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Primase Activity of Human DNA Polymerase α-Primase

DNA polymerase α-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coliexpression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase α-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

After the finding that eukaryotic primase forms an enzyme complex together with DNA polymerase ␣ (8), interest arose to determine the subunits of the complex that carry the catalytic center of the primase. Each of the four subunits of DNA polymerase ␣-primase (pol-prim) 1 was proposed to contain primase activity by biochemical and immunological methods (9 -16).
When the mouse primase was purified to near-homogeneity, it was found to contain two proteins of 55 and 49 kDa, which were comparable to the molecular masses of the two small subunits of Drosophila pol-prim that showed primase activity after separation from the complex (17,18). Thereafter, it was proposed that the 48-kDa subunit of the bovine pol-prim is sufficient for primase activity (14). Soon this result was questioned, since cross-linking studies with photoreactive ribonucleotides suggested that the 58 -60-kDa subunit of pol-prim might also be involved in the synthesis of oligoribonucleotides (19,20). The characterization of a 48-kDa protein from bakers' yeast that had primase activity and that was immunologically related to the 48-kDa subunit of pol-prim seemed to provide a satisfactory solution to the issue (21). However, the discussion concerning the function of the primase subunits was revitalized when the cloning and subsequent expression of pol-prim subunits from different organisms led to a controversy whether p48 alone can synthesize the first dinucleotide (initiation activity) and then elongates it or whether it has only elongation activity and requires p58 for the synthesis of the first dinucleotide (22)(23)(24)(25)(26).
To address the activity of p48 again, we modified the T7 RNA polymerase promoter system of Escherichia coli (27) to allow the co-expression of multiprotein complexes from a single plasmid. The p58 and p48 subunits of human pol-prim were expressed either as wild type or as fusion proteins with oligohistidines at their N terminus. Thus, human primase that contained p58 and p48 was expressed and purified in high yields as a stable and active protein complex. Furthermore, for the first time the vectors that are described here allowed expression and purification of eukaryotic p48 in E. coli as a soluble protein and a fully active primase with regard to initiation and elongation. The addition of magnesium and manganese stabilized its primase activity. The bacterial and baculovirus expressed proteins could be assembled in vitro and formed a four-subunit pol-prim complex that is active in DNA replication.

MATERIALS AND METHODS
Enzymes were obtained from Amersham Pharmacia Biotech (Freiburg, Germany), Boehringer Mannheim (Mannheim, Germany), or New England Biolabs (Schwalbach, Germany). Reagents were purchased from Merck unless otherwise indicated (Darmstadt, Germany).
Protein Manipulations-Protein concentrations were determined according to Bradford (28) using a commercial reagent with bovine serum albumin as a standard (Bio-Rad, Munich, Germany). SDS-gel electrophoresis was carried out as described (29) with a 10-kDa ladder or prestained molecular mass marker proteins (Life Technologies GmbH, Eggenstein, Germany). After polyacrylamide gel electrophoresis, proteins were detected either by staining with Coomassie Brilliant Blue or by Western blot analysis as described (26). DNA polymerase ␣ and DNA primase assays were performed according to Grosse and Krauss (30,31). Bovine pol-prim was purified according to Nasheuer and Grosse (32) and was a generous gift of Hella Förster (Abteilung Biochimie, Institut für Molekulare Biotechnologie). One unit of DNA polymerase is defined as the amount that catalyzes the incorporation of 1 nmol of dAMP into acid-insoluble material in 1 h at 37°C with activated DNA as template primer. One unit of primase is defined as the amount that leads to an incorporation of 1 nmol of dNMP/h at 37°C using the Klenow fragment of E. coli DNA polymerase I for elongation of the newly synthesized RNA primers on poly(dT) as template (30 -32).
Plasmids-The EcoRI restriction site of pET11a and pET15b (AGS, Heidelberg, Germany) was deleted by cutting each plasmid with EcoRI and filling in the ends with Klenow DNA polymerase followed by ligation (33). This yielded the plasmids pET11-⌬EcoRI and pET15-⌬EcoRI.
After digestion of each vector with NdeI and BamHI the two nonphosphorylated oligonucleotides (5Ј-TATGATCATCGGATCCCGGGT-ACCGCGGCCGCGTCGACTAGTAGAAT TCTCGA-3Ј and 5Ј-GATC-TCGAGAATTCTACTAGTCGACGCGGCCGCGGTACCCGGGATCCG-ATGATCA-3Ј) were added. The mixture was heated to 65°C and slowly cooled to room temperature, and the DNA was ligated at 15°C overnight. The plasmids containing the new oligonucleotides were called pET11-MCS and pET15-MCS, respectively. They were used for the expression of single proteins and multisubunit protein complexes (Fig.  1, A and B).
To express the cDNAs that code for human primase subunit p48 and p58, each cDNA was amplified with primer pairs that contain an NdeI site at its first ATG or a BamHI after its stop codon according to standard procedures (26). The cDNAs were cloned into the vectors pET11-MCS after verification of their sequence and pET15-MCS to express the primase subunits as unmodified proteins or fusion proteins with 6xHis, respectively.
To co-express the proteins the vector pET11-Hp48 was digested with SpeI and EcoRI, and the vector pET15-Hp58 was cut with XbaI and EcoRI. The DNAs were separated on agarose gels and purified according to standard procedures (33). The fragment containing the coding sequence of human p58 was ligated into vector pET11-Hp48, and the plasmid pET-Hp48-HisHp58 was created that expressed p48 as an unmodified protein and p58 as a fusion protein.
Expression and Purification of Primase Proteins-E. coli cells BL21(DE3) containing the expression vectors either pET15-Hp48, pET15-Hp58, or pET-Hp48-HisHp58 were grown in terrific broth at 37°C (33) to an optical density of 2 measured at 600 nm. After lowering the growth temperature to 23°C, the expression of recombinant proteins was induced by the addition of 1 mM isopropyl-1-thio-␤-D-galactopyranoside. The cells were harvested 3.5 h after induction, collected at 2000 ϫ g, and washed twice with phosphate-buffered saline (33). Alternatively, p48 was expressed and purified in the presence of divalent cations, as indicated.
Cells that expressed p48 or p58 as single proteins were immediately homogenized in lysis buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 3 mM 2-mercaptoethanol, 0.05 mM leupeptin, and 0.01 mg/ml Trasylol) by sonication. After sonication the homogenate was adjusted to 1% Triton X-100, and then DNA and insoluble proteins were removed by centrifugation at 5000 ϫ g. The supernatant was applied to metal chelate chromatography using Talon resins (CLONTECH, Heidelberg, Germany). Then the resin was washed extensively with buffer 1 (20 mM Tris/HCl, pH 8, 250 mM NaCl, 3.5 mM 2-mercaptoethanol, 1% Triton X-100; about 50 column volumes). After that the resin was washed with 5-column volumes of each buffer 2 (100 mM (NH 4 ) 2 SO 4 , pH 8.2) plus 10 mM imidazole and buffer 2 plus 20 mM imidazole. Proteins were eluted with 100 mM imidazole in buffer 2. For the purification in the presence of divalent cations, lysis buffer, buffer 1, and buffer 2 additionally contained 0.1 mM MnSO 4 (Sigma) or 1 mM MgCl 2 , as indicated. To purify p48 further, the eluted fractions of the metal chelate column were dialyzed against buffer 3 (50 mM Tris/HCl, pH 7.5, 3 mM 2-mercaptoethanol, 1 mM MnSO 4 , and 1 mM MgCl 2 ) plus 80 mM NaCl overnight and applied to phosphocellulose chromatography (buffer 3 and a gradient from 80 to 1000 mM NaCl) using the ÄKTA system (Amersham Pharmacia Biotech). For long term storage of p48, the active fractions were dialyzed against 50 mM potassium phosphate, pH 7.5, 3 mM 2-mercaptoethanol, 0.1 mM MnSO 4 , 1 mM MgCl 2 , and 30% glycerol. The half-life of the primase activity of the dialyzed enzyme was about 2 months at 4°C.
In the case of co-expressed Hp58-Hp48, the cells were frozen and stored at Ϫ80°C until purification. Frozen cells were quickly thawed, and the heterodimer was purified as described above but without divalent cations. The purification yielded a stable protein complex.
The pol-prim was purified from insect cells co-infected with four baculovirus vectors as described (26,34). Alternatively pol-prim was prepared from crude insect cell extracts that contained p180 and p68 mixed with crude E. coli extracts that included recombinant p58 plus p48. After incubation of the combined extracts for 1 h on ice, the four-subunit pol-prim was purified according to Stadlbauer et al. (34).
SV40 DNA Replication in Vitro-S100 extracts were prepared from logarithmically growing mouse FM3A cells as described previously (26,34). The replication of SV40 DNA in vitro was performed as described (34). Briefly, the SV40 assay (60 l) contained 0.6 g of SV40 T antigen, 0.25 g of SV40 origin DNA (pUC-HS), 0.6 g of human RP-A, 300 g of S100 or depleted S100 extract from FM3A cells in 30 mM HEPES-KOH, pH 7.8, 0.5 mM dithiothreitol, 7 mM magnesium acetate, 1 mM EGTA, pH 7.8, 4 mM ATP, 0.3 mM CTP, GTP, and UTP, 0.1 mM dATP and dGTP, 0.05 mM dCTP and dTTP, 40 mM creatine phosphate, 80 g/ml creatine kinase, and 5 Ci each of [␣-32 P]dCTP and [␣-32 P]dTTP (3000 Ci/mmol). Comparisons between different enzyme preparations were made by adjusting the amounts of enzyme added to equal primase activity as indicated. The primase activity was determined at the time the replication assays were performed. The incorporation of radioactive deoxynucleoside monophosphate (dNMP) was measured by acid-precipitation of DNA and scintillation counting.

Expression and Purification of Recombinant Human
Primase in E. coli-The cDNAs of p48 and p58 were expressed separately fused to six histidine residues at their N terminus with the E. coli vectors pET15-Hp48 and pET15-Hp58. After metal chelate chromatography, the purified recombinant p48 subunit reproducibly showed primase activity as determined in the primer elongation assay using Klenow DNA polymerase (Fig.  3A, column 1), whereas the recombinant p58 was inactive (Fig.  3A, column 2). Typically about 1 mg of soluble p48 was purified from 500 ml of bacterial culture ( Fig. 2A, lanes 1-3). Then affinity purified p48 was applied to phosphocellulose chromatography which yielded a single protein and activity peak (data not shown).
Primase preferentially synthesizes products of short size typically 5-10 nucleotides long (8,17,18). To verify the primase activity of the bacterially expressed proteins, the incorporation of NMP was directly determined by separating the synthesis products on denaturating gels and visualizing them by autoradiography ("direct assay"; Figs. 3B and 7). In the direct assay, p58 did not show any primase activity (Fig. 3B, lane 5), and no radioactive products were detectable above background (Fig.  3B, compare lanes 5 and 7). In contrast to this result, p48 efficiently initiated oligoribonucleotide synthesis on different templates such as poly(dC) (Fig. 3B, lanes 1-4; Fig. 7A), poly(dT) (Fig. 7D), poly(dC,dT), and M13ssDNA (data not shown). In addition, in the presence of p48, as well as p58 -48 and pol-prim, long products were produced on poly(dC) that were not fully resolved by the gel electrophoresis (Figs. 3B and 7, A-C). The observed high amounts of oligo/poly(G) primase products that were detected at the top of the gel might be due to a lower solubility of oligo/poly(G) products. Second, the poly(dC)⅐oligo/poly(G) products are extremely stable and they might quickly reanneal after heating to 95°C and loading on the gel. Thus, a great portion of the primase products did not move into gel. These interpretations are consistent with the observation made by other investigators. 2 By using the plasmid pET-Hp48-HisHp58, co-expression of p48 and p58 yielded high amounts of purified two-subunit primase with high specific primase activity using metal chelate chromatography (8 -10 mg per 500-ml culture of bacteria with a specific activity of about 10,000 units/mg; Fig. 2A, lanes 4 and  5; Fig. 3, lane 6; data not shown). Interestingly, the size of products synthesized on poly(dC) by p48 alone and p58-p48 heterodimer varied. The p48 primase synthesized preferentially short products in the range of 5-10 nucleotides and some products longer than 17 nucleotides, whereas the p58-p48 heterodimer predominantly synthesized products that were longer than 17 nucleotides (Fig. 3B, compare lanes 1-4 with lane 6, respectively). The activity of heterodimeric primase was stable for several months at 4°C, and after dialysis against 30% glycerol it could be stored at Ϫ20 or Ϫ80°C. Freezing and thawing was without detectable influence on the activity of p58-p48 primase (data not shown). 2 R. Kuchta, personal communication. FIG. 2. Purification of recombinant proteins. A, the subunit p48 and the heterodimer p58-p48 were expressed in E. coli BL21 (DE3) using the vectors pET-HisHp48 and pET-HisHp58-Hp48. The p48 protein was expressed and purified in the presence of 1 mM Mg 2ϩ by metal chelate chromatography, and the protein-containing fractions were subjected to SDS-gel electrophoresis (lanes 1-3). The two-subunit primase was expressed and purified in the absence of divalent cations, and the eluted fractions 1 and 2 were analyzed by SDS-gel electrophoresis (lanes 4 and 5). P48 (lanes 1-3) and p58 (lanes 4 and 5) have slightly lower mobilities due to the additional histidine residues. B, the p180 and p68 subunits were expressed in insect cells, and Hisp58 and p48 were expressed in E. coli. After the assembly of pol-prim, the foursubunit complex was purified by phosphocellulose and immunoaffinity chromatography (lanes 1 and 2). Lanes M and M1 represent a 10-kDa ladder molecular mass marker and the prestained molecular mass marker, respectively. Divalent Cations Stabilize Primase Activity of p48 -However, the enzymatic activity of p48 expressed in E. coli was extremely unstable and could only be determined if expression, purification, and enzyme assay occurred within a few hours. Lengthy purification, freeze-thawing of bacterial cell pellets, or low concentrations of p48 resulted in a rapid loss of enzyme activity that could not be prevented by 1 mg/ml BSA, 10% glycerol, or 10% ethylene glycol (data not shown). Within 16 h of storage at 4°C, the primase activity of p48 decreased by more than 85% of the starting activity (Fig. 4A, compare columns 1 and 2). At concentrations of p48 that were lower than about 0.4 mg/ml, the half-life of primase activity was even shorter, and the activity decreased to background levels within 3 h (data not shown).
The primase activity of p48 was reproducibly stabilized by the presence of 1-10 mM Mg 2ϩ or 0.1-10 mM Mn 2ϩ (Fig. 4A,  compare column 2 with columns 3-7). In contrast, Zn 2ϩ (1 M to 10 mM; Fig. 4A, columns 8-11), and low concentrations of Mg 2ϩ or Mn 2ϩ (1 M to 0.1 mM or 1-10 M, respectively, and data not shown) were ineffective. On the other hand, with 10 mM Mg 2ϩ or Mn 2ϩ the enzyme activity of p48 was even detected after 14 days of storage at 4°C (data not shown).
Since divalent cations stabilized the primase activity of p48, we compared the influence of Mg 2ϩ on the primase activity of recombinant p48, heterodimeric p58-p48, and heterotetrameric pol-prim (Fig. 4B). The enzymes were diluted in buffer without (open bars 1-3) or with (hatched bars 4 -6) 10 mM Mg 2ϩ , and the activity was measured after incubation at 37°C. As expected the activity of p48 diminished within 10 min in the absence of Mg 2ϩ , and only about 5% activity of the starting activity that was determined before preincubation could be detected (Fig. 4B, column 1). In the presence of Mg 2ϩ the activity of p48 and of the other enzymes was equally stable (Fig. 4B, compare columns 4 -6). In contrast to p48, the presence of 10 mM of Mg 2ϩ did not stabilize the primase activity of either p58-p48 or pol-prim (Fig. 4B, compare columns 2 and 3 with 5 and 6, respectively).
These results prompted us to optimize the production of active p48. The primase activity was measured by the primer elongation assay or directly by analyzing the primase products on denaturing gels. The addition of divalent cations to the purification buffers increased specific primase activity of p48 by a factor of 3 to 4 (Fig. 5, compare columns 1 and 2). The presence of Mg 2ϩ during protein expression and purification raised the specific primase activity by a factor of up to 10 in comparison with the procedure without divalent cations (Fig. 5,  compare columns 1 and 3). These findings were supported by direct assays that were performed in parallel (Fig. 3B, lanes  1-4). It is worth mentioning that the addition of divalent cations to the bacteria decreases the level of expression of p48 by FIG. 3. Primase activity of recombinant human p48. A, the subunits of human primase, p48 and p58, were expressed separately in E. coli BL21 (DE3) using the expression vectors pET-HisHp48 and pET-Hisp58. The recombinant proteins were purified in parallel by metal chelate chromatography. The primase activity of each purified protein fraction was measured by incorporation of radioactive dAMP in the coupled primer elongation assay. B, the products that were synthesized on poly(dC) template by recombinant proteins were separated by denaturing gel electrophoresis and determined by autoradiography using x-ray films (direct assay). Primase activities of these proteins (p48, columns 1 and 4; p58-p48, columns 2 and 5; pol-prim, columns 3 and 6) were measured before and after incubation for 10 min at 37°C using the Klenow elongation assay. The primase activity of each protein fraction that was determined before preincubation was defined as starting activity (100%). a factor of 2 to 3 (data not shown).
Mg 2ϩ Dependence of Primer Synthesis Performed by Primase-To compare the dependence of the recombinant enzymes on Mg 2ϩ , the activity of the primase was investigated in the presence of increasing Mg 2ϩ concentration using the direct assay. The addition of 2.5 mM Mg 2ϩ was sufficient for the synthesis of oligoribonucleotides on poly(dC) by p48 and the p58-p48 heterodimer. The primase activity of these enzymes increased in a concentration-dependent manner until an optimal concentration of 10 mM was reached; 15 mM Mg 2ϩ resulted in a slight decrease of primase activity (Fig. 6).
On the poly(dT) template magnesium dependence of the p48 subunit and the heterodimer p58-p48 differed slightly, since p48 required 5 mM Mg 2ϩ to synthesize oligoribonucleotides efficiently, and with 2.5 mM Mg 2ϩ , primase activity was hardly detectable (Fig. 6). In contrast to these findings, p58-p48 already efficiently synthesized oligoribonucleotides with 2.5 mM Mg 2ϩ . Increasing the Mg 2ϩ concentration to up to 10 mM did not change the length distribution of products synthesized by p58-p48 (data not shown) and only slightly increased the amount of primase products (Fig. 6).
Primase Activity of p48 Is Influenced Differently on Poly(dC) and Poly(dT) Template by Potassium Ions-Activity of primase is inhibited by monovalent cations (7). Therefore, we wanted to compare the primase activity of p48 to that of other primase complexes in the presence of potassium acetate. On both templates p48 synthesized products in the range of 5-12 and 17-22 nucleotides. On poly(dC) the primase activity of p48 was stimulated, and the maximal activity was determined in the presence of 100 mM K ϩ (Fig. 7A, lanes 1-5), whereas on poly(dT) the activity of p48 was readily inhibited by K ϩ in a concentrationdependent manner (Fig. 7D, lanes 1-5). In the presence of 150 mM K ϩ the primase activity was nearly abolished (Fig. 7D, lane  5).
The heterodimer p58-p48 synthesized products on poly(dC) that were longer than 17 nucleotides. The activity was hardly influenced by potassium ions with a maximum of its activity in the presence of 100 mM K ϩ (Fig. 7B, lane 1-5). On poly(dT) recombinant p58-p48 preferentially synthesized products in the range of 5-11 nucleotides in length and some products in the range of 20 and 30 nucleotides. Its activity was reproducibly stimulated by K ϩ up to a concentration of 150 mM (Fig. 7E,  lanes 1-5). The bovine pol-prim synthesized products with com-parable lengths to p48 (Fig. 7, compare C and F with A and D). The activity of pol-prim was stimulated by K ϩ on poly(dC) and on poly(dT) (Fig. 7, C and F, respectively).
The E. coli-expressed Primase Forms a Complex with p180 and p68 That Is Active in SV40 DNA Replication in Vitro-Using a cell-free DNA replication system is a stringent test for the function of a recombinant replication protein. The heterodimeric primase p58-p48 alone cannot initiate SV40 DNA replication and requires the large subunits of pol-prim (data not shown). Therefore, a four-subunit complex was formed by mixing crude bacterial extracts with insect cell extracts that contain the recombinant human p58-p48 and p180-p68 complexes, respectively. Then the assembled four-subunit pol-prim complex was purified by ion exchange and immunoaffinity chromatography to near-homogeneity (Fig. 2B, lanes 1 and 2). The pol-prim assembled in vitro had high specific DNA polymerase and primase activity (7,000 and 4,500 units/mg, respectively) which was comparable to those of recombinant pol-prim from insect cells that were infected with four baculoviruses (7,600 and 5,100 units/mg DNA polymerase and primase activity, respectively).
The replication activity of the in vitro assembled pol-prim complex was tested in extracts from mouse cells that are nonpermissive for SV40. In the absence of human pol-prim the incorporation of radioactive dNMP was just 4 pmol in these extracts (Fig. 8, column 1), and the addition of 1 unit of purified heterodimeric primase did not significantly change the incorporation of dNMPs (Fig. 8, column 2). In the mouse cell extracts the addition of human pol-prim formed in vitro allowed the replication of SV40 DNA in a concentration-dependent manner (Fig. 8, columns 3-5). The incorporation of dNMPs rose from 4 pmol to 13.5, 28, and 35 pmol in the presence of 0.25, 0.5, and 1 unit of pol-prim, respectively (Fig. 8). These results show that human primase that was expressed in E. coli is functional in a eukaryotic DNA replication assay in vitro. p48-and p58-p48-synthesized products of poly(dC) and poly(dT) in the presence of increasing Mg 2ϩ concentrations. After 1 h at 37°C, the reaction was stopped, and the products were precipitated ("Materials and Methods"), resolubilized, and separated by denaturing gel electrophoresis and analyzed with a PhosphorImager. To calculate the amount of synthesized products, the nonspecific background was subtracted from each lane. The amount of product that was synthesized with the specified protein and template in the presence of 10 mM magnesium (optimal concentration) was determined and arbitrarily displayed as 10. The arbitrary units of each template and protein are shown: (q, p48 on poly(dC); E, p48 on poly(dT); ࡗ, p58-p48 on poly(dC); छ, p58-p48 on poly(dT).

DISCUSSION
The initiation activity of primase is an essential function to start DNA replication de novo (2). The exact mechanism of initiation is as yet unknown, and it is still under discussion whether p48 alone can initiate primer synthesis on ssDNA or whether it requires a second subunit p58 for the synthesis of the first dinucleotide (14,(21)(22)(23)(24)(25)(26).
To study their functions the primase subunits were expressed using modified bacterial vectors that also allow the co-expression of protein complexes. With these plasmids, we were able to express and purify the p48 subunit of human pol-prim that is soluble and shows both initiation and elongation activities. However, depending on the expression and purification conditions the enzymatic activity of p48 is highly labile, since low concentrations of p48 or the absence of diva-lent cations leads to a fast loss of enzyme activity which could not be stopped by the addition of 1 mg/ml BSA, 10% glycerol, or 10% ethylene glycol. The instability of p48 primase activity observed here confirms earlier findings with bovine and yeast p48 that had highly unstable primase activity (14,21). The specific requirement of divalent cations for the stability of enzyme activity is underlined by the presence of a metal binding motif in human p48 that is also present in primases from mouse, Drosophila, and bacteriophages T4 and T7 (22, 26, 36 -40). In biochemistry the function of divalent cations during catalysis is well characterized and documented (for reviews see Refs. 2 and 41), but only since quite recently is an additional view of their function becoming more and more accepted. Divalent cations like calcium, magnesium, manganese, and zinc might also influence the stability of enzyme activities inde- FIG. 7. Influence of potassium ions on the primase activity. To compare the influence of the monovalent cation potassium on the activity of recombinant primase and primase from a cellular source, increasing amounts of potassium were added to the assay containing recombinant p48 (A and D), recombinant p58-p48 (B and E), and bovine pol-prim (C and F) that was purified from thymus (A-C using poly(dC), and D-F using poly(dT)). Primase products were synthesized in the presence of 0, 25, 50, 100, and 150 mM potassium acetate (lanes 1-5, respectively). Lane M shows 5Ј-end-labeled oligo(dT) 12-18 markers as indicated on the right. The top of the gels is marked by arrows.
The determined stability of p48 activity allowed us to start characterization of its enzymatic activity and compare it with that of the two-subunit primase and the four-subunit pol-prim. Interestingly, on poly(dT) the cations Mg 2ϩ and K ϩ modulated the primase activity of single subunit p48 differently from that of two-and four-subunit enzyme complexes (Figs. 6 and 7, D-F), whereas on poly(dC), the different primase assemblies behaved comparably and had a broad optimum for both cations (Figs. 6 and 7, A-C; Ref. 14). On poly(dT), efficient primer synthesis by p48 required Mg 2ϩ concentrations higher than 2.5 mM, whereas the two-subunit primase was highly active with 2.5 mM Mg 2ϩ . These results suggest that on poly(dT) p48 might have to overcome a rate-limiting step in the formation of the dinucleotide or its elongation that requires higher Mg 2ϩ concentration. Alternatively, the affinity of p48 to Mg 2ϩ is too low for the initiation with 2.5 mM Mg 2ϩ on poly(dT), and the p58 primase subunit might increase the affinity of p48 for the divalent cation. The further characterization of p48 primase showed that the presence of K ϩ inhibited primase activity of p48 on poly(dT), even concentrations as little as 25 mM were effective, whereas the addition of the monovalent cation stimulated activity of the two-and four-subunit enzyme complexes. Several mechanisms could be the cause of the high sensitivity of p48 primase toward K ϩ , e.g. the binding of p48 to the template poly(dT) or to the substrate ATP might be salt-sensitive. Alternatively, a specific step during the polymerization reaction (either the synthesis of the first nucleotide or the elongation step) could be inhibited by K ϩ . Since recent findings indicate that primase forms a ternary enzyme-template-NTP complex that preferentially contains GTP (52), the different influence of cations on the activity of p48 using poly(dC) or poly(dT) suggests that the p48-poly(dC)-GTP complex might be less sensitive to the concentration of cations than the p48poly(dT)-ATP complex. Taken together these findings indicate that p48 has a complete primase activity that is able to initiate and elongate. The different salt sensitivity of p48 in compari-son to p58-p48 and pol-prim suggests that this activity might be modulated by p58 and the other subunits of the pol-prim complex.
Co-expression of both primase subunits resulted in a highly active and stable primase that could be purified in large quantities. These findings reproduced earlier results that co-expression of mammalian primase subunits had yielded an active primase that was stable for months (23,24,26). The observed lengths of products that were synthesized by the two-subunit primase on poly(dC) prompted us to ask whether the recombinant enzyme was able to support DNA replication in vitro. To this end, we produced a chimeric four-subunit DNA polymerase ␣-primase in vitro that contained the bacterially expressed primase subunits and baculovirus-expressed p180 and p68. This in vitro assembled enzyme complex supports DNA replication in a cell-free system which indicates that the bacterially expressed primase is competent to support DNA replication reactions that require primase. The results presented here suggest some functions for the p58 subunit of pol-prim as follows: p58 might modulate and stabilize the primase activity of p48 either by increasing the affinity of p48 to divalent cations (which would explain the higher Mg 2ϩ requirements of the p48 primase in comparison to that of the two-and four-subunit complex). In addition, p58 and divalent cations might selectively protect the inactivation of p48 activity by a specific mechanism, e.g. oxidative stress, which would resemble the selective inactivation of the 3Ј 3 5Ј exonuclease activity of T7 DNA polymerase (44,50). During rate-limiting steps of oligoribonucleotide synthesis p58 might also act as a stimulatory factor for the catalysis by p48, since the specific activity of the heterodimeric primase was significantly higher than that of p48 alone.
In summary, the vectors pET11-MCS and pET15-MCS allow the simple expression of single proteins and multiprotein complexes in E. coli. Our data showed that p48 can be expressed as a soluble protein, which requires Mg 2ϩ and Mn 2ϩ for stabilization of its enzyme activity. Furthermore, putative new functions for the p58 subunit of pol-prim emerged. The protein might increase the affinity of p48 to divalent cations; it might act as a stimulatory factor during the rate-limiting step of the dinucleotide synthesis, and p58 as well as specific divalent cations might selectively protect the inactivation of p48 activity by a specific mechanism. In the future, the expression and purification of large amounts of eukaryotic primase will allow detailed biochemical, biophysical, and structural analysis of the initiation reaction and DNA replication.