Structure and Processivity of Two Forms of Saccharomyces cerevisiae DNA Polymerase δ
1998; Elsevier BV; Volume: 273; Issue: 31 Linguagem: Inglês
10.1074/jbc.273.31.19756
ISSN1083-351X
AutoresPeter Burgers, Kimberly J. Gerik,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoYeast DNA polymerase δ (Polδ) consists of three subunits encoded by the POL3, POL31, andPOL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1–10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30-fold overproduction of Polδ, which was identical in enzymatic properties to Polδ isolated from a wild-type yeast strain. Whereas overproduction of POL3 together withPOL32 did not lead to an identifiable Pol3p·Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 andPOL31. This two-subunit complex, designated Polδ*, is structurally and functionally analogous to mammalian Polδ. The properties of Polδ* and Polδ were compared. A gel filtration analysis showed that Polδ* is a heterodimer (Pol3p·Pol31p) and Polδ a dimer of a heterotrimer, (Pol3p·Pol31p·Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Polδ* showed a processivity of 2–3 on poly(dA)·oligo(dT) compared with 5–10 for Polδ. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Polδ* on a natural DNA template was dependent on PCNA and on replication factor C. However, Polδ*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Polδ was achieved upon addition of Pol32p to Polδ*. Yeast DNA polymerase δ (Polδ) consists of three subunits encoded by the POL3, POL31, andPOL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1–10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30-fold overproduction of Polδ, which was identical in enzymatic properties to Polδ isolated from a wild-type yeast strain. Whereas overproduction of POL3 together withPOL32 did not lead to an identifiable Pol3p·Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 andPOL31. This two-subunit complex, designated Polδ*, is structurally and functionally analogous to mammalian Polδ. The properties of Polδ* and Polδ were compared. A gel filtration analysis showed that Polδ* is a heterodimer (Pol3p·Pol31p) and Polδ a dimer of a heterotrimer, (Pol3p·Pol31p·Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Polδ* showed a processivity of 2–3 on poly(dA)·oligo(dT) compared with 5–10 for Polδ. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Polδ* on a natural DNA template was dependent on PCNA and on replication factor C. However, Polδ*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Polδ was achieved upon addition of Pol32p to Polδ*. The subunit structure of eukaryotic DNA polymerase δ (Polδ) 1The abbreviations used are: Polδ, DNA polymerase δ; Polδ*, Polδ lacking Pol32p; SS, single-stranded; PCNA, proliferating cell nuclear antigen; RFC, DNA replication factor C; DTT, dithiothreitol; BuPhdGTP,N 2-(p-n-butylphenyl)-2′-deoxyguanosine-5′-triphosphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; nt, nucleotide; kb, kilobase(s). remains ambiguous (for a review, see Ref. 1Hindges R. Hubscher U. Biol. Chem. 1997; 378: 345-362Crossref PubMed Google Scholar). The most thoroughly characterized form of mammalian Polδ is that isolated from calf thymus, a two-subunit enzyme with a 125-kDa catalytic subunit and a 48-kDa accessory subunit (2Lee M.Y.W.T. Tan C.-K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (194) Google Scholar). The accessory subunit is required for efficient stimulation of Polδ by the proliferating cell nuclear antigen (PCNA) (3Zhou J.Q. He H. Tan C.K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1094-1099Crossref PubMed Scopus (57) Google Scholar, 4Sun Y. Jiang Y. Zhang P. Zhang S.-J. Zhou Y. Li B.Q. Toomey N.L. Lee M.Y.W.T. J. Biol. Chem. 1997; 272: 13013-13018Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Likewise, mouse Polδ can be purified in two forms, the single catalytic subunit form which is not stimulated by PCNA and the two-subunit enzyme which is stimulated by PCNA (5Goulian M. Herrmann S.M. Sackett J.W. Grimm S.L. J. Biol. Chem. 1990; 265: 16402-16411Abstract Full Text PDF PubMed Google Scholar). The subunit composition of the two-subunit enzymes is that of a heterodimer (2Lee M.Y.W.T. Tan C.-K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (194) Google Scholar, 5Goulian M. Herrmann S.M. Sackett J.W. Grimm S.L. J. Biol. Chem. 1990; 265: 16402-16411Abstract Full Text PDF PubMed Google Scholar). In contrast, Polδ isolated from the two yeasts is more complex with the enzyme from Saccharomyces cerevisiae having three subunits and that from Sshizosaccharomyces pombe at least four, and perhaps five subunits (6Zuo S.J. Gibbs E. Kelman Z. Wang T. Odonnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (73) Google Scholar, 7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The three subunits of S. cerevisiae Polδ have apparent sizes by SDS-PAGE of 125, 58, and 55 kDa and are encoded by thePOL3, POL31, and POL32 genes, respectively (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The 125-kDa catalytic subunit encoded by thePOL3 (CDC2) gene is very highly conserved in all eukaryotes (1Hindges R. Hubscher U. Biol. Chem. 1997; 378: 345-362Crossref PubMed Google Scholar). The 58-kDa second subunit is encoded by thePOL31 (HYS2, SDP5) gene (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Mutations in this essential gene can cause sensitivity to the replication inhibitor hydroxyurea, for the hys2-1 allele, or suppress the temperature sensitivity of mutations in the catalytic subunit, for thesdp5-1 allele (8Sugimoto K. Sakamoto Y. Takahashi O. Matsumoto K. Nucleic Acids Res. 1995; 23: 3493-3500Crossref PubMed Scopus (42) Google Scholar, 9Giot L. Chanet R. Simon M. Facca C. Faye G. Genetics. 1997; 146: 1239-1251Crossref PubMed Google Scholar). Pol31p shows 23–28% sequence similarity to the 48-kDa subunit of human Polδ and to S. pombe Cdc1. The essential cdc1 + gene encodes the second subunit of S. pombe Polδ (6Zuo S.J. Gibbs E. Kelman Z. Wang T. Odonnell M. MacNeill S.A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11244-11249Crossref PubMed Scopus (73) Google Scholar). The 55-kDa subunit is encoded by the POL32 gene. Mutants deleted for POL32 are viable, but show both replication and repair defects (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Although the sequence similarity between Pol32p and Cdc27, which is the essential third subunit of S. pombe Polδ, is very low, other considerations including the presence of a PCNA-binding motif in both subunits denote these two as functional homologues (discussed in Ref. 7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In the previous paper (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) we have described the cloning of the two small subunit genes of Polδ and their characterization. Here we describe the overproduction in yeast of the three-subunit form of Polδ, and of a two-subunit form, called Polδ*, which is analogous to mammalian Polδ. The yeast strains used in this work are the protease-deficient galactose-inducible strains BJ2168 (MATa,ura3-52, trp1-289, leu2-3, 112,prb1–1122, prc1-407, pep4-3), PY116 (MAT a ura3-52 trp1-Δ his3-11, 15 leu2-3, 112 pep4-3 prb1–1122 nuc1::LEU2) and its pol32Δderivative PY117 (MAT a ura3-52 trp1-Δ his3-11, 15 leu2-3, 112 pep4-3 prb1–1122 nuc1::LEU2 pol32Δ::HIS3) (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The overproduction plasmids used in this study are based upon the pRS420 series plasmids into which the GAL1–10 upstream activating sequence (GAL1–10 upstream activating sequence) including the transcriptional start sites for the GAL1 andGAL10 genes, as a 678-nt BamHI-EcoRI fragment, was inserted into the corresponding plasmid polylinker sites, resulting into vectors pRS424-GAL (TRP1), pRS425-GAL (LEU2), and pRS426-GAL (URA3) (10Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene ( Amst. ). 1992; 110: 119-122Crossref PubMed Scopus (1463) Google Scholar). All vectors have in addition the yeast 2 μm origin for high copy maintenance in yeast and the Bluescript SKII+ backbone for propagation in E. coli. The transcriptional start site of the GAL1 gene is 60 nt upstream of the BamHI cloning site and the transcriptional start site of the GAL10gene is 10 nt upstream of the EcoRI cloning site. Both promoters are of similar strength. Coordinates are with reference to the translational start sites. pBL336 (TRP1 GAL1-POL3) has a 3.6-kb HgiAI (trimmed)-HindIII fragment (coordinates: −45 to 3543) cloned into the BamHI (filled)-HindIII sites of pRS424-GAL. pBL338 (LEU2 GAL1-POL31) has a 1.6-kb NcoI (filled)-ClaI (filled) fragment (coordinates: 2 to 1567) from pBL361 cloned into theSacII site of pRS425-GAL (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). pBL340 (URA3 GAL10-POL32) has a 1.7-kb HpaI-SalI fragment (coordinates: −20 to 1688) from pBL384 cloned into theEcoRI (filled)-SalI sites of pRS426-GAL (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). A single colony of a plasmid-containing strain from a selective SCGL plate was grown in an air shaker at 30 °C in 100 ml of selective SCGL medium. SCGL medium contains per liter: 1.7 g of yeast nitrogen base without amino acids and ammonium sulfate, 5 g of ammonium sulfate, 30 ml of glycerol, 20 ml of lactic acid, 1 g of glucose, 20 g of agar for solid media, 20 mg each of adenine, uracil, histidine, tryptophan, proline, arginine, and methionine, 30 mg each of isoleucine, tyrosine, and lysine, 50 mg of phenylalanine, and 100 mg each of leucine, glutamic acid, aspartic acid, valine, threonine, and serine. Uracil, tryptophan, and/or leucine were omitted when appropriate to ensure the selective maintenance of plasmids. Prior to autoclaving, the pH of the media was adjusted to 5–6 with concentrated sodium hydroxide. After 2–3 days when the OD660 had reached 0.8–1, the culture was used to inoculate 1200 ml of SCGL media. After overnight growth, when the OD660 was about 1, 1200 ml of YPGL were added. YPGL contains per liter: 10 g of yeast extract, 20 g of peptone, 30 ml of glycerol, 20 ml of lactic acid, 2 g of glucose, and 20 mg of adenine. Prior to autoclaving, the pH of the media was adjusted to 5–6 with concentrated sodium hydroxide. The culture was equally divided over two 4-liter flasks and grown at 30 °C for 3 h. Solid galactose (2% final concentration) was then added to each flask and after 4 h of continuous shaking the cells were harvested. All steps were carried out at 0–4 °C. The following buffers were used: buffer A: 0.1 m Tris-HCl, pH 7.8, 5% (v/v) glycerol, 175 mm ammonium sulfate, 2 mm EDTA, 1 mm EGTA, 3 mm DTT, 0.025% Nonidet P-40, 5 μm pepstatin A, 5 μm leupeptin, 2 μg/ml chymostatin, 0.5 mm p-methylphenylsulfonyl fluoride, 5 mm benzamidine,10 mmNaHSO3. Buffer B consisted of 25 mmKH2PO4, pH 7.5, 10% glycerol, 2 mmEDTA, 1 mm EGTA, 3 mm DTT, 0.01% Nonidet P-40, 5 μm pepstatin A, 5 μm leupeptin, 2 μg/ml chymostatin, 0.5 mm p-phenylsulfonyl fluoride. Buffer C was 30 mm triethanolamine-HCl, pH 7.3, 1 mm EDTA, 0.5 mm EGTA, 10% glycerol, 0.01% Nonidet P-40, 3 mm DTT, 5 μm pepstatin A, 5 μm leupeptin, 5 mm NaHSO3. Buffer D was 30 mm HEPES-NaOH, pH 7.4, 1 mm EDTA, 0.5 mm EGTA, 10% glycerol, 0.01% Nonidet P-40, 5 mm DTT, 2 μm pepstatin A, 2 μmleupeptin, 0.1% (v/v) ampholytes 3.5–9. Salt concentrations (as NaCl) are indicated by a suffix, e.g. BufferA500 = Buffer A + 500 mm NaCl. Buffers were precooled on ice water. Bead beating was carried out in a 350-ml chamber containing 175 ml of glass beads (0.4–0.5 mm diameter) and 80–100 g, wet weight, of cells, resuspended in an equal volume of 2 × buffer A. The chamber was cooled in ice water and the beater turned on for 45 s, followed by a cooling period of 2 min, for a total beating time of 5 min. The lysate was poured in a cold graduated cylinder and the beads were washed with 50 ml of extraction buffer. An aliquot was centrifuged at 45,000 × g for 20 min (cleared lysate, see Table I). The volume of the crude lysate was measured and 40 μl of 10% Polymin P were added per ml of lysate. After 5 min of mixing, the lysate was spun for 40 min at 13,000 rpm in a GSA rotor. Solid ammonium sulfate (0.28 g/ml) was added to the supernatant and dissolved by stirring. The precipitate was collected at 13,000 rpm for 45 min. The pellet was resuspended in 20 ml of Buffer B. After dialysis against 2 × 500 ml of buffer B for 8 h each, the dialysate was cleared by centrifugation at 18,000 rpm for 20 min.Table IOverproduction of PolδPOL genesRelative DNA polymerase activityLysate(NH4)2SO4PCMonoQaPolδ activity only.None (vector)1111POL31 + POL321.2POL3821.52.2POL3 + POL3114121317bIncludes Polδ* activity.POL3 + POL3292.52.22.6POL3 + POL31 + POL3220221832DNA polymerase assays on activated DNA were carried out in the presence of BuPhdGTP to eliminate most of the contribution due to Polα. Activities were determined in the cleared lysate (lysate), after dialysis of the redissolved ammonium sulfate precipitate ((NH4)2SO4), after dialysis following phosphocellulose chromatography (PC), and after MonoQ HPLC. Activities are relative to that of the strain lacking the overexpression plasmid.a Polδ activity only.b Includes Polδ* activity. Open table in a new tab DNA polymerase assays on activated DNA were carried out in the presence of BuPhdGTP to eliminate most of the contribution due to Polα. Activities were determined in the cleared lysate (lysate), after dialysis of the redissolved ammonium sulfate precipitate ((NH4)2SO4), after dialysis following phosphocellulose chromatography (PC), and after MonoQ HPLC. Activities are relative to that of the strain lacking the overexpression plasmid. The cleared ammonium sulfate fraction was loaded on a 20-ml phosphocellulose column, equilibrated in buffer B. The column was washed with 40 ml of B25 and eluted with B750. The protein-containing fractions were combined and dialyzed for 2 × 3 h against 150 ml each of buffer C until the conductivity of the dialysate was equal to that of C25. The dialyzed fraction was injected onto a 8-ml MonoQ column, equilibrated in buffer C25, washed with 10 ml of C25, and eluted with a 120-ml linear gradient from C25 to C500. Fractions of 2.5 ml were collected, Polδ* eluted at ∼C150 and Polδ at ∼C200. Individual Mono Q fractions were diluted with 2 volumes of buffer D and injected onto a 1-ml MonoS column, equilibrated in buffer D50. The column was washed with 2 ml of D50 and eluted with a 15-ml linear gradient from D50 to D500. Polδ* eluted at ∼D250 and Polδ at ∼D400. Samples of 200 μl were injected onto a 20-ml Superose 6 column in 40 mm Hepes-NaOH, pH 7.5, 10% ethylene glycol, 1 mm EDTA, 0.02% Nonidet P-40, 0.2 m NaCl, 5 mm DTT, 5 mm NaHSO3, and 2 μm each of leupeptin and pepstatin A. The column was run at 0 °C at 0.2 ml/min. Fractions of 300 μl were collected and analyzed by 10% SDS-PAGE and for DNA polymerase activity. The DNA polymerase assay on activated DNA is described in the previous paper (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The 10-μl reaction contained 20 mm Tris-HCl, pH 7.8, 8 mm MgAc2, 0.2 mg/ml bovine serum albumin, 1 mm dithiothreitol, 25 μm[α-32P]dTTP, 100 ng of poly(dA)-(dT)22(40:1, nucleotide ratio, 0.4 pmol of primer termini), 0.02 pmol of Polδ or 0.03 pmol of Polδ*, and PCNA as indicated. Incubations were for 5 min at 37 °C. The assays were stopped with 7 μl of 95% formamide, 20 mm EDTA and electrophoresed on a 12% denaturing polyacrylamide gel. The standard 30-μl reaction contained 40 mm Tris-HCl, pH 7.8, 8 mmMgAc2, 0.2 mg/ml bovine serum albumin, 1 mmdithiothreitol, 100 μm each of dATP, dCTP, and dGTP, and 25 μm [3H]dTTP (100 cpm/pmol dNTP), 0.5 mm ATP, 40 fmol (100 ng) of singly primed SS mp18 DNA (the 36-mer primer is complementary to nt 6330–6295), 850 ng ofEscherichia coli single stranded-binding protein, 75 mm NaCl, 100 fmol of RFC, and Polδ, Polδ*, and PCNA as indicated in the legend to the figures. Incubations were at 37 °C for the times indicated. The reactions were stopped and acid insoluble radioactivity was determined as described above. When an electrophoretic analysis of the replication products was carried out, [α-32P]dTTP replaced [3H]dTTP, and the assays were stopped with 10 μl of 60% glycerol, 50 mmEDTA, 1% SDS, and electrophoresed on a 1 or 1.5% alkaline-agarose gel. An inducible system for the overexpression of the Polδ genes allows normal cell growth without possible deleterious effect on cells due to constitutive high levels of the Polδ subunits. The strain used in this study is the protease-deficient strain BJ2168. In this strain, the expression of genes placed under control of the GAL1–10 upstream activating sequence is appropriately induced by addition of galactose to the media, but the strain grows very poorly on galactose as sole carbon source. Satisfactory cell growth was obtained on media containing as carbon source 3% glycerol, 2% lactate, and a non-repressing concentration of glucose (0.1%). Galactose was added to this media to induce expression. The three Polδ genes were cloned under control of the bi-directional GAL1–10 upstream activating sequence as described under "Materials and Methods." Constitutive overproduction of Polδ is inhibitory to yeast cell growth as a strain carrying the three overexpression plasmids grew less well on galactose medium than on raffinose, a non-inducing carbon source (data not shown). Cells containing one or more of the Polδ genes underGAL1–10 control were grown up to mid-logarithmic phase, induced with galactose, and crude extracts were made as described under "Materials and Methods." These extracts were assayed for DNA polymerase activity. However, because Polα constitutes the major polymerase activity in yeast extracts, a moderate overproduction of Polδ would not be readily apparent when assayed in crude extracts. Therefore, Polα activity was specifically inhibited with the dGTP analog BuPhdGTP. Under those conditions approximately one-half of the total DNA polymerase activity in crude extracts from a protease-deficient strain can be ascribed to Polδ (34Burgers P.M.J. Bauer G.A. J. Biol. Chem. 1988; 263: 925-930Abstract Full Text PDF PubMed Google Scholar). Overexpression of the POL3 gene alone resulted in an over 100-fold increase in POL3 mRNA levels, a strong increase in Pol3p polypeptide levels, and an 8-fold increase in BuPhdGTP-resistant DNA polymerase activity in cleared lysates (TableI, Fig. 1, data not shown). However, this polymerase activity was unstable and most of the increased activity was lost upon further fractionation by ammonium sulfate precipitation and phosphocellulose chromatography. A Western analysis showed that most of the Pol3p polypeptide precipitated during dialysis of the redissolved ammonium sulfate pellet, and some precipitated during dialysis after phosphocellulose chromatography (data not shown). These data indicate that the catalytic polypeptide of Polδ is active but unstable. The same results were obtained when overexpression of POL3 together with POL32 was carried out. Overproduction of polymerase activity in the cleared lysates was 9-fold, but the activity also decayed upon further fractionation (Table I). Again, by Western analysis, Pol3p was found in the insoluble pellet whereas most of the overproduced Pol32p remained soluble. A gel filtration analysis of the enzyme fraction after phosphocellulose chromatography failed to identify an activity which contained both Pol3p and Pol32p but lacked Pol31p (data not shown). These data suggest that Pol3p does not form a stable active complex with Pol32p. In contrast, overexpression of POL3 together withPOL31, or of POL3 together with POL31and POL32 resulted in a much larger increase in polymerase activity in crude extracts (Table I). Yet, the polypeptide levels in these extracts were not higher than when Pol3p was overproduced alone or together with Pol32p (Fig. 1). In addition, the polymerase activity from these strains remained stable during ammonium sulfate fractionation, phosphocellulose chromatography, and MonoQ HPLC (TableI). Overexpression of POL31 together with POL32gave an increase in the levels of these subunits, but no significant increase in DNA polymerase activity, indicating that the level of Pol3p limits the level of Polδ in the cell (Fig. 1, Table I). The partially purified preparations were fractionated on a strong anion exchanger (MonoQ), which separates Polδ from Polα and Polε. In comparison to the control strain a 2.2-fold increase in Polδ activity was measured in the MonoQ fractions when Pol3p alone was overproduced, and a 2.6-fold increase when Pol3p and Pol32p were overproduced together (Fig. 2 A and TableI). Interestingly, two poorly separated peaks of DNA polymerase activity resulted from overproduction of Pol3p together with Pol31p. Whereas the elution position of the minor peak coincides with that of Polδ, the earlier eluting major peak represents a novel activity (Fig. 2 B). The elution position is similar to that observed during fractionation of extracts from a mutant strain lacking Pol32p (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). To show that this novel peak represents a two-subunit form of Polδ, we overproduced Pol3p together with Pol31p in a protease-deficient pol32Δ strain. Only the early eluting peak, and no Polδ peak, was observed, and this activity was increased more than 10-fold in fractions from the overproducing strain (Fig.3). For the purpose of this study we identify the two-subunit form of Polδ as Polδ*.Figure 3MonoQ fractionation of DNA polymerases from overproducing strains lacking Pol32p. The profiles from a wild-type strain (■), from the isogenic pol32Δ mutant (○) and from the mutant strain overexpressing POL3 +POL31.(•, shown with ↑) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both Polδ and Polδ* were further purified by chromatography on a strong cation exchanger. Interestingly, Polδ* again eluted prior to Polδ, indicating that the highly charged nature of Pol32p promotes retention of Polδ on either ion-exchange matrix. After the MonoS step, Polδ was about 95% pure and Polδ* 50% pure (data not shown). They were further purified by gel permeation chromatography as described below. Enzyme from the MonoS step was injected on a Superose 6 column. Polδ eluted at a position consistent with that of a 520-kDa complex (Fig.4). This is in agreement with our initial studies of Polδ in which we noted that the apparent size of Polδ was >300 kDa (11Bauer G.A. Heller H.M. Burgers P.M.J. J. Biol. Chem. 1988; 263: 917-924Abstract Full Text PDF PubMed Google Scholar). In contrast, the elution position of Polδ* indicated a size of 180 kDa for that complex (Fig. 4). The data shown in Fig. 4 were obtained when concentrated enzyme at 0.4 mg/ml was injected onto the column. Dilution of the injected enzyme to 0.05 mg/ml did not change the respective elution positions of Polδ and Polδ* (data not shown). To obtain an estimate of the subunit stoichiometry, the peak activity fractions of the Superose 6 column were analyzed on a 10% SDS-polyacrylamide gel. The proteins were stained with Coomassie Brilliant Blue and the stained gels were digitized and quantitated. Scanning results indicate a stoichiometry for Polδ* of Pol3p:Pol31p = 1.0:0.9, and for Polδ of Pol3p:Pol31p:Pol32p = 1.0:1.05:1.1 (Fig. 5). Together with the gel filtration results, these data give as the most likely structure for Polδ* that of a heterodimer (Pol3p·Pol31p), and for Polδ that of a hexamer: a dimer of a heterotrimer (Pol3p·Pol31p·Pol32p)2. The specific activity of Polδ and Polδ* was measured on activated DNA (data not shown). Calculated on a weight basis Polδ had a 1.3-fold higher specific activity than Polδ*. This equates to a 3.2-fold higher specific activity for Polδ if calculated on a molar basis, or 1.6-fold higher if both catalytic cores in the hexameric Polδ are active. The processivity of both enzymes was determined on poly(dA)500·oligo(dT)22. Whereas Polδ* incorporated 2–3 dTMP residues per binding event, Polδ had a processivity of 6–12, in agreement with previous studies (Fig.6 and Ref. 12Bauer G.A. Burgers P.M.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7506-7510Crossref PubMed Scopus (138) Google Scholar). Upon addition of PCNA to the assay, both enzymes were fully processive. However, a much higher level of PCNA was necessary to stimulate processive DNA synthesis by Polδ* than by Polδ (Fig. 6). DNA synthesis by Polδ* or Polδ on extended SS DNA templates is virtually completely inhibited by the presence of 75 mm NaCl in the assay. On the other hand, these conditions are optimal for replication by a complex of Polδ with PCNA (13Yoder B.L. Burgers P.M.J. J. Biol. Chem. 1991; 266: 22689-22697Abstract Full Text PDF PubMed Google Scholar). PCNA was loaded onto singly primed single stranded-binding protein-coated SS mp18 DNA by RFC and ATP. Addition of Polδ resulted in extremely fast and efficient DNA synthesis (Fig. 7 A). Only marginal differences in replication efficiency were observed between Polδ purified from a wild-type strain of yeast through a six-column procedure and Polδ purified from the overproducing strain through a three-column procedure. The fastest complexes complete replication of the 7,250-nt mp18 circle within 1.5 min at 37 °C, a rate of more than 80 nt/s. In comparison, Polδ* is a very inefficient enzyme, replicating only ∼2 kb of DNA during the 20-min assay (Fig.7 A). Replication is still dependent on PCNA as no synthesis was observed in its absence. Pause sites with Polδ* are much more pronounced than with Polδ, indicating that sites of secondary structure form major replication barriers for this two-subunit enzyme (Fig. 7). The poor replication efficiency of Polδ* may be due to the frequent disassembly of replication complexes, perhaps at sites of secondary structure. In that case, rapid reassembly should be stimulated by providing excess PCNA or excess Polδ* resulting in more efficient synthesis. Indeed, high levels of PCNA stimulated Polδ*-mediated replication (Fig. 7 B). In comparison, because Polδ holoenzyme is a very stable complex, a slight molar excess of PCNA over primer termini already allowed maximally efficient synthesis by Polδ (Fig. 7 B). Similarly, a 10-fold molar excess of Polδ* over primer termini also greatly stimulated PCNA-dependent replication of SS mp18 DNA by this enzyme (data not shown). Pol32p overproduced in E. coli is found in inclusion bodies. The protein was efficiently renatured from a 6 m urea extract. Its properties indicate that Pol32p is a homodimer (7Gerik K.J. Li X. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Polδ* was incubated with renatured E. coli expressed Pol32p, and assayed in a holoenzyme assay on single-stranded mp18 DNA, in order to determine whether in vitro reconstitution of the three-subunit Polδ could be performed. The result in Fig.8 A shows that reconstitution of Polδ as measured by the formation of a processive holoenzyme proceeded quite efficiently. Controls, a dialyzed urea extract fromE. coli cells or from cells overproducing Pol31p, show that reconstitution is specific for Pol32p. To determine whether Pol32p would restore the dimeric structure of Polδ upon reconstitution, the E. coli produced subunit was purified to ∼60% homogeneity by MonoQ HPLC, preincubated with an equimolar quantity of Polδ*, and subjected to Superose 6 gel filtration. DNA polymerase activity, as measured on activated DNA, eluted as two poorly separated peaks at ∼500 and ∼200 kDa (Fig. 8,B and C). When the fractions were assayed for activity in a Polδ holoenzyme assay on SS mp18 DNA, the low molecular weight peak showed up as a mere shoulder to the high molecular weight peak (Fig. 8 B). An SDS-PAGE analysis showed that the peak at 500 kDa contained all three subunits of Polδ including Pol32p (Fig.8 C). The peak at 200 kDa also contained low amounts of Pol32p, but considering its low replication activity on SS mp18 DNA, functional Polδ was likely not reconsituted. Overproduction of Polδ in yeast was easily accomplished by cloning the genes for its three subunits under control of the galactose-inducible GAL1–10promoter (Table I). The enzyme isolated and purified from such an overproduction strain did not show marked differences with the enzyme isolated from a w
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