane 2). The mixture of Pfup41 and Pfup46 was also applied to a gel filtration column (Superdex G-200), and a clear single peak, in which both Pfup41 and Pfup46 were included, was obtained. From the elution time, the apparent molecular mass of the complex was calculated to be about 90 kDa. This result indicates that the two proteins form a stable complex with a one to one molar ratio. A densitometric analysis of the band intensities of the Coomassie Brilliant Blue-stained gel for Pfup41 and Pfup46 in the complex also supports the 1:1 stoichiometric ratio of the primase complex. To confirm the complex formation by Pfup41 and Pfup46 in the cells, immunoprecipitation experiments were performed using the P. furiosus cell extract. As shown in Fig. 3, Pfup41 and Pfup46 were coprecipitated with either anti-Pfup41 or anti-Pfup46 antiserum. The DNA polymerase I of P. furiosus, which is the only DNA polymerase having similar sequence to that of eukaryotic pol , was not precipitated with either Pfup41 or Pfup46. Unlike the eukaryotic machinery, it seems likely that the primase does not form a stable complex with the DNA polymerase.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Immunoprecipitation analysis of Pfup41, Pfup46, and pol I (pol BI). The total cell extract was precipitated with anti-Pfup41, anti-Pfup46, and anti-Pol I, and the precipitates were detected by Western blot analysis with each antiserum shown on the left side. The cell extracts without immunoprecipitation or precipitated with PBS-treated protein A-Sepharose were also loaded as positive and negative controls, respectively.

DNA Binding Ability of the Primase Complex-- We tried primase and polymerase reaction assays using the purified Pfup46 protein, as measured for Pfup41 in our previous study (22). However, neither activity was detected (data not shown). To investigate the role of Pfup46 in the primase complex, we first measured the DNA binding activity of the proteins using a gel-mobility shift assay (Fig. 4). The Pfup41 protein did not have a strong DNA binding activity (Fig. 4A, lane 2). In contrast, with the same amount of the Pfup46 protein, two clear shifted bands were observed in the cases of single-stranded (ss) and primed (pri) DNAs, probably derived from the complexes of the DNA with one and two Pfup46 molecules, respectively (Fig. 4A, lane 3). The double-stranded DNA (ds) was less appropriate for Pfup46 binding, as expected. When the p41-p46 complex was used for the binding assay, one shifted band with a different mobility, showing the complex formation of (p41-p46)-DNA, was observed (Fig. 4A, lane 4). To measure the difference of the DNA binding abilities among the primase molecules more precisely, a titration analysis was carried out. However, as shown in Fig. 4B, Pfup41 seems to aggregate at higher concentrations. This characteristic of Pfup41 made it difficult to obtain the quantified data suitable for calculating K_d values. However, these results clearly indicate that Pfup46 protein increases the affinity of the primase to DNA by forming a complex with the catalytic Pfup41 subunit.

View larger version (57K): [in this window] [in a new window]   Fig. 4.   DNA binding activity of Pfup41, Pfup46, and the p41-p46 complex. A, the primase proteins (300 nM) were incubated with 32P-labeled DNAs (50 nM): the single-stranded DNA (left), the primed DNA (middle), and the double-stranded DNA (right), at 55 °C for 5 min. The reaction products were analyzed by 1% AGE followed by autoradiography. Lanes: 1, no protein; 2, Pfup41; 3, Pfup46; 4, p41-p46. B, the increasing amount of proteins (50, 100, 250, 500, and 750 nM) were incubated with the 32P-labeled single-stranded DNA (50 nM). The conditions of reaction and detection are the same as those in A.

De Novo Synthesis of DNA Strands by p41-p46-- We previously found that the Pfup41 protein synthesizes long DNA segments (up to several kilobases) on the template DNA without a primer (22). Therefore, we tried this reaction using the p41-p46 complex and found that it also synthesizes DNA strands de novo. The amount of the synthesized products was drastically increased than that from Pfup41 alone. Quantification of the signals of these products showed ten times difference. However, the sizes of the DNA strands were less than 700 bases, which are notably shorter than those from Pfup41 (Fig. 5A). No increase of the product size was observed with longer reaction times (Fig. 5B). To investigate whether the synthesized primers can be extended by DNA polymerases from P. furiosus, pol I and pol II were added with their auxiliary proteins to the primase reaction mixtures containing M13 mp18 single-stranded DNA, dNTP (containing [-³²P]dCTP), and p41-p46 complex. As shown in Fig. 5C, in the presence of PfuPCNA and PfuRFC, pol I and pol II extended the primers. DNA (primer) synthesis activity of p41-p46 complex was not stimulated by PfuPCNA and PfuRFC. Furthermore, no product was observed when the p41-p46 complex was omitted from the reaction mixtures. These results indicate that the p41-p46 complex synthesized the primers, which can be extended in the combination with other replication proteins from P. furiosus.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   A, de novo DNA synthesis activity of Pfup41 and the p41-p46 complex. The reaction mixture, containing M13 single-stranded circular DNA, dNTP including [-32P]dATP, and Pfup41 or the p41-p46 complex, was incubated at 70 °C for 20 min, and the reaction products were analyzed by 1% alkaline AGE followed by autoradiography. Lane M, the size marker was prepared by 5' labeling by 32P of the BstPI-digested lambda DNA. B, a time course experiment of the primer synthesis on M13 single-stranded DNA by the p41-p46 complex in vitro. The reaction mixture indicated in A was incubated at 70 °C, and aliquots were taken after 3, 10, 30, 45, and 60 min, respectively, and the reaction products were analyzed by 1% alkaline AGE followed by autoradiography. C, DNA synthesis reactions on M13 single-stranded DNA were carried out using p41-p46, pol I, and pol II at 70 °C for 20 min. PfuPCNA and PfuRFC were added to stimulate the reactions of pol I and pol II as described previously (18).

RNA Primer Synthesis by the p41-p46 Complex-- Primases from Bacteria and Eukarya synthesize ribooligonucleotides as the primer. However, in our previous study little activity to synthesize the ribooligomer was detected in Pfup41 in vitro (22). To investigate whether the p41-p46 complex can synthesize an RNA primer, we performed the assay using M13 mp18 single-stranded DNA and an NTP mixture containing [-³²P]UTP and found that the p41-p46 complex synthesized 12-40-base-long RNA primers. The RNA primers were extended by pol I, pol II, and also by the p41-p46 complex itself, when the dNTP mixture was added after the synthesis reaction of the RNA primers (data not shown). In an attempt to determine which type of primer (DNA or RNA) is synthesized de novo in the P. furiosus cells, we tried the primer synthesis assay in the presence of both dNTPs and NTPs. The primase complex synthesized RNA segments only when NTPs (containing [-³²P]UTP), but not dNTPs, were added as the substrates, as described above. However, we detected a dose-dependent decrease in labeled RNA products with increasing dNTP concentrations of the reactions. When the product amount from the reaction with only NTPs is 100%, the products were decreased to 57%, 41%, and less than 15% in the presence of dNTPs at 1/1000, 1/100, and 1/10, respectively, relative to the amount of NTPs. When both dNTPs and NTPs existed at equal concentrations, no RNA products were observed from the reaction containing [-³²P]UTP (data not shown). This result indicates that the p41-p46 primase preferentially uses dNTPs. However, in the opposite case of detecting a DNA primer using [-³²P]dCTP, the DNA strand synthesis was stimulated by the addition of NTPs. By addition of NTPs to 1, 10, and 100 µM, the amounts of the reaction products were increased to 1.7-, 4.0-, and 5.3-fold, respectively, relative to the product from the reaction without NTPs. This stimulation of DNA strand synthesis was also observed with ATP to the same level as NTPs (data not shown). Furtheremore, the in vitro replication assay of the M13 mp18 single-stranded DNA using P. furiosus replication proteins, p41-p46, pol I, PfuPCNA, and PfuRFC, as described above, revealed that the amount of full-length product was increased by the addition of ATP, but not by the other three nucleotides (Fig. 6, A and B). When a synthetic DNA primer was annealed to M13 DNA template instead of de novo primer synthesis by p41-p46 in the reaction, no enhancement of production was observed by either ATP or NTPs (Fig. 6C). These results suggest that the primer synthesis possibly starts by RNA using ATP de novo in the initiation step of DNA replication in P. furiosus, as found in other organisms of the different biological domains. To investigate the initiation step of the primer synthesis more directly, the reactions using [-³²P]ATP and cold dNTPs were performed. As shown in Fig. 7, synthesized strands of 700 bases in length were detected by autoradiography. The amount of the labeled products was clearly decreased with increased additions of cold ATP. The labeled products were slightly decreased by addition of cold GTP, and no effect was observed by cold CTP and UTP. The amount of the ATP-labeled products was not decreased by addition of increasing amount of cold dATP in the reaction mixtures (data not shown). These results suggest that the p41-p46 primase can discriminate ATP from other NTPs to start primer synthesis.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   ATP stimulates the in vitro replication of M13 single-stranded DNA by P. furiosus proteins. The full-length product (7.2 kilobases) of the reactions containing different concentrations of NTPs were separated by 1% alkaline AGE (A), and the protein bands were quantified from the autoradiogram (B). The reaction product did not increase by addition of NTP when a synthetic DNA oligomer was annealed to M13 single-stranded DNA as a primer (C).

View larger version (104K): [in this window] [in a new window]   Fig. 7.   Synthesis of the 5'-terminal labeled primers by [-32P]ATP was clearly inhibited by addition of cold ATP, but not other NTPs. The reaction mixture containing M13 mp18 ssDNA, p41-p46 complex, [-32P]ATP, and dNTPs was incubated at 70 °C for 45 min with increasing amount of NTPs (0.5, 5, and 50 µM, respectively). The reaction products were analyzed by 8% PAGE containing 8 M urea followed by autoradiography. Lane  indicates the reaction without cold NTPs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We identified the DNA primase complex in the hyperthermophilic archaeon, P. furiosus. Pfup41 and Pfup46, which have some sequence similarity to the eukaryotic primase subunits, exist as a stable complex and may work as the primase in the replication machinery of P. furiosus. The eukaryotic primases exist as a complex with pol . By analogy with this case, we investigated whether the P. furiosus p41-p46 complex directly interacts with P. furiosus pol I (pol BI), because pol I is the only DNA polymerase that belongs to the family B DNA polymerases, including eukaryotic pol , in P. furiosus (8, 11). No coprecipitation of Pfup41 and pol I was observed with either anti-Pfup41 or anti-pol I (Fig. 3). The results suggest that pol I is not the functional counterpart of the eukaryotic pol  in P. furiosus. In the eukaryotic replication machinery, it is believed that the de novo synthesis of RNA primer is performed by p48, and then the primers are extended by pol  by translocation of the active sites from p48 to p180 in the pol -pri complex. Based on the evidence that the P. furiosus p41-p46 complex can synthesize short RNA segments and long DNA segments by itself in vitro, the p41-p46 complex may have dual functions in Archaea: de novo primer synthesis and its elongation. The synthesized DNA strands are then presumably further extended by the replicative DNA polymerases, pol I and/or pol II. The interactions of the PfuRPA with other replication proteins (pol I, pol II, PfuPCNA, and PfuRFC), including the primase, as we showed previously by the immunoprecipitation experiments (19, 22), support the idea that the p41-p46 complex is involved in the replisome in P. furiosus cells. In eukaryotes, both pol  and pol  have proofreading activity and express high processivity when combined with PCNA. Our preliminary results showed that neither Pfup41 nor the p41-p46 complex has 3'-5' exonuclease activity, which suggests that the archaeal primase has very low fidelity.² It would be detrimental to the cells that an enzyme without proofreading activity synthesizes long DNA strands by itself. The Pfup41 subunit synthesizes long DNA strands up to several kilobases in vitro, and therefore, Pfup46 may have an essential function to suppress this long DNA synthesis. In the eukaryotic system, after the primase synthesizes a primer, pol  and primase become coordinated such that further primer synthesis is negatively regulated by the formation of the stable primer-template formation, which is likely to be associated with pol -primase (25, 26). Moreover, it has been suggested that the eukaryotic p58 subunit mediates the transfer of primers from the primase active site to pol  (26). The results of our study imply that the archaeal p46 subunit has similar functions as those of the eukaryotic p58, although the archaeal primase may extend the strand by using dNTPs from the RNA primer by itself without switching to any other polymerase, as described above.

It would be very interesting and important to determine which primer (RNA or DNA) is synthesized de novo in the DNA replication process in P. furiosus cells. Our in vitro studies could not reach this conclusion. It is known that the eukaryotic primase forms a primase-ssDNA-NTP-NTP quaternary complex to start the RNA primer on the template, and purine nucleotides are more preferable than pyrimidines to form this initiation complex. This initiation of primer synthesis is the rate-determining step (27). In the case of the human primase, the p48-p58 complex, but not p48 alone, can initiate the dinucleotide synthesis using ribonucleotide triphosphates (24). Our data showing that the p41-p46 complex, but not p41 alone, can synthesize RNA primer are consistent with the idea that the role of the second subunit is to assist in primer initiation. Furthermore, the fact that ATP stimulated the strand synthesis reaction of the p41-p46 primase (Figs. 6 and 7) implies that the archaeal primer starts with ribonucleotide(s) in the cells. Isolation and characterization of the Okazaki fragments from the archaeal cells are necessary for further clarification.

The sequence search for the primases in the total genomes of several archaeal organisms has yielded some interesting aspects. All of the euryarchaeal organisms, including M. jannaschii, Methanothermobacter thermoautotrophicus (Methanobacterium thermoautotrophicum), Archaeoglobus fulgidus, and Halobacterium sp. NRC-1, as well as Pyrococcals, have open reading frames homologous to Pfup41 and Pfup46. The two ORFs are located in tandem in the genomes of the Pyrococcals and the M. thermoautotrophicus and probably form an operon. It is very interesting that M. thermoautotrophicus, A. fulgidus, and Halobacterium sp. NRC-1 have two p41-like ORFs in their genomes. In the crenarchaeal organisms, one ORF similar to p41 was found in the total genome sequences of Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, respectively. On the other hand, a p46-like ORF is found in Sulfolobus, but not in A. pernix from our analyses. The p46 subunit of A. pernix DNA primase may be highly divergent from other archaeal primases. The sequence analysis of the eukaryotic type DNA primases are also shown on a website (chem-mgriep2.unl.edu/replic/EukPri1AA.html). The COG (clusters of orthologous groups of proteins) data base (www.ncbi.nlm.nih.gov/COG/) includes one ORF from A. pernix in the multiple alignment of the eukaryotic p58-like proteins. Further analyses are necessary to understand the conservation and the diversity of the archaeal primases.

The isolation of the novel Pfup46 protein and demonstration of archaeal primase function presented in this study significantly contributes to the understanding of the DNA priming process in archaeal DNA replication. Furthermore, due to the similar structure and function of the primase proteins, detailed studies on the archaeal primase function will give further insights into the molecular mechanism of more complicated eukaryotic DNA replication.

	ACKNOWLEDGEMENTS

We thank M. Yuasa and T. Ishigaki for technical assistances. We are grateful to Dr. Y. Shimura, the director of Biomolecular Engineering Research Institute, for continuous encouragement.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Environmental Health, University of Washington, Seattle, WA 98195.

Present address: Faculte de Medicine, Universite Laval, Quebec G1K 7P4, Canada.

^** Present address: Dept. of Animal Sciences, University of Illinois, Urbana, IL 61801.

To whom correspondence should be addressed: Dept. of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan. Tel.: 81-6-6872-8208; Fax: 81-6-6872-8219; E-mail: ishino@beri.jp.

Published, JBC Papers in Press, October 2, 2001, DOI 10.1074/jbc.M106391200

² S. Ishino and Y. Ishino, unpublished results.

	ABBREVIATIONS

The abbreviations used are: pol -pri, polymerase -primase complex; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; RPA, replication protein A; ORF, open reading frame; ss, single-stranded; ds, double-stranded; AGE, agarose gel electrophoresis; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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