©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Active Site Mapping of the Catalytic Mouse Primase Subunit by Alanine Scanning Mutagenesis (*)

(Received for publication, September 21, 1994; and in revised form, December 18, 1994)

William C. Copeland (§) Xiaohong Tan

From the Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase alpha. The eukaryotic primase is composed of two subunits, p49 and p58, where the p49 subunit contains the catalytic active site. Mutagenesis of the cDNA for the p49 subunit was initiated to demonstrate a functional correlation of conserved residues among the eukaryotic primases and DNA polymerases. Fourteen invariant charged residues in the smaller catalytic mouse primase subunit, p49, were changed to alanine. These mutant proteins were expressed, purified, and enzymatically characterized for primer synthesis. Analyses of the mutant proteins indicate that residues 104-111 are most critical for primer synthesis and form part of the active site. Alanine substitution in residues Glu, Asp, and Asp produced protein with no detectable activity in direct primase assays, indicating that these residues may form part of a conserved carboxylic triad also observed in the active sites of DNA polymerases and reverse transcriptases. All other mutant proteins showed a dramatic decrease in catalysis, while mutation of two residues, Arg and Arg, caused an increase in K. Analysis of these mutant proteins in specific assays designed to separately investigate dinucleotide formation (initiation) and elongation of primer indicates that these two activities utilize the same active site within the p49 subunit. Finally, mutations in three active site codons produced protein with reduced affinity with the p58 subunit, suggesting that p58 may interact directly with active site residues.


INTRODUCTION

DNA primase initiates DNA replication by the synthesis of small ribonucleotides called primers(1) . The mammalian primases are composed of two subunits, p49 and p58, which purify as a complex tightly bound to DNA polymerase alpha(2, 3, 4) . The tight association of primase with DNA polymerase alpha implicates the DNA polymerase alpha as the lagging-strand DNA polymerase in replication(1, 4) . In the in vitro SV40 replication system, DNA polymerase alpha elongates the RNA primer to complete the synthesis of Okazaki fragment(5) . Okazaki fragments are then extended by either DNA polymerase or , allowing DNA polymerase alpha-primase to recycle and initiate another Okazaki fragment on the lagging strand(6) . This essential role of the DNA polymerase alpha-primase makes it a key component for regulation and inhibition of the initiation of DNA replication.

One unique property of primases (as well as RNA polymerases) is the ability to synthesize nucleotides de novo on a template by the formation of an initial dinucleotide. Primase initiates synthesis with a triphosphate purine moiety at the 5`-end(7, 8, 9) . Relatively few errors are made during the formation of the dinucleotide, whereas the primase readily misincorporates ribonucleotides during elongation of this dinucleotide(10, 11) . After synthesis of 7-10 ribonucleotides, the primer-template is translocated intramolecularly to the active site of the DNA polymerase alpha subunit(12, 13) .

The mechanism of dinucleotide and primer formation by primase is not well understood but may be similar to the mechanism utilized by DNA polymerases. Even less is known about the functional roles of specific amino acids within the primase subunits. Superimposition of the derived polymerase crystal structures of the Klenow fragment, human immunodeficiency virus, type 1 reverse transcriptase, T7 RNA polymerase and DNA polymerase beta demonstrates that, although there is little amino acid homology between such diverged polymerases, the overall folding in the active sites is conserved, bringing certain key residues into the correct position for catalysis or substrate binding(14, 15, 16, 17) . These key residues are highly conserved in nearly all DNA and RNA polymerases and reverse transcriptases, making up motifs A, B, and C (18) . Central to these three motifs are the aspartic and glutamic acids, which make up a catalytic triad within the structures(14) . Mutagenesis experiments on DNA polymerases demonstrate the importance of these residues in metal binding and catalysis(19, 20) . Although the eukaryotic DNA primases do not display any obvious amino acid sequence similarity to motifs A, B, and C in the DNA polymerases, it is reasonable to propose that the active site of the primases will fold similarly to those of the DNA polymerases, reverse transcriptases, and T7 RNA polymerase. Hence, it would be valuable to identify critical amino acid residues in the DNA primase that may make up the equivalent of motifs A, B, and C. This identification would also help to define structural elements responsible for the low fidelity observed with the primases compared with the higher fidelity of DNA polymerases.

Analysis of the recombinant mouse primase subunits demonstrates that both subunits are required for initiation while only the smaller subunit is necessary for elongation(12) . In contrast, the smaller subunit of the yeast primase complex appears to be sufficient for primer synthesis(21) . This discrepancy may be the consequence of the expression and purification procedures used or may represent a difference between species. Indeed, the mouse subunits do not complement temperature-sensitive and deletion alleles of the yeast primase genes(22) . Alternatively, it has been shown with both the yeast and mouse that the larger subunit stabilizes the activity of the p49 subunit(12, 21) . Perhaps, the higher mammalian primase subunits require this stabilization for initiation.

Amino acid alignment between the yeast (23) and the mouse p49 reveals a 34% identity, where the greatest similarity occurs in the N-terminal half of the two proteins(24) . This homology shows five regions of highly conserved amino sequences(24) . Several temperature-sensitive mutants in the primase of Saccharomyces cerevisiae were generated, and their mutations were determined to be in close proximity to or within these conserved regions(25) . Yeast cells carrying these mutant alleles have reduced primase activity and display a hyper-recombination and mutator phenotype, similar to other DNA replication mutants(26) .

On the basis of the homology between the yeast and mouse p49 primase subunits, we have generated a series of site-specific mutations in invariant charged amino acids in the mouse primase. Enzymatic characterization of these mutant proteins showed many of these charged residues to be critical for primer synthesis. We discuss the roles of these residues in the context of the two activities of primases, initiation and elongation of the primer.


EXPERIMENTAL PROCEDURES

Materials

Nickle nitriloacetic acid cross-linked to agarose was purchased from Qiagen Inc. Glutathione-Sepharose, calf intestine phosphatase, poly(dT), poly(dC), oligo(A), and ultrapure NTPs and dNTPs were purchased from Pharmacia Biotech Inc. [alpha-P]dATP, [alpha-P]dGTP, [alpha-P]ATP, and [alpha-P]GTP were purchased from DuPont NEN. All oligonucleotides were synthesized on an Applied Biosystems 392 DNA synthesizer. A Molecular Dynamics PhosphorImager with the program ImageQuant was used for all quantitation of radioactivity in gels.

Mutagenesis

The 1254-base pair BamHI fragment from pQE9/p49 (12) was subcloned into M13mp18 for generating single-stranded closed circular DNA. Site-directed mutagenesis was performed as described in (27) . Mutant inserts were fully sequenced on one strand. Mutant p49 inserts from M13 RF DNA were ligated into pQE9 (Qiagen Inc.), which had been digested with BamHI and treated with calf intestine phosphatase. Plasmids containing the mutant insert where checked for the mutation either by double-stranded sequencing or by the presence of the new restriction site introduced through mutagenesis.

Expression and Purification of Proteins

The histidine-tagged p49 (H-p49) and p58 (H-p58) subunits were expressed and purified as described in (12) . For expression of the mouse primase subunits as glutathione S-transferase fusion proteins (GST-p49 and GST-p58), the 1254-base pair BamHI restriction fragment of the mouse p49 subunit was ligated into the BamHI site of pGEX-2T (Pharmacia). The 1500-base pair BamHI-EcoRI fragment of the mouse p58 subunit was ligated into pGEX-3X (Pharmacia), digested with BamHI and EcoRI for expression as a glutathione S-transferase fusion protein. Expression and purification of the glutathione S-transferase fusion proteins were carried out essentially as described(28) . The single-subunit human DNA polymerase alpha (p180) was purified from baculovirus-infected insect cells as described(20, 29) .

Single-stranded DNA Binding

50-100 µg of purified p49 protein was loaded onto a 1-ml single-stranded DNA cellulose column (Sigma) equilibrated in buffer A (25 mM Tris-Cl, pH 8.0, 10% glycerol, and 1 mM EDTA) and packed into a fast protein liquid chromatography HR 5/5 column. Bound protein was eluted with a linear 0-200 mM KCl gradient in buffer A, and 0.25-ml fractions were collected. Protein fractions were detected by A absorbance and verified by SDS-PAGE (^1)followed by silver staining. KCl concentrations in the fractions were determined by conductivity measurements.

Subunits Interaction Assay

The interaction of p58 and p49 was tested using differentially tagged proteins. Six micrograms of GST-p58 were added to 4.4 µg of H-p49 protein (wild-type or mutant) in 150 µl of binding buffer (500 mM NaCl, 10% glycerol, 50 mM Tris-Cl, pH 7.5, 1% Triton X-100, and 0.2 mg/ml BSA). Proteins were incubated on ice for 30 min followed by the addition of 15 µl of glutathione-Sepharose beads. After 10 min on ice, the beads were washed 3 times in 1 ml of binding buffer without BSA by quick spin and aspiration. Beads were washed once more in Tris-buffered saline before adding an equal volume of 2 times protein loading dye (2% SDS, 2% beta-mercaptoethanol, 20% glycerol, 125 mM Tris-Cl, pH 6.8, and 10 ng/ml bromphenol blue). Samples were boiled 5 min and loaded onto a 10% SDS-PAGE. Proteins were visualized in the gel by Coomassie Blue staining.

Thermostability Assay

The thermostabilities of the mutant p49 primase subunits were tested by the thermolysin assay as described by Polesky et al.(19) . One microgram of p49 protein in the absence of p58 was incubated with 20 ng of thermolysin (Sigma) in a 30-µl reaction volume containing 50 mM Tris-Cl, pH 7.5, 2 mM MgCl(2), 2 mM CaCl(2), and 0.2 mM dithiothreitol at the following temperatures: 4, 16, 25, 30, 37, 42, 48, 55, and 65 °C. After 15 min, the samples were boiled in SDS protein sample buffer and loaded onto a 12% SDS-PAGE. Gels were stained with silver to visualize the digested proteins.

Coupled Primase-Polymerase Assay

Primase activity was tested in the coupled assay using either poly(dT) or poly(dC) as template as described(12) . Four picomoles each of p49 (wild-type or mutant), p58, and human p180 were combined in a 30-µl reaction containing 50 mM Tris-Cl, pH 8.0, 20 mM KCl, 200 µg/ml acetylated BSA, 4 mM MgCl(2), 2 mM dithiothreitol, 2 mM either ATP or GTP, 25 µM either [alpha-P]dATP or [alpha-P]dGTP, and 20 µg/ml either poly(dT) or poly(dC). Reactions were incubated for 30 min at 30 °C before adding 1 ml of stop solution (0.5 N NaOH, 44 mg/ml sodium pyrophosphate, 100 µg/ml carrier DNA, and 0.5 mg/ml BSA) and 1 ml of 20% trichloroacetic acid. DNA was allowed to precipitate on ice and filtered through Whatman GF/C glass fiber filters, washed with 1 N HCl and counted in a liquid scintillation counter. Control reactions containing only p180 and p58 produced no measurable activity over background.

Steady-state Kinetics

Steady-state kinetic parameters were determined from gel analysis of products using poly(dC) as template. One pmol of p49bulletp58 complex was added to a 10-µl reaction containing 0.2 µg poly(dC), 4 mM MgCl(2), 25 mM Tris-Cl, pH 8.0, 20 mM KCl, 0.1 mg/ml acetylated BSA, 4 mM dithiothreitol, and varying concentrations of [alpha-P]GTP (0.05-1.5 mM). Reactions were allowed to proceed for 15 min at 30 °C before quenching on ice followed by the addition of an equal volume of 95% formamide loading dye. Samples were boiled for 5 min, and 3 µl were separated through a 15% sequencing gel. The full spectrum of products in the gel was quantitated using a Molecular Dynamics PhosphorImager. Three microliters of a 50 µM [alpha-P]GTP reaction mix containing no enzyme was loaded onto the gel for the last 5 min and used as a standard for quantitating the picomoles of product formed.

Primer Elongation Assay

The extension of oligo(A)-primed poly(dT) was assayed essentially as described(12) . Oligo(A), 250 pmol, was annealed to 84 pmol of poly(dT) in 10 mM Tris-Cl, pH 8.0, 20 mM KCl. Five pmol (with respect to oligo(A)) of this annealed primer template was added to a 10-µl reaction containing 25 mM Tris-Cl, pH 8.0, 20 mM KCl, 0.1 mg/ml acetylated BSA, 4 mM dithiothreitol, 2 mM MgCl(2), 900 µM [alpha-P]ATP, and 0.5 pmol of p49 protein. Reactions were incubated for 15 min at 30 °C and stopped by the addition of an equal volume of 95% formamide sequencing dye. Samples were boiled, and 3 µl were separated through an 18% sequencing gel. The gel was then subjected to autoradiography and quantitated using a Molecular Dynamics PhosphorImager.

Dinucleotide Product Analysis

Dinucleotides were synthesized by the wild-type and mutant primase complexes using a (ATC) template in the presence of [alpha-P]ATP and GTP. One picomole of primase complex was incubated in a 10-µl reaction containing 0.4 µg of (ATC) template, 5 mM MgCl(2), 25 mM Tris-Cl, pH 8.0, 20 mM KCl, 0.1 mg/ml acetylated BSA, and 4 mM dithiothreitol with varying concentrations of GTP (0.05-1.5 mM) and [alpha-P]ATP (0.05-1.5 mM). Reactions were incubated for 15 min at 30 °C and then terminated by heating at 70 °C for 5 min. One microliter containing 0.6 units of calf intestine phosphatase, 50 mM Tris acetate, 50 mM magnesium acetate, and 250 mM potassium acetate was added and incubated for 1 h at 37 °C, followed by the addition of an equal volume of 95% formamide loading dye. Samples were boiled for 5 min and loaded onto a 1 mm times 15 cm times 20 cm 20% sequencing gel. The bromphenol blue was allowed to migrate 10 cm into the gel before it was stopped, and the wet gel was subjected to autoradiography and quantitated using a Molecular Dynamics PhosphorImager. Three microliters of a 250 µM [alpha-P]ATP reaction mix containing no enzyme was loaded onto the gel for the last 5 min and used as a standard for quantitating the pmol of product formed.


RESULTS

Analysis of the amino acid sequence homology between the mouse, human, and S. cerevisiae catalytic primase subunit reveals five conserved regions (Fig. 1) containing numerous charged residues. Of these residues, 14 are found within clusters of conserved amino acids in regions IV and V(24, 30) . Fig. 1shows the locations of these residues within the p49 sequence. These 14 charged residues were changed to alanine to address their role in primer synthesis. Mutations were screened by dideoxy sequence analysis and by the presence of the new restriction site. Mutant and wild-type p49 proteins were expressed in and purified from Escherichia coli as histidine-tagged proteins (H-p49). Fig. 2shows a Coomassie-stained gel of the purified p49 mutants as well as the mouse p58 and human p180 subunits used throughout this study. These mutant proteins had the same apparent molecular weight as the wild-type p49 and were more than 90% homogeneous.


Figure 1: Schematic diagram of the locations of the alanine mutations in the linear amino acid sequence. Shown as boxes in the linear diagram are the five conserved regions in the p49 subunit(24) . Ticmarksbelow the boxes represent the location of the residues mutated in this study and the Cs above region IV represent Cys residues that lie in a putative zinc finger. In the lowerpart of the figure is the alignment of these regions from mouse, human, and yeast. Invariant residues are listed below the alignments, and boldfaced amino acids represent amino acids that were altered to alanine. The mouse p49 sequence is from (24) ; the human sequence is from (39) , and W. C. Copeland, unpublished observations. The yeast sequence is from (23) .




Figure 2: SDS-PAGE analysis of mutant 49 subunits. Coomassie Blue stained gel of the purified wild-type and mutant p49 subunits. Also shown are the mouse p58 subunit and single subunit human DNA polymerase alpha (p180) used in the assays throughout this work. Approximately 0.5-2 µg of protein were loaded in each lane.



Subunit Interaction

Initiation of primer synthesis requires the interaction of the larger primase subunit, p58, with p49. The first question we addressed is whether the alanine substitutions can disrupt the subunit interface between the p49 and p58. To test this affinity, we expressed the primase subunits as glutathione S-transferase fusion proteins (GST-p49 and GST-p58, Fig. 3, lanes1 and 2, respectively). These GST-p58 and GST-p49 fusion proteins were active in primase assays when the respective H-p49 and H-p58 subunits were added (data not shown). The subunit interaction was tested by mixing GST-p58 and H-p49 proteins followed by precipitation with glutathione-Sepharose beads. Preliminary experiments demonstrated that H-p49 protein bound weakly to the glutathione-Sepharose matrix. Thus, a stringent wash containing 500 mM NaCl and 1% Triton X-100 was needed to reduce the binding of H-p49 with the glutathione-Sepharose matrix (Fig. 3, lanes1 and 3). Under these high salt conditions, the wild-type and most of the mutant proteins bound the p59 subunit. However, mutant proteins D105A, E148A, and D149A had a reduced association for the p58 subunit (Fig. 3, lanes7, 13, and 14). A reverse assay, in which GST-p58 and H-p49 were precipitated with nickle nitriloacetic acid-agarose beads, showed similar results with D105A, E148A, and D149A proteins, while the rest of the mutant proteins bound GST-p58 like the wild-type p49 (data not shown), demonstrating that these three mutant proteins had reduced affinity with the p58 subunit as compared with the wild-type p49.


Figure 3: Interaction of p58 with wild-type and mutant p49 subunits. Coomassie-stained gel containing co-precipitated GST-p58 protein with bound wild-type and mutant H-p49 subunits. Glutathione-Sepharose beads were mixed with preformed samples and washed as described under ``Experimental Procedures.'' Beads were then boiled in SDS sample buffer and loaded onto a 9% SDS-PAGE gel. Protein combinations that were mixed with the glutathione-Sepharose beads are listed on top of each lane of the gel. Lanes1-3 are control samples. GST-p49 (68 kDa) and H-p49 (49 kDa) were precipitated in lane1 to show that only the GST-p49 binds the glutathione-Sepharose beads. GST-p58 (82 kDa) was precipitated in lane2, while H-p49 was precipitated in lane3 with the glutathione-Sepharose beads to show nonspecific binding. Samples in lanes4-18 were co-precipitated with the GST-p58 protein where lane4 contains the wild-type p49. H-p49 signifies the histidine-tagged p49 protein while GST- signifies the glutathione S-transferase fusion proteins.



DNA Binding Affinity

Because initial screening of the alanine mutant proteins revealed several mutant proteins with little or no activity (see below), we utilized a physical assay over an enzymatic one to probe DNA binding. This affinity was tested by the interaction of the p49 subunit with single-stranded DNA cellulose. Fifty to one hundred micrograms of wild-type or mutant protein were loaded onto a single-stranded DNA cellulose column and eluted with a linear 0-200 mM KCl gradient. The results of this assay demonstrated that all the mutant proteins co-eluted at the same concentration of KCl, 100 mM, as did the wild-type protein. Fig. 4shows an example of the elution profile for the wild-type and E105A proteins.


Figure 4: DNA binding assay. Elution profile of wild-type and E105A p49 subunits from single-stranded DNA cellulose. Shown are silver-stained gels of the eluted fractions from the wild-type (top) and E105A protein (bottom). The scale on the left shows the migration of the molecular weight standards. The heavyline represents the KCl concentration at that particular fraction according to the rightscale where the arrow represents 100 mM KCl.



Thermostability

In addition to the DNA binding assay, a thermostability assay was also utilized to test large structural changes. Mutant and wild-type proteins were treated with thermolysin as a function of increasing temperature and degradation patterns analyzed by SDS-PAGE as described(19) . This assay probes for structural changes in the mutant proteins by testing the availability of proteolytic sites in the protein as a function of temperature. Thus, if a mutation caused a global structural change in the protein, then it may start to denature at a lower temperature, exposing protease sites differently as compared with the wild-type. All mutant proteins displayed similar proteolysis patterns by thermolysin compared with wild-type at a given temperature, indicating that the mutant proteins retained the same overall structural geometry as that of the wild-type (data not shown).

Activity Profile of Mutant Proteins

Wild-type and mutant p49 proteins were initially screened for activity using the coupled primase-polymerase assay(12) . Incorporation of labeled deoxynucleotide was measured on poly(dT) and poly(dC) homopolymers in the presence of the single subunit human DNA polymerase alpha and the recombinant mouse p58 subunit. This assay is highly sensitive for primer formation since the DNA polymerase amplifies the incorporated label. However, one requirement is that primers be long enough to be recognized by the DNA polymerase alpha. Fig. 5illustrates the relative activity in this coupled reaction by the mutant proteins and wild-type. Incorporation of dGTP in the wild-type reaction was about 2-fold higher than dATP incorporation, 910 pmol/h versus 480 pmol/h, respectively. This could be a consequence of DNA polymerase alpha's affinity for dGTP and dATP or a real difference in primer synthesis by the primase since the eukaryotic primases prefer poly(dC) over poly(dT) (31) . Most all mutant proteins synthesized primers more efficiently on poly(dC). In fact, many of the mutant proteins did not show any significant incorporation on poly(dT). The exceptions were the E148A and D149A, mutant proteins that incorporated more label with poly(dT) as template than with poly(dC). The D114A mutant displayed the largest difference in template preference showing a low but detectable level of activity on poly(dT) but near wild-type level of activity on poly(dC). Activity by the K130A mutant was similar with both templates. The D111A mutant had no measurable activity over background, while E105A and D109A had less than 1% activity relative to wild-type.


Figure 5: Coupled primase-polymerase assay on poly(dT) and poly(dC). Relative activities of the mutant proteins and wild-type primase complexes as measured in the coupled primase-polymerase assay. Solidbars represent activity with poly(dT) as template and ATP/[alpha-P]dATP as label, while stripedbars represent activity with poly(dC) as the template and GTP/[alpha-P]dGTP as label. Results represent the average of two or more experiments.



Steady-state Kinetics

Steady state kinetic parameters for de novo synthesis were determined using poly(dC) and [alpha-P]GTP for each of the active p49 mutant proteins. Products of the reactions were separated on a 15% sequencing gel and quantitated using a Molecular Dynamics PhosphorImager. Fig. 6A shows the results of this experiment for the wild-type and several of the mutant proteins. The major products by the wild-type and mutant proteins formed were 15-20 nucleotides long with a minor product of 9 nucleotides. The kinetics of formation of this 9-mer, formed either by the wild-type or mutant proteins, displayed a linear curve over the entire substrate concentration range, indicating a very high K(m). The longer products observed are primer dimers that primase forms in the absence of the associated DNA polymerase alpha(12) . The primase products of 15-20 nucleotides and higher were quantitated, and kinetic constants were determined. Fig. 6B illustrates the K(m), k, and k/K(m) parameters for these active mutant proteins. The wild-type K(m) value correlated closely to that previously described for the calf thymus primase(32) . Most all active mutant proteins displayed a dramatic decrease in k with the exception of E103A, D114A, and K130A, which had only a modest decrease in k The overall efficiency of the mutant proteins is estimated by the k/K(m) ratio. These k/K(m) values indicated that all mutant proteins except E103A, D114A, and K130A displayed activity well below 5% of the wild-type level. The two arginine mutant proteins at positions 162 and 163 showed a significant increase in K(m), suggesting a role for these amino acids in nucleotide binding. Interestingly, the major product formed by R162A and R163A was the 9-mer with very little product fully extended to the 15-20 nucleotide range (Fig. 6A). This 9-mer was also observed in other mutant proteins with very low activity and turnover rates.


Figure 6: Steady state kinetic analysis of primase mutant proteins on poly(dC). A, autoradiogram of 15% sequencing gel showing the de novo synthesis of primers on poly(dC) template under increasing [alpha-P]GTP concentrations. Names of the mutant proteins are listed below the lanes. Lanes1-7 represent the primers formed with 10, 25, 50, 100, 250, 500, and 750 µM [alpha-P]GTP, respectively. Lane0 represents no enzyme control with 750 µM GTP. Molecular weight markers on the left side were determined from P-end-labeled DNA oligonucleotides. B, bar graphs representing the individual kinetic parameters as determined by PhosphorImager analysis of the reaction products in gels. Only those mutant proteins that gave measurable product are shown. Results represent the average of three or more experiments.



Dinucleotide Formation

Primase activity is the combination of two catalytic activities: 1) formation of a dinucleotide and 2) extension of the dinucleotide to form full-length primers. Assays specific for the two distinct phases of primer synthesis were applied to study the p49 mutant proteins. The formation of the dinucleotide was tested utilizing a trinucleotide repeat oligonucleotide including only two of the ribonucleotides in an assay similar to that utilized by Sheaff and Kuchta(33) . Therefore, an oligonucleotide of 39 bases containing an ATC repeat was used as template. Dinucleotides were formed by the primases with the addition of GTP and [alpha-P]ATP. This forced the formation of a 5`-pppGpA-3` dinucleotide where the label is between the two nucleotides. This dinucleotide product was visualized by the gel assay developed by Scherzinger et al.(34) where the products of the reaction were treated with alkaline phosphatase and separated on a 20% sequencing gel. Removal of the 5`-phosphate residues caused the dinucleotide to migrate as a function of charge allowing resolution from the unincorporated label(34) . Fig. 7A shows the dinucleotides formed by wild-type and some of the mutant proteins under three nucleotide concentrations. The results from this assay for mutant proteins producing detectable dinucleotides are quantitated in Fig. 7B. Steady-state kinetic analysis of this reaction for the wild-type primase complex produced high K(m) values for both ATP and GTP, 250-800 µM, and a slow k, indicating that this is the rate-limiting step in primer synthesis (data not shown). The E105A, D109A, and D111A mutant proteins did not form any detectable dinucleotide. Wild-type and mutant p49 proteins showed the general trend of more dinucleotides produced with a higher concentration of the second nucleotide, ATP, versus the first nucleotide. This probably represents the greater affinity of the primase for GTP versus ATP as shown in Fig. 5. The relative levels of dinucleotide produced correlated closely to the kinetics on poly(dC) (Fig. 6) and the coupled primase- polymerase assay (Fig. 5). In addition, the wild-type and all active mutant proteins produced only a dinucleotide with no trace of trinucleotide or greater. Thus, even at the high NTP concentrations, initiation by primase to form the dinucleotide must be a highly accurate event as previously demonstrated(10) .


Figure 7: Analysis of dinucleotides formed by the wild-type and mutant primase complexes. Reactions were performed as described under ``Experimental Procedures.'' A, dinucleotide products were treated with alkaline phosphatase and separated on a 20% sequencing gel followed by autoradiography and PhosphorImager analysis. Lane1 represents the products with 250 µM GTP and 1.5 mM [alpha-P]ATP. Lane2 represents the products with 1.5 mM GTP and 1.5 mM [alpha-P]ATP. Lane3 represents the products with 1.5 mM GTP and 250 µM [alpha-P]ATP. Reactions in lane4 contained only 1.5 mM [alpha-P]ATP as substrate. B, graph showing the PhosphorImager quantitation of the dinucleotide produced with 1.5 mM GTP and 1.5 mM [alpha-P]ATP. Only those mutant proteins that produced detectable dinucleotide are shown. Values shown are the average of three or more experiments. The photograph of the autoradiogram in A does not reflect the detection limit of this assay and does not show the dinucleotide produced by the K104A mutant.



Oligo(A) Extension

Extension of a preformed riboprimed template was addressed using oligo(A)-primed poly(dT) as described previously(12) . In this assay, p49 proteins were incubated with and without the addition of the p58 subunit. An autoradiogram of this assay without the p58 subunit is shown in Fig. 8A and quantitated in the graph in Fig. 8B. K104A, E105A, D109A, D111A, D116A, D149A, D305A, and K317A mutant proteins displayed no measurable extension with or without the presence of the p58 subunit. E148A, R162A, and R163A mutant proteins showed only trace amounts of extension. E103A, D114A, and K130A gave the highest level of extension below the wild-type. Addition of p58 to this assay increased the amount of extension product by 5-fold for all mutant proteins showing extension activity. The lower extension observed may be due to the lower affinity for poly(dT) and ATP versus poly(dC) and GTP. In general, although a lower activity was observed in this assay, the relative levels of oligo(A) extension correlated with the results of the poly(dC) kinetics and dinucleotide formation, indicating a tightly coupled mechanism for these two activities.


Figure 8: Extension of Oligo(A)-primed poly(dT) by wild-type and mutant p49 proteins. Products from the reaction containing oligo(A)-primed poly(dT), [alpha-P]ATP and individual p49 proteins were separated on a 15% sequencing gel and subjected to autoradiography as described under ``Experimental Procedures.'' A, photograph of autoradiogram; B, PhosphorImager quantitation of gel containing oligo(A) extension products where products are represented as percent incorporation relative to the wild-type. Results represent the average of three or more experiments.




DISCUSSION

We describe here the first analysis of the structure-function relationships in the eukaryotic primase. Invariant charged amino acids within highly conserved regions of the mouse p49 subunit were altered to alanine to address their role in primer synthesis. These mutant proteins were characterized on the basis of what is required for primer synthesis. Previous characterization of the calf thymus primase and overexpressed mouse primase subunits has demonstrated that in vitro primer synthesis by the eukaryotic primase can be divided into six separate steps including two distinct enzymatic reactions: 1) association of the p49 subunit with the p58 subunit; 2) binding of the p49bulletp58 complex to single-stranded DNA; 3) binding of the incoming ribonucleotides to the primase-single-stranded DNA complex; 4) initiation of primer synthesis by formation of a dinucleotide; 5) elongation of the dinucleotide to make full-length primers; and 6) an intramolecular switch of the full-length primer and template from the primase active site to the DNA polymerase alpha active site(12, 13, 33) .

The activities of these mutant proteins were tested on a wide variety of substrates designed to obtain steady-state kinetics parameters and to separately investigate the initiation and elongation reactions of primer synthesis (Table 1). The E103A mutant behaved similarly to the wild-type enzyme in most all assays. Hence, the importance and high conservation of Glu are unclear. Perhaps more definitive experiments are necessary to elucidate the function of Glu. Three mutant proteins, E105A, D109A, and D111A were the least active. E105A and D109A proteins had only trace amounts of activity in the coupled primase-polymerase assay using poly(dC), while D111A had no detectable activity. However, these three mutant proteins displayed wild-type like elution from single-stranded DNA cellulose and gave a similar digestion pattern with thermolysin as a function of temperature, indicating that the loss of activity was not due to any gross aberration in structure.



The D114A mutant demonstrated a dramatically altered substrate preference in the coupled primase-polymerase assay relative to the wild-type. The lower activity detected with poly(dT) in this assay by D114A was unusual since a relatively high activity was observed in the extension and initiation assays. Taken together, these data suggest that the D114A mutant may have difficulty initiating de novo synthesis with poly(dT), while initiation with the (ATC) oligonucleotide may be more efficient due to the GTP requirement at the 5`-end. This difference in initiation by the D114A mutant suggests that Asp may participate in the recognition of the 5` purine residue.

Steady state kinetic analysis demonstrated that the greatest effect observed with the active mutant proteins was on k with only a slight change in apparent K(m). The exceptions were the two arginine mutant proteins at positions 162 and 163 in which the 5-10-fold increase in K(m) indicates a role in nucleotide binding. The positively charged side chain of these arginines may bind the negatively charged phosphate moiety of the incoming nucleotide. Lysine and arginine residues in motif B of the Klenow fragment also have a similar role(35, 36) . In addition, the major product formed by these two arginine mutant proteins with poly(dC) was the 9-mer, whereas all other active mutant proteins produced 15-20 nucleotide products. This may indicate a role for these arginine residues in processivity.

The lack of activity in direct primase assays with the E105A, D109A, and D111A mutant proteins suggests that these residues play an essential role in primer synthesis. In DNA polymerases, conserved aspartic and glutamic acid residues make up the triad of negative charges in the active site and are critical for metal binding and catalysis(14, 19, 20) . A similar motif may exist in primase with Glu, Asp, and Asp to make up part of the core active site. Given the close proximity and affect of alanine changes at residues Asp and Asp, it is tempting to speculate that these two residues make up the equivalent of the two negative charges found in motif C of the DNA polymerases. However, a clearer definition of the role of these residues requires additional experiments (analysis of mutant proteins that show partial activity).

The assignment of the other putative motifs, namely A and B, is difficult to make at this time. There is no obvious homology between motifs A, B, and C of the DNA polymerases (18) and the eukaryotic primases, which is probably a consequence of the extreme divergence of the primases from the polymerases. The recently determined structures of the DNA polymerase beta (16, 17) demonstrate that these motifs are not always co-linear. Thus, if residues 105-111 have a motif C-like function, then it is quite possible that motif A or B is C-terminal to this region and contains some of the residues mutated in this study.

Three mutant proteins, E105A, E148A, and D149A, demonstrated a reduced affinity with the p58 subunit. However, since neither wild-type (histidine-tagged or glutathione S-transferase-tagged) nor mutant proteins displayed any de novo primase activity in the absence of the p58 subunit, the primase activity observed by the E105A, E148A, and D149A mutant proteins in the coupled primase-polymerase assay do demonstrate an association with the p58 subunit under the lower salt assay conditions. Therefore, the apparent reduced affinity with p58 by the E105A, E148A, and D149A mutant proteins only occurred under the high salt conditions. The E148A and D149A mutant proteins were also interesting since they produced more product with poly(dT)/ATP than with poly(dC)/GTP in the coupled primase-polymerase assay. Surprisingly, these mutant proteins performed very poorly with oligo(A)-primed poly(dT).

It is noteworthy that these studies identify two regions, 105 and 148-149, to interact with the p58, especially since Glu is proximal to two other essential amino acid side chains, Asp and Asp, and probably deep in the active site. This suggests that the role of p58 during initiation may occur by interacting with active site residues. The requirement of p58 in initiation and interaction with the active site residues is very different than what would be expected of a DNA polymerase. However, the architecture of a primase or RNA polymerase must be somewhat different from a DNA polymerase since it is required to hold two nucleotides in the active site instead of one during initiation. Studies of two other RNA polymerases show the involvement of other subunits or domains in initiation. Mutagenesis and structural analysis of the T7 RNA polymerase implicates the N-terminal region outside of the palm-fingers-thumb active site region in binding the initiating ribonucleotide(15, 37) . More recently, it was shown that a highly conserved region in the E. coli RNA polymerase factor cross-links the initiating nucleotide and forms a face of the active site with the beta subunit of the E. coli RNA polymerase (38) . We previously demonstrated that the p58 subunit of the mouse primase also weakly cross-links labeled ATP(12) . Hence, the weaker affinity of the E105A protein to bind the p58 subunit and the close proximity of Glu within the active site suggest that, as with the subunit of RNA polymerase(38) , the p58 subunit may form a binding surface for an initiating nucleotide within the active site.

For all the mutant proteins studied, we observed similar levels of activities between both the initiation and elongation assays as compared with the kinetic and coupled polymerase-primase assays. In general, these mutations failed to separate the two activities, which suggest a tightly coupled mechanism between the dinucleotide formation and elongation, possibly and most likely utilizing the same active site residues. In addition, the precise involvement of the p58 subunit in the initiation still remains obscure. Clearly a mutagenesis study in the larger subunit is warranted.


FOOTNOTES

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

§
To whom correspondence should be addressed: Laboratory of Molecular Genetics, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-4792; Fax: 919-541-7613; copelan1{at}niehs.nih.gov.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Miriam Sander, Ken Tindall, and Philip Ropp for critical reading of this manuscript.


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