©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Catalytic Subunit of Human DNA Polymerase in Mammalian Cells Using a Vaccinia Virus Vector System (*)

(Received for publication, October 24, 1994)

Peng Zhang Isabelle Frugulhetti Yunquan Jiang Geraldine L. Holt (1) Richard C. Condit (1) Marietta Y. W. T. Lee (§)

From the Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101 Department of Microbiology and Immunology, University of Florida, Gainesville, Florida 32610

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The catalytic polypeptide of human DNA polymerase was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3` to 5` exonuclease activity. Recombinant pol was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol may be located in this region of the N terminus.


INTRODUCTION

DNA polymerase (pol ) (^1)was first isolated from the rabbit reticulocyte (Byrnes et al., 1976). Calf thymus (Lee et al., 1984) and human (Lee et al., 1991) pol have been isolated to near homogeneity and consist of two subunits of 125 and 50 kDa. The 125-kDa polypeptide was demonstrated to be the catalytic polypeptide by activity staining (Lee et al., 1991). A central role of this enzyme activity in mammalian DNA replication has been established through the investigation of the in vitro replication of the SV40 genome. A complex array of accessory proteins are also involved. The functional insertion of pol at the replication fork requires the actions of replication factor C, which leads to the recruitment of PCNA to the primer template terminus followed by binding of pol . Replication protein A, a single-stranded DNA binding protein, further potentiates the activity of the pol complex. The replication of SV40 has now been reconstituted in vitro using isolated components (Waga et al., 1994).

Because of the importance of the DNA polymerases, they have been intensively studied, and there is now a great deal of information on the primary structures of these enzymes from a wide range of organisms that span a broad evolutionary spectrum. Cloning of the human pol alpha cDNA showed that it is a member of a family of polymerases that include T4 DNA polymerase (Wong et al., 1988; Wang, 1991). cDNAs for the calf thymus (Zhang et al., 1991) and human (Chung et al., 1991; Yang et al., 1992) pol catalytic subunit have been cloned, as have the genes for the yeast homologues of pol (Boulet et al., 1989; Pignede et al., 1991). Mammalian and yeast pol are more highly conserved with each other than with their homologous pol alpha enzymes (Yang et al., 1992). Multiple sequence alignments have shown that there is a highly conserved central core region in the pol alpha family that is thought to contain most of the catalytic site (Wang, 1991). There is less conservation in the C-terminal regions, which, however, do contain two zinc finger regions that are thought to be involved in DNA binding. Analysis of the N-terminal regions show that there is some conservation among the members of the pol group of enzymes, and that these regions are conserved to some extent in herpes simplex virus type I DNA polymerase (Yang et al., 1992). The regions involved in the 3` to 5` exonuclease activity of yeast pol have been identified by site-directed mutagenesis (Simon et al., 1991). Little is known of the regions of pol that are involved in its functional interactions with other proteins. We have attempted to develop a system for the overexpression of pol that will provide a ready means for the preparation of the enzyme and for study of its structure-function relationships. In this report we describe the successful expression of the active catalytic subunit of pol using a vaccinia virus/pTM1 expression system in BSC-40 cells.


EXPERIMENTAL PROCEDURES

Cells

BSC-40 cells were a continuous line of African green monkey cells derived from BSC-1 cells (Hruby, et al., 1979). Cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics.

Vaccinia Virus Vectors

The recombinant vaccinia virus, which expresses the T7 bacteriophage RNA polymerase (vvTF7-3) was grown in BSC-40 cells from plaque-purified stocks (Fuerst et al., 1986). Plasmid pTM1 contains the encephalomyocarditis virus untranslated leader region downstream of pT7. The NcoI site contains the translation initiation codon and can be used for insertion of 5` end of the protein-coding segment (Moss et al., 1990).

Plasmids

Plasmid pALTER-1 was purchased from Promega (Dotto et al., 1981, 1984; Dotto and Zinder, 1983). This plasmid is a phagemid, defined as a chimeric plasmid containing the origin of a single-stranded DNA bacteriophage. This phagemid produces single-stranded DNA upon infection of host cells with a helper phage. pALTER-1 contains two genes for antibiotic resistance. One of them, for tetracycline resistance, is always functional. The other, for ampicillin resistance, has been inactivated. An oligonucleotide restores ampicillin resistance to the mutant strand during the mutagenesis reaction.

Site-directed Mutagenesis for cDNA Sequence Correction

The pol cDNA was inserted in pALTER-1 with BamHI and HindIII cloning sites. The construct (pALTER-pol ) was then used to transform E. coli JM109 competent cells. The single-stranded DNA was obtained after infection of the host cells with the helper phage R408. Two synthetic oligonucleotides (containing the corrected sequences based on the human DNA pol genomic sequence), 5`-ACCATCCCGCGGCTCCGT and 5`-AACTTGGCCATCAGCCGGGACAG, were annealed to the single-stranded DNA template at the same time as the oligonucleotide, which restores ampicillin resistance to the mutant strand during the mutagenesis reaction, and were subsequently treated with T4 DNA polymerase and T4 DNA ligase to produce a double-stranded circular molecule. The DNA was transformed into a repair minus strain of Escherichia coli (BMH 71-18 Mut S), and the cells were grown in the presence of ampicillin. The ampicillin-resistant plasmids were purified and then used to transform E. coli JM109 competent cells. The correctness of the insertion (pALTER-pol ) was confirmed by DNA sequencing using the dideoxynucleotide termination method (Sanger et al. 1977).

Insertion of Human DNA pol Coding Sequence into the pTM1 Vector

PCR amplification was used for the insertion of the coding sequence of human DNA pol into the pTM1 vector at a site between the pT7 and T7 terminator. The template used for PCR was the pALTER-pol cDNA clone. The sense and antisense primers were based on the human DNA pol sequence. The primer 5`-AAGCGCCATGGATGGCAAGCGG was used for 5` end of the coding sequence with an engineered NcoI site (underlined residues) at the initiating methionine codon. The second strand primer was 5`-GGCTGGGAGCTCCAGCCAGTT, which had a SacI site (underlined residues). The primers were phosphorylated with T4 polynucleotide kinase before use. The PCR conditions used were 94 °C for 1 min, 52 °C for 2 min, and 72 °C for 3 min for 25 cycles. The product was a single band of about 0.8 kilobase pairs that provides the 269 amino acid residues of human DNA pol from the initiation site to the SacI site. After purification using a Centricon 100 filter followed by phenol/chloroform extraction, the PCR product was digested with NcoI and SacI and then ligated into the pTM1 vector, which had been previously digested with NcoI and SacI and purified by agarose gel electrophoresis. The construct (pTM1--5`) was then used to transform E. coli DH5alpha-competent cells. The correctness of the inserted DNA was confirmed by DNA sequencing.

The human DNA pol cDNA clone (Yang et al., 1992) was digested with SacI and BamHI. After purification by agarose gel electrophoresis, the 2.7-kilobase pair fraction from SacI to termination codon was selected and then was ligated into pTM1--5`, which had been previously digested with SacI and BamHI. After transformation of E. coli DH5alpha competent cells, the single colony harboring the full-length pol coding sequence in pTM1 (pTM1-) was selected. The DNA sequence of pTM1- was again confirmed by DNA sequencing (Sanger et al., 1977).

Expression of Human DNA Poly Using pTM1-

BSC-40 cells were grown in 20 100-mm dishes with Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. When the cells had grown to 90% confluence, they were infected with vvTF7-3 with multiplicity of infection = 10 for 2 h. Innoculum was removed, and cells were washed with phosphate-buffered saline. Serum-free Dulbecco's modified Eagle's medium (10 ml) containing 5 mg/ml lipofectin (Life Technologies, Inc.) and 15 µg of pTM1- was added to each dish. The culture was incubated at 37 °C for 24 h.

Purification of Recombinant pol

The innoculum was removed, and the cells were washed twice with ice-cold phosphate-buffered saline and then were harvested into phosphate-buffered saline by using a rubber policeman. Cells were combined from all 20 dishes, centrifuged, and then were suspended in 40 ml of ice-cold isotonic buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 8.0) for 10 min. After centrifugation, the cells were resuspended in 18 ml of ice-cold hypotonic buffer (10 mM Tris-HCl, 10 mM KCl, 5 mM EDTA, pH 8.0) and were incubated on ice for 10 min. 50 µl of beta-mercaptoethanol and 2 ml of 10% Triton X-100 were added. The cells were incubated on ice for 10 min and then were centrifuged at 2000 rpm with Sorvall RT 6000 for 5 min. Centrifugation was repeated twice. The supernatant, which contains cytoplasm, was carefully pipetted for loading onto an immunoaffinity column. The pellet, which contains the nuclear portion, was resuspended in 20% ethylene glycol, 100 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 5 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium bisulfate, pH 7.5, and was sonicated at 50 watts for 2 min on ice with intermittent cooling. It was centrifuged at 40,000 rpm in a Beckman TLA 100.3 rotor at 4 °C for 30 min. The supernatant was collected and then was used to load onto the immunoaffinity column.

Immunoaffinity Chromatography

The extract (120 ml) was adjusted to a conductivity equivalent to 40 mM NaCl and was loaded onto a monoclonal DNA polymerase immunoaffinity column (10 times 1 cm), equilibrated with TGEE buffer (50 mM Tris-HCl, 0.5 mM EDTA, 0.1 mM EGTA, 10% glycerol, pH 7.8). The column was washed with the same buffer containing 0.4 M NaCl. This was followed by elution of pol with elution buffer (30% ethylene glycol, 50 mM Tris-HCl, 0.7 M NaCl, 0.5 mM EDTA, 0.1 mM EGTA, pH 7.9). Fractions of 1 ml were collected, and the fractions containing pol were identified by SDS-PAGE, Western blotting with C-terminal monoclonal antibody to pol , and assaying for pol activity.

Single-stranded DNA Cellulose Chromatography

The pooled fractions (30 ml) were dialyzed against HEPES buffer (20 mM HEPES, 5 mM MgCl(2), 10% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.5) and were then loaded on a single-stranded DNA-cellulose column (0.5 times 5 cm) equilibrated with HEPES buffer. The column was washed with same buffer. The enzyme was then eluted with a series of salt concentrations (85, 200, and 600 mM, and 1 M KCl) in the HEPES buffer at a flow rate of 60 ml/h. Fractions of 1 ml were collected and analyzed by SDS-PAGE, Western blotting, and polymerase activity assay.

Assay for pol Activity

Sparsely primed poly(dA)/oligo(dT) and poly(dA-dT) were used as the templates as described by Lee et al.(1984). For the poly(dA)/oligo(dT) assay, the standard reaction (100 µl) contained 0.25 OD units/ml poly(dA)/oligo(dT) (20:1), 200 µg/ml BSA, 5% glycerol, 10 mM MgCl(2), 25 mM HEPES, pH 6.0, 100 cpm/pmol [^3H]TTP, and 0.2-0.4 units of pol in the presence of 0.2 µg of PCNA. Reaction mixtures were incubated for 60 min at 37 °C and were terminated by spotting onto DE81 filter papers. Filters were washed 4 times with 0.3 M ammonium formate, pH 7.8, and once with 95% ethanol and counted as described previously (Lee et al., 1991). For the poly(dA-dT) assay, the standard reaction (100 µl) contained 0.4 OD units/ml of activated poly(dA-dT), 100 µg/ml BSA, 5% glycerol, 1 mM MgCl(2), 40 mM Tris-HCl, pH 7.8, 0.04 mM dATP, and 100 cpm/pmol [^3H]TTP and 10 µl of enzyme. After incubation for 37 °C for 60 min, the reaction was terminated by the addition of 2 ml of cold 5% trichloroacetic acid containing 10 mM sodium pyrophosphate. The precipitates were collected, washed, and counted as described previously (Lee et al., 1991).

Analysis of the Processivity of the pol Reaction

P-5`-end labeled oligo(dT) was annealed to poly(dA) (Midland Co.). The labeled template was purified on a Centricon 100 filter. The reaction mixture (60 µl) contained 0.33 OD units/ml [P]poly(dA)/oligo(dT), 40 mM Tris-HCl, pH 6.5, 5 mM MgCl(2), 2 mM dithiothreitol, 10% glycerol, 0.1 mg/ml BSA, 80 µM dTTP, 0.2 µg of PCNA, and 5 ng (about 1 unit) of pol activity. Reaction mixtures were incubated at 37 °C for 30 min, and terminated by the addition of 10 µl of 20 mM EDTA, 100 µg/ml salmon testes DNA. The DNA was precipitated with ethanol, dissolved in deionized formamide, 10 mM EDTA, 0.1% xylene cyanole. The samples were heated at 100 °C for 2 min, cooled on ice, and subjected to electrophoresis on 8% polyacrylamide gels containing 8% urea.

Assay for 3`-5` Exonuclease Activity

This was assayed by measuring the release of [^3H]dTMP from [^3H]dT as described previously (Lee et al., 1984). Each reaction mixture contained 2 µM [^3H]dT (200-300 cpm/pmol), 25 mM HEPES buffer, pH 7.4, 0.005 mg of BSA, 5 mM MgCl(2), and 0.2-0.4 units of pol in a final volume of 0.06 ml. Reaction mixtures were incubated for 30 min at 37 °C and were terminated by spotting 20 µl onto DE81 filter papers. Filters were washed 4 times with 0.3 M ammonium formate, pH 7.8, and once with 95% ethanol and counted as described previously (Lee et al., 1984).

Immunoblotting

This was performed using pol C-terminal monoclonal antibody (Zeng et al., 1994). After electrophoresis in 5-15% gradient gels, the protein was transferred to nitrocellulose membranes. Prestained protein standards (Sigma) were used as molecular weight markers and also to provide visual confirmation of efficient transfer. The nitrocellulose blot was incubated with 5% BSA in phosphatebuffered saline as a blocking agent. The blot was then incubated with pol C-terminal monoclonal antibody for 12 h at 25 °C. After washing 3 times with 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, the blot was incubated with biotinylated sheep anti-mouse immunoglobulin followed by incubation with streptavidin-biotinylated horseradish peroxides complex. Color development was performed by incubation with 4-chloro-1-naphthol and hydrogen peroxide and was terminated with sodium azide.

Protein Determinations

These were performed by the Bradford method(1976) using bovine serum albumin as a standard.


RESULTS

Construction of the pTM1 Vector for Expression of Human DNA pol in the Hybrid Vaccinia Virus/Plasmid System

We have characterized the gene structure of the 125-kDa catalytic polypeptide of human DNA pol (^2)and have sequenced all of the exons. Comparison of the genomic nucleotide sequence for the coding regions with that of the pol cDNA, which we previously reported (Yang et al., 1992) revealed several differences, two of which result in changes in amino acid sequence. Both of these are in the N terminus; His-119 and Asn-173 in the cDNA sequence are Arg and Ser, respectively, in the genomic sequence. It is possible that these differences could represent polymorphic variations, but we are certain of the genomic sequences because we have now reexamined this region of the sequence using additional cDNA clones from libraries different from those we had previously used. We therefore mutated the cDNA sequence to the genomic sequence by construction of the Arg-119/Ser-173 variant. The corrected coding sequence of pol was inserted into the pTM1 vector. Subsequent reisolation and DNA sequencing showed that the insert contained the correct sequence.

The pTM1 plasmid allows the insertion of a coding sequence for a given protein under the control of the T7 promoter and also contains the 5`-untranslated sequence of the encephalomyocarditis virus untranslated leader region. This plasmid was designed to allow expression of foreign messages in mammalian cells in the presence of T7 RNA polymerase following transfection with the plasmid. Eukaryotic expression is based on simultaneous infection with a recombinant vaccinia virus (vvTF7-3) that synthesizes the bacteriophage T7 RNA polymerase. Efficient translation of the uncapped mRNAs in mammalian cells is due to the presence of the encephalomyocarditis virus untranslated leader region (Nakano et al. 1991). For transient expression in mammalian cells, an infection/transfection protocol is performed with the plasmid and the recombinant vaccinia virus expressing T7 RNA polymerase (Nakano et al., 1991).

Expression and Isolation of Recombinant pol -p125

The insertion of the coding sequence for the 125-kDa catalytic polypeptide of pol into the pTM1 vector was found to allow its expression as a soluble protein in the cytosol of BSC-40 cells. The pTM1 vector without an insert was used as a control, and under the conditions used for Western blotting, pol was undetectable in cytosolic extracts of BSC-40 cells. The SDS-PAGE and Western blotting of crude cytosolic extract protein between control and experimental are shown in Fig. 1. As can be seen, pol was detected only in the BSC-40 cell extracts that had been infected/transfected with vaccinia virus (vvTF7-3) and pTM1-. Further experiments confirmed that the expressed pol was largely present in the cytosol and not in the nucleus, as determined by Western blotting (Fig. 1). Based on the relative intensity of the blots, it is apparent that almost all of the expressed pol polypeptide is in the cytosolic extract. Assays for pol activity (using poly(dA)/oligo(dT) as a template in the presence of PCNA) in the crude extracts of cells that had been transfected with pTM1- and the control plasmid pTM1 showed that only the cytosolic extracts from cells expressing pol displayed activity. Estimation of the degree of expression based on the specific activity of the purified enzyme (see below) indicated that the pol polypeptide represented approximately 1% of the soluble cytosolic protein.


Figure 1: SDS-PAGE and Western blot analysis of BSC-40 cells for the expression of the catalytic subunit of human pol . BSC-40 cells were infected/transfected with vvTF7-3 and pTM1 or pTM1- (see ``Experimental Procedures''). Cell extracts were then prepared and subjected to SDS-PAGE and Western blotted with a monoclonal antibody directed toward the C terminus of human pol . Leftpanel shows the Coomassie Blue staining of SDS-PAGE gels. LaneS, protein standards (alpha(2)-macroglobulin, 180 kDa; beta-galactosidase, 116 kDa; fructose 6-phosphate kinase, 84 kDa; pyruvate kinase, 58 kDa; fumarase, 48 kDa; lactate dehydrogenase, 36 kDa; triosephosphate isomerase, 26 kDa). pTM1- and pTM1 refer to cytosolic cell extracts from cells transfected with either the pTM1- or the pTM1 control plasmid. Centerpanel, Western blots of the cytosolic cell extracts (50 µl) from cells transfected with the pTM1- and the pTM1 plasmids. Rightpanel, Western blots (50 µl) of the nuclear and cytosolic extracts from cells transfected with the pTM1- plasmid. When the nuclear extract was concentrated 20-fold and Western blotted, a weak reaction was detectable (not shown).



Purification of Human DNA Polymerase

Recombinant human DNA pol was purified to homogeneity by a procedure involving chromatography on immunoaffinity chromatography procedure, (^3)followed by chromatography on a single-stranded DNA-cellulose column. The purification was monitored by assay of pol activity, SDS-PAGE, and Western blotting. The elution profile on immunoaffinity chromatography is shown in Fig. 2. It is seen that the eluted enzyme has activity toward both poly(dA-dT) and poly(dA)/oligo(dT) as the templates. In the latter case, the activity was assayed in the presence of PCNA and was approximately 10-fold higher than the activity assayed using the poly(dA-dT) template. The 3` to 5` exonuclease activity, a characteristic of DNA polymerase also co-eluted with the DNA polymerase activity. The SDS-PAGE and the Western blot across the peak of activity is shown in Fig. 3. It is seen that the activity peak is co-incident with the presence of a 125-kDa polypeptide that is strongly Western blotted by an antibody against pol . The peak fractions still contained a number of minor bands.


Figure 2: Purification of recombinant pol by immunoaffinity chromatography. A cell extract from BSC-40 cells that had been transfected with the pTM1- plasmid was chromatographed on an immunoaffinity support (see ``Experimental Procedures''). The diagram shows the assay of the fractions from the column that were eluted with 30% ethylene glycol, 50 mM Tris-HCl, 0.7 M NaCl, 0.1 mM EDTA, pH 7.9. Samples (5 µl) of the fractions were assayed for DNA polymerase activity using poly(dA-dT) as the template (solidtriangles) or with poly(dA)/oligo(dT) in the presence of PCNA (solidcircles), and for 3` to 5` exonuclease activity using 3`-end labeled poly(dT) (solidsquares).




Figure 3: Western blot analysis of the fractions obtained by immunoaffinity chromatography. Samples (50 µl) from the column fractions shown in Fig. 2were subjected to SDS-PAGE and stained for protein with Coomassie Blue (lowerpanel) and Western blotted using a C-terminal antibody against pol (upperpanel; only the 125 kDa band is shown).



The peak fractions of the immunoaffinity column were then passed through a single-stranded DNA cellulose column. After this step, the recombinant protein was near homogeneous and was Western blotted by a monoclonal antibody to the C-terminal region of human pol (Fig. 4). The summary of the purification achieved is shown in Table 1. Approximately a 100-fold purification was required, i.e. the pol polypeptide is expressed at about 1% of the soluble cytosolic protein. The levels of enzyme activity and protein far exceed those present in the BSC-40 cytosol, so that contamination of the expressed protein with endogenous BSC-40 pol , or for that matter, with vaccinia virus DNA polymerase, is unlikely, particularly since it was purified by a specific immunoaffinity chromatography procedure.


Figure 4: SDS-PAGE and Western blot analysis of recombinant pol after single-stranded DNA cellulose chromatography. The diagram shows the SDS-PAGE analysis of pol obtained after single-stranded DNA-cellulose chromatography (see ``Experimental Procedures''). Samples of the peak fractions of activity (50 µl) were subjected to SDS-PAGE and stained with Coomassie Blue (A) and Western blotted using anti-pol monoclonal antibody (B). S refers to protein standards as in Fig. 1.





Characterization of Recombinant Human Polymerase pol

Like the native enzyme, recombinant pol is inhibited by aphidicolin (Fig. 5). The recombinant and native human enzyme have the same sensitivity to KCl (Fig. 6). This is in marked contrast to the bacterial overproduced yeast polymerase , which is markedly stimulated by 250 mM KCl (Brown et al., 1993), whereas the wild-type yeast enzyme is inhibited by KCl. Recombinant and native human polymerase are both resistant to 10 µM BuAdATP (not shown).


Figure 5: Sensitivity of native and recombinant pol to aphidicolin. Purified human placental pol (Lee et al. 1991) and purified recombinant pol -p125 (5 µl each) were assayed in the presence of increasing amounts of aphidicolin. The results were expressed as relative activities (solidcircles, recombinant pol ; opencircles, native pol ).




Figure 6: Inhibition of recombinant pol by KCl. Purified human placental pol (Lee et al. 1991) and purified recombinant pol were assayed using poly(dA)/oligo(dT) as the template in the presence of PCNA and in the presence of increasing amounts of KCl. The results were expressed as relative activities (squares, recombinant pol ; circles, native pol .).



The response of recombinant pol to PCNA was examined. The recombinant enzyme was sensitive to PCNA and was stimulated 4.5-fold by 300 ng of PCNA. A highly purified human placental pol preparation (Lee et al., 1991) purified by conventional means was stimulated 10-fold by the same amount of PCNA (Fig. 7). In the same experiments, examination of the reaction products showed that the stimulation of recombinant pol by PCNA is also due to an effect on processivity.


Figure 7: Response of recombinant pol to PCNA. Purified human placental pol (Lee et al. 1991) and purified recombinant pol -p125 (5 µl each) were assayed using poly(dA)/oligo(dT) as the template in the presence of increasing amounts of PCNA. The results were expressed as relative activities (squares, recombinant pol ; circles, native pol .) The rightpanel shows an analysis of the reaction products to determine effects of PCNA on the processivity of pol (see ``Experimental Procedures''). Lanes1 and 5, human placental pol in the absence and presence of PCNA; lanes2 and 6, recombinant pol in the absence and presence of PCNA; lane3, PCNA alone; lane4, template alone.



Analysis of a PCNA-insensitive pol Mutant

In the course of the construction of the pTM1- vector, we used PCR amplification of the 5` end (see ``Experimental Procedures''). One of the full-length constructs was found to contain two PCR errors, such that nucleotide 467 was changed from A to G and nucleotide 473 was converted from T to C. This changed the amino acid sequence such that His-142 and Phe-144 were changed to Arg and Ser, respectively. The double mutant (H142R/F144S) was expressed and purified by immunoaffinity chromatography and single-stranded DNA cellulose. This mutant exhibited DNA polymerase activity but was found to have lost its sensitivity to PCNA (Fig. 8). Assays in which the replication factor C, replication protein A, and ATP were added together with PCNA also did not restore PCNA sensitivity (not shown). The behavior of this fortuitous mutant in which there are two mutations in the N-2 region strongly suggests that this region in the N terminus is involved in PCNA binding.


Figure 8: Lack of PCNA response by a double mutant of pol . The H142R/F144S mutant (squares) and the wild type recombinant mutant pol (circles) were assayed in the presence of increasing amounts of PCNA using poly(dA)/oligo(dT) as the template (see ``Experimental Procedures'').




DISCUSSION

We report here the first successful overexpression of the catalytic subunit of human pol as a soluble active protein using a vaccinia virus/plasmid vector system. The amounts of protein expressed were estimated to be 1% of the soluble protein. In practical terms, about 22 µg of p125 could be prepared to near homogeneity using a simple 2-step procedure involving immunoaffinity chromatography and single-stranded DNAcellulose chromatography. Isolation of the mammalian pol is extremely tedious, and only microgram amounts of purified enzyme can be obtained (Lee et al., 1984, 1991). Only a handful of laboratories have ever reported preparations of demonstrable purity owing to the difficulties of its isolation, and even highly purified pol preparations commonly consist of multiple polypeptide components (Goulian et al., 1990; Feher and Mishra, 1994). There are no previous reports of the expression of mammalian pol , and we also have encountered extreme difficulty in expression of this cDNA in E. coli. Yeast DNA pol has been overexpressed in E. coli (Brown and Campbell, 1993) as an insoluble and inactive aggregate, and the recombinant enzyme was obtained after solubilization with urea and renaturation.

These studies show that the vaccinia virus (vvTF7-3)/pTM1 system is effective for the expression of the human pol catalytic subunit. The use of the vaccinia virus/pTM1 infection/transfection system has been shown to allow for very efficient overexpression of genes in mammalian cells (Fuerst and Moss, 1989; Moss et al., 1990) and also allows for rapid screening of the mutants. Levels of the foreign transcripts have been estimated to be as high as 30% of the total steady-state RNA in the cytoplasm of the infected cells after 24 h as shown for T7-lac Z transcripts (Fuerst and Moss, 1989). The recombinant vv/T7 hybrid expression system offers certain advantages over the baculovirus system. For example, site-directed mutants of the target gene can be constructed rapidly in the expression vector pTM1, and their biological activities can be assayed by the infection/transfection protocol (Moss et al., 1990). In addition, protein synthesized in insect cells are likely to be different from those synthesized in mammalian cells with respect to some of post-translational modifications (Miller, 1988). Thus, the vaccinia virus system allows for overexpression in a mammalian cell context that provides some assurance that any post-translational modifications would be those normally encountered.

Our studies show that the recombinant p-125 is almost exclusively found in the cytoplasm of the BSC-40 cells. Our findings differ from those of the adenovirus DNA polymerase, which was transiently expressed using the same vv/pTM1 expression system (Nakano et al., 1991) in that the recombinant adenovirus polymerase was distributed both in the cytoplasm and nucleus. It may be possible that translocation of pol -p125 to the nucleus is a relatively slow process, or alternatively, it may be a regulated process.

Recombinant pol was characterized and exhibited essentially similar catalytic properties as the native enzyme. However, we observed that its PCNA response was consistently lower than that of enzyme isolated from human placenta. It has been previously suggested that the catalytic polypeptide is not responsive to PCNA and that this property is conferred by the p50 subunit. The evidence is inferential and is based on isolation of enzyme preparations that consist only of the 125-kDa subunit and do not respond to PCNA. Mouse cells have been suggested to possess of two forms of pol , one consisting of only a 125 kDa, and the second having two subunits of 125 and 50 kDa. The first is not stimulated by PCNA, suggesting that the 50-kDa subunit is required for the interaction of polymerase with PCNA (Goulian et al., 1990). Rabbit reticulocyte pol was reported to have a single subunit of 122 kDa (Goscin and Byrnes, 1982). This form was later reported by Lu and Byrnes(1992) to be a PCNA-independent form. The same authors have also purified a PCNA-dependent DNA polymerase from rabbit bone marrow that is similar to the calf thymus DNA polymerase in consisting of two subunits and being responsive to PCNA. Chiang et al.(1993) purified a pol from Drosophila to near homogeneity, which consisted of a single 120-kDa polypeptide and is not stimulated by PCNA. Our results show that unequivocally the human p125 subunit does respond to PCNA. Our data agree with that of Brown and Campbell(1993), who showed that overexpressed yeast pol in E. coli is stimulated about 2.5-3-fold by PCNA. Nevertheless, our findings do indicate that the p50 subunit may be important in the response of the catalytic subunit to PCNA, since the degree of stimulation by PCNA was consistently lower than that of the native two subunit enzyme consisting of 125- and 50-kDa subunits. In other preliminary studies, we have shown by reconstitution experiments that the addition of the p50 subunit also potentiate the response of the recombinant p125 subunit to replication factor C and replication protein A in the presence of PCNA.

The vv/pTM1 system thus provides a useful means for preparation of the catalytic subunit as well as a means for structure-function studies of pol by mutagenesis. Examination of an adventitious double mutant in which 2 residues in the N terminus, His-142 and Phe-144, were mutated to Arg and Ser, respectively, showed that its response to PCNA was severely attenuated. This strongly suggests that this region is involved in PCNA binding. This is the first report of a site-directed mutant that is affected in its PCNA binding and is consistent with other studies using synthetic peptides that suggest that the PCNA binding region of pol is localized to a region between residues 129 and 149 (Zhang et al., 1995).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM31973. 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: P. O. Box 016960 (R57), University of Miami School of Medicine, Miami, FL 33101. Tel: 305-547-6338; Fax: 305-547-3955.

(^1)
The abbreviations used are: pol , DNA polymerase ; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.

(^2)
L.-S. Chang, L. Zhao, L. Zhu, M.-L. Chen, and M. Y. W. T. Lee, submitted for publication.

(^3)
Y. Jiang, S.-J. Zhang, S.-M. Wu, and M. Y. W. T. Lee, submitted for publication.


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