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Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii
Introduction
Materials And Methods
Materials
Computer analysis of protein sequences
Cloning of MJ0839 ORF
Expression and purification of the MJ0839 ORF-encoded protein
DNA primase activity assay
pH optimum
Thermostability and thermophilicity
Results
Identification of putative DNA primases in archaea
Overexpression and purification of Mjpri
Biochemical characterisation of Mjpri
Thermostability and thermophilicity of Mjpri activity
Discussion
Acknowledgements
References
Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii
Received July 23, 1999; Revised and Accepted September 29, 1999
ABSTRACT We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase [alpha]-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg2+, Mn2+ or Zn2+, and is additionally stimulated by the monovalent cation K+. A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.
INTRODUCTION
DNA primases are essential components of the DNA replication machinery. A primase initiates DNA replication by synthesising short oligoribonucleotides on single-stranded DNA templates, which serve as primers both for the initiation of replication at the origin and for the initiation of the Okazaki fragments on the lagging strand (1).
DNA primases from various viral, bacterial and eukaryotic sources have been isolated and biochemically characterised. In eukaryotes, the DNA primase activity co-purifies with DNA polymerase [alpha]. The polymerase [alpha]-primase complex consists of four subunits with approximate molecular masses of 180, 70, 60 and 50 kDa, which are highly conserved in a wide range of eukaryotic organisms. The p180 polypeptide is responsible for the polymerase activity(2), while the DNA primase is a heterodimer of the 60 and 50 kDa polypeptides. In a number of species the p50 subunits have been shown to be sufficient for RNA primer synthesis (3-5), although in some cases both subunits have been reported to be necessary for the primase activity (6). The function of the p60 subunit is still poorly understood, although it appears to stabilise the enzyme activity and is required for the binding of the primase heterodimer to the p180 polymerase [alpha] polypeptide (7). The 70 kDa protein species has no known enzymatic activity, is possibly involved in the interaction with the other proteins bound to the origin of replication and is phosphorylated in a cell-cycle dependent manner (8,9).
The DNA polymerase [alpha]-primase complex normally catalyses the synthesis of oligoribonucleotides with a length of between 7 and 12 residues. In the absence of dNTPs multiples of these `unit-length' primers are produced (3,4,10,11). Although there is no evidence for sequence specificity in priming, DNA primases show in vitro a strong preference for pyrimidine homopolymers (12,13).
No sequence similarity can be detected between the eukaryotic p50 and p60 subunits and the primases purified from bacteriophages and bacteria.
Recent genomic studies on archaea have revealed a profound evolutionary distinction between the informational and metabolic facets of the cell: while the metabolic aspects are more similar to those seen in bacteria, the processes that involve genomic structure and organisation resemble their eukaryotic counterparts. In particular many proteins involved in eukaryotic DNA replication have close homologues in archaea. Yet the current picture of archaeal DNA replication is fragmentary, and some obviously necessary components have not been identified.
No protein responsible for DNA primase activity has been characterised in archaea. When the Methanococcus jannaschii genome sequence (14) is searched for open reading frames (ORFs) encoding homologues of bacterial or eukaryotic primases, a putative primase can be identified (ORF MJ0839), based on partial sequence homology with the p50 subunit. Despite the low overall similarity (16-20% identity) a number of motifs are present that are conserved in all the known sequences of eukaryotic primases.
Here we report the purification and preliminary biochemical characterisation of the M.jannaschii primase (Mjpri) expressed in Escherichia coli. We have shown that in vitro Mjpri alone is capable of synthesising oligoribonucleotides on single-stranded DNA templates. We have also studied the thermostability of the protein and its optimal temperature, pH and metal requirements.
MATERIALS AND METHODS
Materials
All chemicals were reagent grade. DNA restriction and modifi-cation enzymes were from Roche Diagnostics, unless otherwise stated. Oligonucleotides were synthesised by Primm S. R. L. (Milan, Italy). All the radioactive reagents were purchased from Amersham Life Science Products.
Computer analysis of protein sequences
The M.jannaschii genome database was searched using the BLASTP program (15). Multiple sequence alignments were generated with the ClustalX program (16).
Cloning of MJ0839 ORF
The synthetic oligonucleotide primers: Mjpri5[prime] (5[prime]-CTGG-GATCCGATGAATACTTTTGCTGAGGTTCAAAAATTGTATAGGGAATATTACAACTT-3[prime]) and Mjpri3[prime] (5[prime]-CTGA-AGCTTTTAGGATTTAAGTAATTCAAATTTAATATTAT-GACCAAATAAATATAGTAA-3[prime]) were used in a PCR experiment to amplify the MJ0839 gene and to insert HindIII and BamHI restriction sites at the 5[prime] and 3[prime] ends, respectively. PCR was performed with 0.1 µg of M.jannaschii genomic DNA as the template, 100 pmoles of each primer in a final volume of 50 µl and the Expand High Fidelity PCR system from Roche Diagnostics according to the manufacturer's instructions. Each cycle was set for 1 min of denaturation at 94°C, 1 min of annealing at 58°C and 1 min of elongation at 72°C. Forty reaction cycles were carried out in a thermal cycler. The amplified product was purified from an agarose gel, digested with HindIII and BamHI, and cloned into a pPROEX-Hta expression vector (Gibco-BRL), to obtain the construct pPROEX-Mjpri. This plasmid was sequenced and subsequently used to transform E.coli XL1-blue cells for plasmid preparation and protein expression.
Expression and purification of the MJ0839 ORF-encoded protein
Growth of recombinant E.coli cells. Single colonies of E.coli XL1-blue cells harbouring the pPROEX-Mjpri plasmid were inoculated into 50 ml of LB medium (containing ampicillin at 100 µg/ml). After overnight growth at 37°C, this culture was used to inoculate 6 l of fresh medium supplemented with ampicillin. When the culture reached an optical density of ~0.8 at 600 nm, protein expression was induced by addition of isopropyl-[beta]-D-thiogalactopyranoside (IPTG) at 0.5 mM final concentration. Cells were grown for additional 3 h at 30°C and then collected by centrifugation at 10 000 g for 10 min at 4°C. The pellet was stored at -20°C.
Purification by affinity chromatography. The cell pellet was thawed and resuspended in 100 ml of 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole, 10% glycerol (buffer A). Lysozyme at 1 mg/ml and phenylmethylsulfonylfluoride (PMSF) at 1 mM were added. This suspension was incubated on ice for 30 min and then at room temperature for 5 min. Cell lysis was performed by sonication and the resulting lysate clarified by centrifugation for 30 min at 27 000 g.
The crude extract was passed through a 0.45 µm filter and loaded onto a Hi-trap chelating column (5 ml, Pharmacia) charged with Ni2+ ions, connected to a FPLC apparatus (Pharmacia). The column was extensively washed with buffer A and then a 20 ml linear gradient from 5 to 100 mM imidazole applied, followed by a 10 ml washing step at 100 mM imidazole and a 45 ml linear gradient from 100 to 375 mM imidazole. Fractions containing a 44.7-kDa protein product, as assessed by SDS-polyacrylamide gel electrophoresis, were centred around 200 mM imidazole. These fractions were pooled and dialysed overnight against buffer B (100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 40 mM KCl, 0.5 mM EDTA, 10% glycerol).
(iii) TEV protease cleavage. Recombinant His-tagged TEV protease (0.5 mg) was added to the dialysed protein (~10 mg of His-tagged primase in 35 ml) together with 2-mercaptoethanol at 1 mM. The endoprotease digestion was carried out for 16 h at 4°C. Removal of the tag was tested by denaturing gel electrophoresis on a small aliquot of the reaction mixture. The digested sample was subsequently loaded onto the Ni2+ column to remove the His-tagged TEV protease and any trace of uncleaved primase. The recombinant untagged protein was collected in the flow-through fraction (volume 40 ml). This sample was dialysed overnight against buffer C (50 mM MOPS, pH 7.2, 20 mM KCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 10% glycerol).
(iv) Mono S chromatography. The dialysed sample was loaded onto a Mono S column (HR 5/5, Pharmacia) equilibrated in buffer C. The bound protein was eluted with a 30 ml linear gradient from 0 to 0.75 M NaCl in buffer. Fractions containing the recombinant protein were pooled and dialysed against buffer D (50 mM MOPS, pH 7.2, 100 mM NaCl, 20 mM KCl, 10 mM MnCl2, 1 mM 2-mercaptoethanol). The dialysed sample was concentrated with a Centricon-30 microconcentrator (Amicon) to ~2 mg/ml. One volume of pure glycerol was added and the sample was stored at -20°C. The final yield of purified Mjpri was ~1 mg of protein per litre of cell culture.
DNA primase activity assay
The routine reaction mixture contained 50 mM glycine-NaOH buffer, pH 9.0 (at 25°C; pH 8.06 at 60°C), 20 mM KCl, 10 mM MnCl2, poly(dT)400 or poly(dC)2500 at 40 ng/µl, 0.1 mM [[alpha]-32P]ATP (or 0.1 mM [[alpha]-32P]GTP) with a specific activity 1.5 µCi/nmol, 1-2 µM DNA primase in a final volume of 20 µl. The reactions were carried out for 10 min at 60°C in a thermostatic block (equipped with a heated lid to prevent evaporation) and were stopped by adding 10 µl of a solution containing 97.5% formamide, 10 mM EDTA, pH 7.5, 0.3% xylene cyanole and 0.3% bromophenol blue. Samples were incubated for 5 min at 95°C and loaded onto a 20% polyacrylamide/urea gel. After the electrophoretic run, the wet gel was subjected to autoradiography and the radioactivity quantified using a phosphorimager (Molecular Dynamics).
pH optimum
The dependence of DNA primase activity on pH was assayed at 60°C in the presence of buffers adjusted to the desired pH at 60°C. The buffer systems used (50 mM final concentration) were as follows: imidazole-NaOH (pH 5.5 and 6.0); Tris-HCl (pH 6.71, 7.12 and 7.6); glycine-NaOH (pH 8.06, 8.52 and 9.52). The reaction mixtures (with a final volume of 20 µl) contained 10 mM MnCl2, 20 mM KCl, 40 ng/µl poly(dT) or poly(dC), 0.1 mM [[alpha]-32P]ATP (specific activity 1.5 µCi/nmol) and 2 µM DNA primase. The reactions were carried out for 10 min at 60°C and the products resolved by denaturing gel electrophoresis, as previously described. The amount of radioactivity in each lane was determined by phosphoimagery.
Thermostability and thermophilicity
The thermal stability of the DNA primase activity was measured by incubating enzyme solutions (protein concentration 0.7 mg/ml in buffer D) at 60, 70 and 80°C. At the indicated times, aliquots of the incubated enzymes were withdrawn, cooled on ice, and assayed at 60°C under standard conditions. The dependence of the activity on the temperature was determined by assaying aliquots of the enzyme in the reaction mixture at temperatures ranging from 55 to 80°C, using poly(dT) or poly(dC) as a template.
RESULTS
Identification of putative DNA primases in archaea
Analysis of the M.jannaschii genome database (14) with the program BlastP (15) pinpointed one ORF (MJ0839) coding for a protein with some sequence similarity to the eukaryotic DNA primase subunit p50. The translated sequence (Mjpri) corres-ponded to a polypeptide of 350 amino-acid residues, with a molecular weight of 41.8 kDa. Similar sequences were also identified in the genome sequences of other archaeal species [Methanobacterium thermoautotrophicum (17), Pyrococcus horikoshii (18), Archeoglobus fulgidus (19)].
A multiple sequence alignment of some eukaryotic primase small subunits and their archaeal homologues was produced using the program ClustalX (16) (Fig. 1). While the eukaryotic sequences are well conserved (with >30% of the residues invariant), the homology within the archaeal family is lower (generally 22-24% identity), not much higher than the level of sequence identity found between the archaeal and eukaryotic proteins (16-20%). Despite the low sequence homology, a number of motifs are present that are conserved across all the known sequences of archaeal and eukaryotic primases. No homologues of the large primase subunit (p60) could be detected in any of the archaeal genomes.
Figure 1. Alignment of DNA primase sequences from eukaryotic and archaeal species obtained using the program ClustalX (16). The organisms and the protein accession numbers are: Sce, S.cerevisiae (P10363); Spo, Schizosaccaromyces pombe (O14215); Hsa, Homo sapiens (P49642); Mmu, Mus musculus (P20664); Dme, Drosophila melanogaster (Q24317); Afu, A.fulgidus (O29516); Mth, M.thermoautotrophicum (O26685); Pho, P.horikoshii (O57934); Mja, M.jannaschii (Q58249). Amino acid residues that are identical in at least 7 out of the 9 sequences are identified with a hash sign; amino-acid residues that are identical within the eukaryotic family (Sce, Spo, Hsa, Mmu, Dme) are identified with a plus sign, while residues that are identical within the archaeal family (Afu, Mth, Pho, Mja) are identified with a cross. Residues that are conserved within both the eukaryotic and archaeal families but are not identical between the two groups are identified with a star.
Overexpression and purification of Mjpri
To determine whether the ORF MJ0839 truly encoded a DNA primase, the corresponding protein was produced in E.coli, purified and biochemically characterized. The ORF was directly amplified by PCR using M.jannaschii genomic DNA as a template with primers that contained restriction sites for the cloning of the amplified product into the plasmid pPROEX-Hta. This expression vector is designed to introduce at the N-terminal end of the recombinant protein a hexa-histidine tag, which can be removed by using the highly specific endoprotease from the tobacco etch virus (TEV) (20). The recombinant protein was found to be largely soluble and could be purified from E.coli cell extracts by affinity chromatography using a Ni2+-chelating column. After digestion with recombinant His-tagged TEV protease the protein sample was again loaded onto the metal-chelating column and the untagged primase collected in the flow-through fraction. A final chromatographic step on a Mono-S cation exchange column yielded a highly purified protein, as shown in Figure 2.
Figure 2. Purification of recombinant Mjpri. His-tagged Mjpri was produced in E.coli cells using the vector pPROEX-Hta (Gibco-BRL). After each purification step protein samples (0.5 µg per lane) were subjected to SDS-PAGE. Lane 1, molecular mass markers; lane 2, pool from the nickel affinity chromatography (the slightly lower mobility is due to the additional N-terminal hexa-histidine tag); lane 3, protein sample digested with TEV protease to remove the tag; lane 4, pool from the second metal-chelate chromatography; lane 5, protein sample eluted from the Mono S column.
Biochemical characterisation of Mjpri
To test the DNA primase activity of the recombinant protein, synthesis of radiolabeled oligoribonucleotides on a single-stranded DNA template was monitored by separating the reaction products on denaturing polyacrylamide gels and visualising them by autoradiography and phosphoimagery. Mjpri was able to utilise as templates oligopyrimidine molecules, such as the synthetic homopolymers poly(dT) and poly(dC) (Figs 3 and 4) and single-stranded DNA from phage M13 (data not shown). On the other hand, the enzyme was almost completely inactive on a poly(dA) synthetic template.
Figure 3. The influence of pH on Mjpri activity was studied on poly(dT) as a template, as described in Materials and Methods. The buffer systems, which were adjusted to the desired pH values at 60°C, were as follows: 50 mM imidazole-NaOH; 50 mM Tris-HCl; 50 mM glycine-NaOH. Reaction products were separated on 20% polyacrylamide/urea gel and quantitated by phosphoimagery. The results are reported in (A) and expressed as percentage relative to the maximal value obtained in 50 mM glycine-NaOH, pH 8.06. The autoradiogram of the gel is shown in (B):lane 1, control reaction without enzyme; lanes 2 and 3, reactions in 50 mM imidazole-NaOH pH 5.5 and 6.0, respectively; lanes 4-6, reactions in 50 mM Tris-HCl, pH 5.71, 7.12 and 7.6, respectively; lanes 7-9, reactions in 50 mM glycine-NaOH, pH 8.0, 8.52 and 9.52, respectively. The position of the unit-length monomer (7mer) and dimer (14mer) is shown on the left.
Figure 4. Dependence of Mjpri activity on metal ions. The influence of some divalent cations on Mjpri activity was tested either on poly(dT) (A and C) or on poly(dC) (B and D) templates. Activity assays were carried out at 60°C for 10 min using 20 pmol of DNA primase, which had been dialysed against buffer D without MnCl2. (A and B) Autoradiograms of 20% polyacrylamide gels showing de novo synthesis on poly(dT) in the presence of [[alpha]-32P]ATP and on poly(dC) in the presence of [[alpha]-32P]GTP, respectively. Lanes 1-3 represent reactions with Zn2+, Mg2+ and Mn2+ at 10 mM, respectively. (A) Lane C represents a control reaction with the enzyme omitted. (C and D) Bar graphs representing ATP and GTP incorporation by Mjpri on poly(dT) and poly(dC), respectively. To quantitate the pmoles of nucleotide incorporated by phosphoimagery, [[alpha]-32P]ATP or [[alpha]-32P]GTP (3 nCi) were loaded onto the gels for the last 5 min of the run and used as a standard.
The effect of pH on the enzyme activity was studied in the range 5.5-9.5 using poly(dT) as a template. As shown in Figure 3A, the incorporation of radiolabeled ATP shows a peak at pH 8.06 in 50 mM glycine-NaOH. In the pH range 6.0-7.6 the main reaction products are oligoribonucleotides of 7 and 14 bases (Fig. 3B, lanes 3-6), whereas at pH 8.06 and pH 8.52 longer products were also detected (Fig. 3B, lanes 7 and 8).
The primase activity was dependent on the presence of divalent cations, as shown in Figure 4A. The influence of Zn2+, Mg2+ and Mn2+ ions at 1, 10 and 50 mM on Mjpri synthetic function was tested on poly(dT) and poly(dC) templates. In both cases, maximal activity was measured in the presence of Mn2+ ions at 10 mM (Fig. 4C and D). The Mn2+ dependence of the primase activity was investigated in the presence of increasing MnCl2 concentrations (1, 2.5, 5.0, 10, 20, 50 and 100 mM). The addition of 1 mM MnCl2 was sufficient to promote oligoribonucleotide synthesis, with maximal ATP/GTP incorporation obtained in the presence of 10 mM Mn2+, whereas at higher ion concentrations a noticeable decrease in activity was observed. The length distribution of products did not appear to be influenced by the Mn2+ ion concentration.
A substantial effect of the monovalent cation K+ on Mjpri activity was also observed. On either poly(dT) or poly(dC) templates in the presence of 10 mM MnCl2 the synthetic function was stimulated by potassium ions. The maximal effect was obtained in the presence of 20 mM KCl [5- and 2-fold activation on poly(dT) and on poly(dC), respectively].
Thermostability and thermophilicity of Mjpri activity
The heat resistance of Mjpri activity was investigated at 60, 70 and 80°C, as reported in Figure 5. Samples of the purified enzyme were preincubated at each temperature, withdrawn at the indicated times, quenched on ice, and tested for residual activity under standard conditions. Mjpri activity was found to be relatively stable at 60 and 70°C, with a loss of ~20 and 30% of the initial activity observed after 1 h incubation at these temperatures, respectively. On the other hand, the synthetic function showed a half-life of ~15 min at 80°C (Fig. 5).
Figure 5. Thermal stability of Mjpri activity. The heat resistance of the DNA primase activity was tested at 60, 70 and 80°C, as indicated in Materials and Methods.
The Mjpri activity was greatly influenced by the assay temperature. As shown in Figure 6A, when poly(dT) was used as a template, the activity reached a maximum at 60°C, followed by a rapid decrease at higher temperatures. On the other hand, on a poly(dC) template the enzyme was maximally active in a broader range of temperature (from 60 to 75°C; Fig. 6B). This behaviour may well be a result of the different melting temperatures of the synthesis products. In fact, above 60°C the poly(dC)-oligo(G) molecules are significantly more stable than poly(dT)-oligo(A) duplexes, and therefore the noticeable activity decrease on poly(dT) at temperatures higher than 60°C may be due mainly to the melting of synthesis products and only to a small extent due to enzyme inactivation. This is in agreement with the observation that after 10 min incubation at 70 and 80°C the enzyme loses 20 and 40% of its activity, respectively.
Figure 6. Effect of the temperature on Mjpri activity. The dependence of the primase activity on the temperature was determined by assaying the enzyme in the standard reaction mixture at the indicated temperatures, using poly(dT) (A) or poly(dC) (B) as a template. The reaction products were separated on a 20% polyacrylamide/urea gel and quantitation of the radioactivity carried out by phosphoimagery.
DISCUSSION
Despite the fact that archaeal genomes appear to be more similar to their bacterial counterpart (with circular chromosomes, lack of introns in mRNA, genes arranged in operons and DNA not contained in a nucleus), the recent publication of the complete genome sequence of some archaeal species has revealed that DNA replication machinery of archaea is more similar to eukaryotes than to bacteria (21). Our present knowledge of the DNA replication process in archaea is still poor: the DNA sequence acting as origin of duplication and the factors involved in replication fork formation and progression have not yet been studied in archaeal species. In particular the details of the initial steps in the reaction mechanism, leading to the synthesis of the first oligoribonucleotide at the origin of replication, are still not known.
ORFs that show sequence similarity to the eukaryotic DNA primase small subunit can be identified in the genome of M.jannaschii, and in a number of other archaea, whereas no ORF encoding a protein similar to the large subunit could be detected in any of these organisms. We have overexpressed the p50 M.jannaschi homologue in E.coli and purified to near homogeneity the corresponding polypeptide (Mjpri). In this report we have demonstrated that recombinant Mjpri is able to synthesise de novo oligoribonucleotides on various single-stranded DNA molecules [poly(dT), poly(dC)], in the presence of divalent cations such as Zn2+, Mg2+ and Mn2+.
Although the primase activity in eukaryotic cells is associated with the p50-p60 heterodimer, a large body of evidence points to the p50 subunit as being responsible for the catalytic activity: affinity labelling studies have shown that this subunit binds the ribonucleotide triphosphate (22), free p50 subunit devoid of any detectable p60 contamination has been found in yeast (3) and, in a variety of different eukaryotic systems, purified or recombinant p50 subunits have been shown to be sufficient for RNA primer synthesis (3-5,23). Some conflicting results may be due to the fact that the p50 activity is very labile in the absence of the large subunit (24-26). The fact that the archaeal homologue of the p50 subunit shows primase activity in vitro only confirms that the catalytic activity of the eukaryotic heterodimer resides within the smaller polypeptide.
The sequences of the primases from archaea are far more divergent than their eukaryotic counterparts, with sequence identities of the order of 22%, as compared with 30% in the eukaryotic p50 family. When the amino-acid sequences of the eukaryotic p50 subunits are aligned (Fig. 1), most of the homologous regions occur in the N-terminal half of the protein: these regions are generally well conserved in the archaea sequences. One striking exception is the occurrence of a deletion in the middle of a highly conserved motif (Arg 162 in the mouse sequence).
A comparison of the three-dimensional structures of various DNA and RNA polymerases shows that, despite a lack of overall sequence similarity, certain structural features are conserved. A central role in catalysis is played by three negatively charged amino acids (Asp or Glu), which are positioned in a similar three-dimensional arrangement in all the known crystal structures, co-ordinating two metal ions involved in the nucleotidyl transfer reaction (27). The eukaryotic DNA primases have no detectable sequence similarity with other DNA or RNA polymerases, but site-directed mutagenesis experiments carried out on the mouse p50 subunit (24) have shown that three absolutely conserved negatively charged residues (Glu 105, Asp 109 and Asp 111 in the mouse sequence) are essential for catalysis. These three residues are part of a motif (ELVFDID) that is well conserved in the archaea primases. Of the other residues that have been mutated in the mouse protein, Arg 162 and Arg 163 have been implicated in nucleotide binding but only the latter is conserved across all the sequences. Asp 114 (possibly involved in recognition of the 5[prime] purine residue) is conserved only in M.thermoautotrophicum and P.horikoshii.
The activity of eukaryotic primases is dependent on the presence of Mg2+ ions, with a broad optimum varying between 5 and 10 mM (3,4). The stability of the catalytic activity of the smaller subunit has been shown to be strongly dependent on the presence of the divalent cations Mg2+ and Mn2+, while for the p50-p60 heterodimer the dependence on the concentration of ions was less critical (26). Similarly, we found that the activity of Mjpri is dependent on the presence of the divalent cations Zn2+, Mg2+ and Mn2+. The maximal activity is measured with 10 mM MnCl2, although it is probable that Mg2+ ions are physiologically more important, due to the higher concentration of these cations in the cell. The primase activity is also stimulated by the presence of the monovalent cation K+ with a maximum at a concentration of ~20 mM. A similar effect of monovalent cations on the activity of the human primases has been reported, with optimal concentration of KCl of ~10-20 mM (6), while a more complicated pattern has been described for the p50 subunit alone by Schneider et al. (26), with K+ having an inhibitory effect on poly(dC) and a stimulatory effect on poly(dT) templates.
Zinc binding motifs have been identified in a number of proteins involved in DNA and RNA recognition. The only conserved motif that has been postulated across all the primase sequences is a potential zinc binding site, which includes cysteine and histidine amino-acid residues, but only in a few primases from bacteria (28-30) and bacteriophages (31,32) has the presence of zinc atoms and the role of the conserved residues been well established. In the eukaryotic primases the proposed Zn-binding motif is located at residues 128-171 in the mouse p50 subunit, and includes the sequence C X2C X35C X2C (where in the Saccharomyces cerevisiae sequence the last cysteine residue is replaced by a serine). Of this motif, only the first two cysteines are conserved in the available archaea sequences (with the exception of the A.fulgidus sequence).
In eukaryotes the DNA primase is stably associated with DNA polymerase [alpha] in the pol [alpha]-primase 4-subunit complex. Multiple DNA polymerases have been identified in several archaeal species, but their biological roles have not yet been elucidated. M.jannaschii, which belongs to the archaeal Euryarcheota subdomain, possesses a monomeric [alpha]-like DNA polymerase (Pol I) and a recently identified dimeric DNA polymerase (Pol II) (33-35). Pol II is made up of a 69 kDa polypeptide, with sequence homology to the small subunit of eukaryotic DNA polymerase [delta], and of a 143 kDa polypeptide with no obvious sequence similarity to other known proteins. In Pyrococcus furious a homologue of Pol II has been expressed and shown to have primer-extension ability and strong 3[prime]-5[prime] exonuclease activity, suggesting that Pol II may be the replicative enzyme (34,36). Due to our lack of knowledge of the details of the DNA replication machinery in archaea, it is unclear whether Mjpri interacts directly with either polymerase (Pol I or Pol II) or if other essential factors involved in the switch between the priming and elongation steps have not yet been identified.
ACKNOWLEDGEMENTS
This work received financial support in the form of grants from Consiglio Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie), from the European Union (Contract B 104-CT96-0488), and from Ministero dell' Università e della Ricerca Scientifica e Tecnologica (Progetto Murst, `Biomolecole per la salute umana') to M.R. G.D. is a recipient of a Marie Curie Research Training Fellowship.
REFERENCES
*To whom correspondence should be addressed. Tel: +39 081 725 7316; Fax: +39 081 725 7240; Email: pisani{at}dafne.ibpe.na.cnr.it
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