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• W2000480897 abstract "In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase α. 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 Glu105, Asp109, and Asp111 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, Arg162 and Arg163, caused an increase in Km(NTP). 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. In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase α. 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 Glu105, Asp109, and Asp111 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, Arg162 and Arg163, caused an increase in Km(NTP). 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. INTRODUCTIONDNA primase initiates DNA replication by the synthesis of small ribonucleotides called primers(1Kornberg A. Baker T.A. DNA Replication. 2nd. Ed. W. H. Freeman and Co., New York1992: 275-298Google Scholar). The mammalian primases are composed of two subunits, p49 and p58, which purify as a complex tightly bound to DNA polymerase α(2Conaway R.C. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2523-2527Crossref PubMed Scopus (115) Google Scholar, 3Kaguni L.S. Rossignol J.-M. Conaway R.C. Banks G.R. Lehman I.R. J. Biol. Chem. 1983; 258: 9037-9039Abstract Full Text PDF PubMed Google Scholar, 4Wang T.S.-F. Annu. Rev. Biochem. 1991; 60: 513-552Crossref PubMed Scopus (451) Google Scholar). The tight association of primase with DNA polymerase α implicates the DNA polymerase α as the lagging-strand DNA polymerase in replication(1Kornberg A. Baker T.A. DNA Replication. 2nd. Ed. W. H. Freeman and Co., New York1992: 275-298Google Scholar, 4Wang T.S.-F. Annu. Rev. Biochem. 1991; 60: 513-552Crossref PubMed Scopus (451) Google Scholar). In the in vitro SV40 replication system, DNA polymerase α elongates the RNA primer to complete the synthesis of Okazaki fragment(5Stillman B. Annu. Rev. Cell Biol. 1989; 5: 197-245Crossref PubMed Scopus (282) Google Scholar). Okazaki fragments are then extended by either DNA polymerase δ or ε, allowing DNA polymerase α-primase to recycle and initiate another Okazaki fragment on the lagging strand(6Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (493) Google Scholar). This essential role of the DNA polymerase α-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(7Tseng B.Y. Erikson J.M. Goulian M. J. Mol. Biol. 1979; 129: 531-545Crossref PubMed Scopus (34) Google Scholar, 8Shioda M. Nelson E.M. Bayne M.L. Benbow R.M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7209-7213Crossref PubMed Scopus (40) Google Scholar, 9Gronostajski R.M. Field J. Hurwitz J. J. Biol. Chem. 1984; 259: 9479-9486Abstract Full Text PDF PubMed Google Scholar). Relatively few errors are made during the formation of the dinucleotide, whereas the primase readily misincorporates ribonucleotides during elongation of this dinucleotide(10Sheaff R.J. Kuchta R.D. J. Biol. Chem. 1994; 269: 19225-19231Abstract Full Text PDF PubMed Google Scholar, 11Zhang S. Grosse F. J. Mol. Biol. 1990; 216: 475-479Crossref PubMed Scopus (20) Google Scholar). After synthesis of 7-10 ribonucleotides, the primer-template is translocated intramolecularly to the active site of the DNA polymerase α subunit(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar, 13Sheaff R.J. Kuchta R.D. Ilsley D. Biochemistry. 1994; 33: 2247-2254Crossref PubMed Scopus (44) Google Scholar).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 β 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(14Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 15Sousa R. Chung Y.J. Rose J.P. Wang B.C. Nature. 1993; 364: 593-599Crossref PubMed Scopus (338) Google Scholar, 16Davies J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 17Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (398) Google Scholar). These key residues are highly conserved in nearly all DNA and RNA polymerases and reverse transcriptases, making up motifs A, B, and C (18Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (573) Google Scholar). Central to these three motifs are the aspartic and glutamic acids, which make up a catalytic triad within the structures(14Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar). Mutagenesis experiments on DNA polymerases demonstrate the importance of these residues in metal binding and catalysis(19Polesky A.H. Steitz T.A. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1990; 265: 14579-14591Abstract Full Text PDF PubMed Google Scholar, 20Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 11028-11040Abstract Full Text PDF PubMed Google Scholar). 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(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). In contrast, the smaller subunit of the yeast primase complex appears to be sufficient for primer synthesis(21Santocanale C. Foiani M. Lucchini G. Plevani P. J. Biol. Chem. 1993; 268: 1343-1348Abstract Full Text PDF PubMed Google Scholar). 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(22Santocanale C. Locati F. Falconi M.M. Piseri A. Tseng B.Y. Lucchini G. Plevani P. Gene (Amst.). 1992; 113: 199-205Crossref PubMed Scopus (5) Google Scholar). Alternatively, it has been shown with both the yeast and mouse that the larger subunit stabilizes the activity of the p49 subunit(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar, 21Santocanale C. Foiani M. Lucchini G. Plevani P. J. Biol. Chem. 1993; 268: 1343-1348Abstract Full Text PDF PubMed Google Scholar). Perhaps, the higher mammalian primase subunits require this stabilization for initiation.Amino acid alignment between the yeast (23Plevani P. Francesconi S. Lucchini G. Nucleic Acids Res. 1987; 15: 7975-7989Crossref PubMed Scopus (12) Google Scholar) and the mouse p49 reveals a 34% identity, where the greatest similarity occurs in the N-terminal half of the two proteins(24Prussak C.E. Almazan M.T. Tseng B.Y. J. Biol. Chem. 1989; 264: 4957-4963Abstract Full Text PDF PubMed Google Scholar). This homology shows five regions of highly conserved amino sequences(24Prussak C.E. Almazan M.T. Tseng B.Y. J. Biol. Chem. 1989; 264: 4957-4963Abstract Full Text PDF PubMed Google Scholar). 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(25Francesconi S. Longhese M.P. Piseri A. Santocanale C. Lucchini G. Plevani P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3877-3881Crossref PubMed Scopus (37) Google Scholar). Yeast cells carrying these mutant alleles have reduced primase activity and display a hyper-recombination and mutator phenotype, similar to other DNA replication mutants(26Longhese M.P. Jovine L. Plevani P. Lucchini G. Genetics. 1993; 133: 183-191Crossref PubMed Google Scholar).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 PROCEDURESMaterialsNickle nitriloacetic acid cross-linked to agarose was purchased from Qiagen Inc. Glutathione-Sepharose, calf intestine phosphatase, poly(dT)272, poly(dC)423, oligo(A)8-12, and ultrapure NTPs and dNTPs were purchased from Pharmacia Biotech Inc. [α-3232P]dATP, [α-3232P]dGTP, [α-3232P]ATP, and [α-3232P]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.MutagenesisThe 1254-base pair BamHI fragment from pQE9/p49 (12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar) was subcloned into M13mp18 for generating single-stranded closed circular DNA. Site-directed mutagenesis was performed as described in (27Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar). 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 ProteinsThe histidine-tagged p49 (H-p49) and p58 (H-p58) subunits were expressed and purified as described in (12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). 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(28Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5034) Google Scholar). The single-subunit human DNA polymerase α (p180) was purified from baculovirus-infected insect cells as described(20Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 11028-11040Abstract Full Text PDF PubMed Google Scholar, 29Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1991; 266: 22739-22748Abstract Full Text PDF PubMed Google Scholar).Single-stranded DNA Binding50-100 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 A280 absorbance and verified by SDS-PAGE 1The abbreviations used are: PAGEpolyacrylamide gel electrophoresisBSAbovine serum albumin. followed by silver staining. KCl concentrations in the fractions were determined by conductivity measurements.Subunits Interaction AssayThe 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 × protein loading dye (2% SDS, 2%β-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 AssayThe thermostabilities of the mutant p49 primase subunits were tested by the thermolysin assay as described by Polesky et al.(19Polesky A.H. Steitz T.A. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1990; 265: 14579-14591Abstract Full Text PDF PubMed Google Scholar). 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 MgCl2, 2 mM CaCl2, 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 AssayPrimase activity was tested in the coupled assay using either poly(dT) or poly(dC) as template as described(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). 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 MgCl2, 2 mM dithiothreitol, 2 mM either ATP or GTP, 25 μM either [α-3232P]dATP or [α-3232P]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 KineticsSteady-state kinetic parameters were determined from gel analysis of products using poly(dC) as template. One pmol of p49•p58 complex was added to a 10-μl reaction containing 0.2 μg poly(dC), 4 mM MgCl2, 25 mM Tris-Cl, pH 8.0, 20 mM KCl, 0.1 mg/ml acetylated BSA, 4 mM dithiothreitol, and varying concentrations of [α-3232P]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 [α-3232P]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 AssayThe extension of oligo(A)(12-18)-primed poly(dT) was assayed essentially as described(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). Oligo(A)(12-18), 250 pmol, was annealed to 84 pmol of poly(dT)272 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 MgCl2, 900 μM [α-3232P]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 AnalysisDinucleotides were synthesized by the wild-type and mutant primase complexes using a (ATC)13 template in the presence of [α-3232P]ATP and GTP. One picomole of primase complex was incubated in a 10-μl reaction containing 0.4 μg of (ATC)13 template, 5 mM MgCl2, 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 [α-3232P]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 × 15 cm × 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 [α-3232P]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.RESULTSAnalysis 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(24Prussak C.E. Almazan M.T. Tseng B.Y. J. Biol. Chem. 1989; 264: 4957-4963Abstract Full Text PDF PubMed Google Scholar, 30Miyazawa H. Izumi M. Tada S. Takada R. Masutani M. Ui M. Hanaoka F. J. Biol. Chem. 1993; 268: 8111-8122Abstract Full Text PDF PubMed Google Scholar). Fig. 1 shows 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. 2 shows 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 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 α (p180) used in the assays throughout this work. Approximately 0.5-2 μg of protein were loaded in each lane.View Large Image Figure ViewerDownload (PPT)Subunit InteractionInitiation 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.View Large Image Figure ViewerDownload (PPT)DNA Binding AffinityBecause 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. 4 shows 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.View Large Image Figure ViewerDownload (PPT)ThermostabilityIn 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(19Polesky A.H. Steitz T.A. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1990; 265: 14579-14591Abstract Full Text PDF PubMed Google Scholar). 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 ProteinsWild-type and mutant p49 proteins were initially screened for activity using the coupled primase-polymerase assay(12Copeland W.C. Wang T.S.-F. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). Incorporation of labeled deoxynucleotide was measured on poly(dT) and poly(dC) homopolymers in the presence of the single subunit human DNA polymerase α 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 α. Fig. 5 illustrates 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 α'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) (31Roth Y.-F. Eur. J. Biochem. 1987; 165: 473-481Crossref PubMed Scopus (35) Google Scholar). 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/[α-3232P]dATP as label, while stripedbars represent activity with poly(dC) as the template and GTP/[α" @default.
• W2000480897 created "2016-06-24" @default.
• W2000480897 creator A5063750217 @default.
• W2000480897 creator A5075186269 @default.
• W2000480897 date "1995-02-01" @default.
• W2000480897 modified "2023-10-14" @default.
• W2000480897 title "Active Site Mapping of the Catalytic Mouse Primase Subunit by Alanine Scanning Mutagenesis" @default.
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