©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Further Characterization of HeLa DNA Polymerase (*)

(Received for publication, September 16, 1994; and in revised form, January 25, 1995)

Gloria Chui (§) Stuart Linn (¶)

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

DNA polymerase (pol ) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol preparation showed two polypeptides of >200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol (as well as pols alpha and ) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl(2). Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol activity, processivity, or substrate specificity. The size of the pol transcript for the catalytic subunit (>200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol by immunoblotting, but not that of pol alpha, beta, or . In addition, mouse polyclonal antiserum raised against column-purified pol was able to recognize and to neutralize pol , and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol .


INTRODUCTION

DNA polymerase (pol ) (^1)from HeLa cells, unlike yeast pol (1) , contains two apparent subunits of >200 and 55 kDa (2) and appears to be required for long patch DNA repair in permeabilized cells(3) . Unlike pol , pol is highly processive in the absence of proliferating cell nuclear antigen (PCNA). Studies with primed, single-stranded DNA observed that human single-stranded DNA binding protein (HSSB; also known as replication factor-A (RF-A)) and activation inhibitor (also known as A1, or replication factor-C (RF-C)) (^2)stimulate pol alpha and pol activities(4, 5) . These replication factors are required for sequential initiation of lagging and leading strand DNA synthesis in reconstituted SV40 replication systems(6, 7) . It has also been suggested that inhibition of HeLa pol by NaCl can be overcome by the presence of HSSB, A1, and PCNA(8) . We have investigated the effect of these replication factors on HeLa DNA pol processivity and activity under a variety of salt conditions.

Previous studies of template specificity among the three HeLa DNA pols suggested that pol alpha prefers activated DNA as template, and pol prefers alternating poly(dA-dT) as template, whereas pol prefers long stretches of single-stranded regions in poly(dA)bulletoligo(dT) as template(9) . We have further investigated the template specificities of these polymerases, particularly with polyribonucleotide primers.

It has been reported that yeast pol is involved in DNA replication (1) , and our current interest is to determine whether HeLa pol is involved in either or both DNA replication and DNA repair. To address the question of DNA replication, we compared the mRNA levels of pol in quiescent and proliferating HeLa cells as well as in untreated and UV-irradiated HeLa cells and normal diploid fibroblasts.

We have also begun an immunogenic study for which a fusion protein (staphylococcal protein A fused to a pol fragment) was used to produce antiserum that is able to recognize denatured pol but can neither immunodeplete nor neutralize enzymatic activity. Purified pol was also used to raise a mouse antiserum that is able to recognize different pol subunits and neutralize pol activity and shows no cross-reaction with HeLa pols alpha, beta, and . We have now produced monoclonal antibodies from this mouse and have obtained purified IgGs, one of which is described here. It specifically recognizes the pol catalytic subunit.


EXPERIMENTAL PROCEDURES

Materials

Nucleotides and Nucleic Acids

Unlabeled deoxy- and ribonucleotides were purchased from Pharmacia Biotech Inc.; [alpha-P]dTTP and [alpha-P]dATP were from Amersham Corp. Poly(dA) and poly(dT) were purchased from Midland Certified Reagent Co., Midland, TX. Activated calf thymus DNA and M13 single-stranded DNA were prepared as described(10, 11, 12) . Oligo(dT), oligo(dA), oligo(rA), and a synthetic 15-nucleotide primer (map positions 6323- 6309) for M13mp18DNA were from New England Biolabs. M13mp18 RF DNA was kindly provided by Dr. C. Kane of this department. The primer was annealed to M13mp18 single-stranded DNA using a 10:1 molar ratio of primer to template except that processivity assays used a 1:1 primer to template molar ratio. Annealing was carried out at 65 °C by heating in a water bath for 10 min and then switching off the heat and allowing the sample to cool slowly to room temperature. RNA-primed M13 template was made by incubating Escherichia coli RNA polymerase holoenzyme and the single-stranded M13 template at 37 °C for 5 min to incorporate an average of 107 ribonucleotides/M13 DNA molecule. CTP was ^3H labeled so that the number of ribonucleotides incorporated could be estimated. The reaction mixture was extracted with phenol and chromatographed on Sephadex G-150 to remove residual NTPs.

Enzymes, Proteins, and Plasmids

DNA pols alpha, , and and PCNA were purified from HeLa cells as described(9) . Pol was purified further by Mono S FPLC column chromatography to remove residual pol alpha. HeLa pol beta was provided by Jeff VanDehy of our laboratory. It was approximately 50% pure. RF-A and RF-C were kind gifts of Drs. Tom Melendy and Bruce Stillman (Cold Spring Harbor Laboratory). HSSB and A1 were kind gifts of Drs. Suk-Hee Lee and Jerard Hurwitz (Memorial Sloan-Kettering Cancer Center). E. coli SSB was purchased from Pharmacia. T4 DNA ligase, isopropyl-1-thio-beta-D-galactopyranoside, and restriction enzymes were from Boehringer Mannheim.

Antibodies

IgG-neutralizing monoclonal antibody against human DNA polymerase alpha (anti-pol alpha) was made from the hybridoma cell line SJK 132-20 obtained from the American Type Culture Collection. IgG was purified either by protein A-Sepharose or by DEAE-Sephacel chromatography.

The protein A gene fusion vector pRIT31 was originally constructed by Uhlen and co-workers (13) and maintained in E. coli RSB571 (derived from E. coli 697). This vector has both the lac^q and the temperature-inducible C1 promoters. The glutathione S-transferase (GST) fusion vector, pGEX-1T, was purchased from Pharmacia with the correct reading frame for the pol fragment and maintained in E. coli DH5alpha. The pol fragment used for constructing the Staphylococcus aureus protein A (SpA-) fusion protein was a 435-bp BamHI/EcoRI fragment of a 1.2-kb clone obtained in this laboratory. This pol fragment (nucleotides 1611-2045) (14) contains the conserved region II of the alpha-class DNA polymerases(15, 16) .

Monoclonal Antibody Production

Approximately 6-month-old retired breeder Swiss-Webster female mice were used for immunization. For injection, column-purified pol antigen was mixed with the adjuvant RIBI (monophosphoryl lipid A synthetic Trehalose dicorynomydolate and cell wall skeleton in squalene and Tween 80) purchased from RIBI Immunochem Research Inc., Hamilton, MT. The mouse myeloma cell line used for fusion was P3X63 AG8-653. The fused cells were selected with HAT medium (10M hypoxanthine, 10M aminopterin, 1.2 times 10M dT) and then grown in Iscove's modified Dulbecco's medium with 20% fetal calf serum (FCS), 10% macrophage-conditioned medium, and half-strength HT (5 times 10M hypoxanthine, 6 times 10M dT). The fusion process was carried out using a Shimadzu SSH-1 somatic hybridizer. ELISA screening was conducted employing a multiscan at 405 nm using 96-well Immulon 2 plates from Dynatech.

Probes for Pol and Actin Transcripts

The probe for Northern analysis contained the conserved regions IV and II corresponding to nucleotides 1022-1835 of the cDNA for human pol (14) . A plasmid containing a 800-bp chicken actin cDNA fragment was obtained from Dr. Richard Harland of our department to use as an actin mRNA probe.

BA-S NC nitrocellulose membranes for Northern blots were from Schleicher & Schuell. Randomly primed DNA kits for labeling probes and RNA molecular markers II (7.4, 5.3, 2.8, 1.9, and 1.6 kb) were purchased from Boehringer Mannheim. GeneClean kits were purchased from U. S. Biochemical Corp.

Methods

Separation of Pol from Pol alpha by Mono S Chromatography

HeLa DNA pol was purified as described (9) except that the final glycerol gradient separation was replaced by Mono S FPLC chromatography, which resulted in a complete separation of pol alpha from pol . The pol , hydroxylapatite fraction (9) was adjusted to 50 mM potassium phosphate (pH 7.5), containing 10% (v/v) glycerol and 1 mM DTT and passed onto the Mono S column, which had been equilibrated with the same buffer. The column was washed with 5 column volumes of the buffer and then eluted with a 12-column volume gradient from 0 to 500 mM NaCl in the same buffer. The pol peak eluted in 130 mM NaCl, pol alpha activity eluted in 250 mM NaCl, and only the pol alpha fractions contained detectable primase activity, indicating that the two polymerases had been completely separated. Denaturing protein gel electrophoresis also confirmed the separation.

DNA Polymerase Trap Experiment

A sequence-specific oligonucleotide was used to form a hairpin template as described by Insdorf and Bogenhagen(17) . HeLa pol (0.5-1 unit) and 7.5 pmol of the hairpin template were incubated in a 25-µl reaction mixture containing 4 nmol of BrdUTP, 25 pmol of [alpha-P]dATP (3,000 Ci/mmol), 50 mM Hepes/KOH (pH 7.5), 5 mM DTT, 7.5 mM MgCl(2), 0.05% (v/v) Triton X-100, 10% (v/v) glycerol, and 0.25 mg/ml bovine serum albumin (BSA) for 10 min at 22 °C. For the control experiment, 5 units of E. coli pol I was used. (One unit incorporates 10 nmol of total nucleotide in 30 min.) After preincubation, the reaction was stopped by chilling on ice, and 1 µl was checked for acid-insoluble counts. The remaining reaction mixtures were irradiated, positioned 6 cm from two 15-watt Fotodyne UV 300 nm bulbs for 10 min while on ice. DNase I (1.5 µg) and 10 µg of micrococcal nuclease were added, and then the mixtures were incubated at 37 °C for 30 min. After incubation, the reaction was precipitated with 12% (g/100 ml) trichloroactetic acid, resuspended into SDS-gel buffer, and run on a 6% SDS-polyacrylamide denaturing gel where the photolabeled polypeptide was visualized by autoradiography.

DNA Polymerase Assays

Standard reaction mixtures (25 µl) for measuring pol activity contained 50 mM Hepes/KOH (pH 7.5), 15 mM MgCl(2) (unless otherwise indicated), 100 mM potassium glutamate (pH 7.8), 10 mM DTT, 0.03% (v/v) Triton X-100, 20% (v/v) glycerol, 0.2 mg/ml BSA, 50 µM^3H- or P-labeled dTTP, and 40 µM (dA) and 4 µM oligo(dT) to give an interprimer distance of about 135 nucleotides. Pol alpha activity was measured with 12.5 µg of activated calf thymus DNA primer-template in a total volume of 50 µl containing 50 mM Tris-HCl (pH 7.5); 7.5 mM MgCl(2); 0.5 mM DTT; 0.2 mg/ml BSA; 50 µM dATP, dGTP, dCTP; and [^3H]dTTP (203 cpm/pmol). Pol was assayed with 40 µM poly(dA) and 4 µM oligo(dT) in a total volume of 50 µl containing 40 mM Hepes/KOH (pH 7.5), 6 mM MgCl(2), 10% (v/v) glycerol, 40 µg/ml BSA, 1 mM DTT, and 40 µM [^3H]dTTP either in the presence or absence of PCNA (100 ng/reaction). Incubations were performed at 37 °C for 30 min for all three polymerases. One unit of polymerase is defined as the amount that catalyzes the incorporation of 1 nmol of total nucleotide into an acid-insoluble form per h(18) .

Processivity assays used either single primed (dA)bulletoligo(dT) or single primed M13mp18 DNA. Reactions were as above except that [alpha-P]TTP (about 5,000-8,000 cpm/pmol) was used. The incorporation was controlled so that on average roughly one nucleotide or less was incorporated per primer terminus. Thus the average length of the product in each case corresponds to a single elongation event and reflects the processivity of the enzyme.

Primase activity was assayed by coupling oligoribonucleotide synthesis to polymerization of dNTPs by E. coli DNA polymerase I Klenow fragment(19) . The rate of the reaction was determined by monitoring the incorporation of labeled dNTPs into acid-insoluble material. Reaction mixtures (50 µl) contained 50 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.5 mg/ml BSA, 7.5 mM MgCl(2), 1 mM ATP, 80 µM (dT), 20 µM [^3H]dATP (200 cpm/pmol), and 0.5 unit of Klenow fragment. Incubations were for 30 min at 30 °C.

DNA Gel Analyses

Two kinds of gels were used to analyze DNA products. Processivity studies utilized 8% polyacrylamide, 7 M urea slab gels prepared as described (20) and run at 300 mV for 3 h and then dried under vacuum. Autoradiography was performed by exposing the gel to Kodak XAR-5 film at -80 °C using an intensifying screen for an appropriate length of time. To determine the maximum length, DNA products were analyzed on 0.8% alkaline agarose gels containing 50 mM NaOH, 2 mM EDTA and were run at 40 mA for 12 h. At the end of the run, the gels were washed in 90 mM Tris borate (pH 8.3), 2.5 mM EDTA for 30 min to remove the alkali and were then vacuum-dried without heat for 20 min to remove water. They were then dried under vacuum with heat for 2 h and visualized by autoradiography. Products were precipitated with ethanol in the presence of 10 µg of salmon sperm DNA and resuspended before analysis.

Analysis of HeLa DNA Pol alpha, , and Preparations with Neutralizing Pol alpha Antibody

Anti-pol alpha antibody (SJK 132-20) was preincubated with HeLa DNA pol alpha in 50 mM Tris-HCl (pH 7.5), 2.5 mM DTT, and 0.5 mg/ml BSA in 15 µl at 0 °C for 2-4 h, and then 35 µl of a mixture was added to bring the reaction to 50 mM Tris-HCl (pH 7.5); 0.5 mg/ml BSA; 50 µM each dATP, dCTP, dGTP; 50 µM [^3H]dTTP (225 cpm/pmol); 0.25 mg/ml activated calf thymus DNA; and 7.5 mM MgCl(2). Pol was similarly preincubated in 50 mM Hepes/KOH (pH 7.5), 0.5 mg/ml BSA, 10 mM DTT, and then 10 µl of a mixture was added to bring the reaction to 50 mM Hepes/KOH (pH 7.5), 50 µM [^3H]dTTP, 0.03% (v/v) Triton X-100, 20% (v/v) glycerol, 15 mM MgCl(2), 10 mM DTT, 40 µM (dA), and 4 µM oligo(dT). Likewise, the preincubation buffer for pol was 40 mM Hepes/KOH (pH 7.5), 0.5 mg/ml BSA, 1 mM DTT, and 35 µl of a solution was added to bring the reaction to 40 mM Hepes/KOH (pH 7.5), 6 mM MgCl(2), 10% (v/v) glycerol, 0.2 mg/ml BSA, 1 mM DTT, 40 µM (dA), and 4 µM oligo(dT), 40 µM [^3H]dTTP, and 1-2 ng/ml PCNA where indicated.

Northern Blot Analysis

Fragments of the cloned pol cDNA as described were used to make probes using a random primed DNA kit. The specific activity of these probes was in the range of 1-8 times 10^8 cpm/µg. Total RNA was prepared from 5-10 times 10^8 HeLa cells or 3-5 times 10^7 F65 normal fibroblasts by guanidium thiocyanate extraction followed by pelleting of the RNA through 5.7 M CsCl(21) . For serum-starved HeLa cells, early to mid-log phase cells were suspended in media containing 0.1% FCS instead of 10% FCS and incubated for 96 h. Half of the cells were utilized for the RNA preparation, and the other half were supplemented with medium containing 10% FCS for an additional 30 h to yield proliferating cells. To assess the effect of UV exposure, 5-10 times 10^8 mid-log phase HeLa cells were saved as the untreated control while 1-2 times 10^9 cells were suspended in 10 ml of phosphate-buffered saline solution. Half of these cells were immediately transferred back to the original medium as the mock treatment, and the other half were layered onto a Petri dish that had been coated previously with phosphate-buffered saline containing 5% FCS and then UV irradiated for 7 s at 2 J/m^2. After UV irradiation, cells were transferred back to the original medium and incubated for 4 h before harvesting. Since F65 fibroblasts adhere to the plates, the supernatant was removed, and UV or mock treatment was performed on the original plates. RNA concentrations were estimated by A.

Thirty µg of the total RNA was denatured and electrophoresed through 0.7% agarose, 6.3% formaldehyde, 50% formamide gels at 40 volts for about 3 h. The gels were electroblotted onto a nitrocellulose membrane for 8-10 h in 20 times SSC and then fixed at 80 °C under vacuum for 1-2 h. The membrane was prehybridized at 42 °C for a minimum of 6 h and then hybridized with a P-labeled probe at 42 °C in 50% formamide for at least 12 h in the presence of 50% formamide. After hybridization, the membrane was washed twice in 2 times SSC, 0.5% SDS for 1 h at room temperature and then twice in 0.1 times SSC, 0.5% SDS for 1 h at 50 °C and finally twice at 65 °C with Ultra Blot Wash (50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.5% sodium pyrophosphate, 1 times Denhardt's solution, 1% SDS, 0.05% N-lauroylsarcosine). Membranes were exposed on Kodak XAR-5 film at -80 °C for appropriate times with an intensifying screen, and then band intensities were quantitated with a Hoefer gel scanner.

For pol activity measurements corresponding to the mRNA levels, HeLa cells or F65 fibroblasts were treated as for the RNA preparation and then harvested. Extracts were prepared as described for the HeLa pol preparation(9) , and then protein was fractioned with ammonium sulfate. The material that precipitated between 30 and 50% saturation was resuspended and dialyzed against 4 liters of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10% (v/v) glycerol. All autoradiograms were quantitated with a Hoefer gel densitometer.

Preparation of the Fusion Proteins for Antibody Production

A SpA fusion vector (pRIT 31) as well as a pol cDNA fragment were cut by BamHI and EcoRI, and the linearized plasmid and a DNA fragment of the pol cDNA from nucleotides 1611-2045 (14) were purified by agarose gel electrophoresis and ligated overnight at 16 °C at a molar ratio of 3 inserts to 1 vector. Colonies were screened by restriction analysis to obtain plasmid SpA-BE2. A second fusion vector, pGEX-1T, containing the glutathione S-transferase gene, was fused to nucleotides 1611-2045 of the pol cDNA to produce a second fusion plasmid, GST-G5. The SpA-pol fusion protein was induced by incubating at 42 °C for 2 h, and the GST-pol fusion protein was induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside. Cultures were grown overnight in 10 ml of Luria broth containing 100 µg/ml ampicillin (LB+amp) and then transferred to 1 liter of LB+amp and allowed to grow about 2 h to an A of 0.5-1.0 before induction. Following centrifugation, cell pellets were washed and resuspended in 50 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml apotinin, 1 mM EDTA, 0.25% (v/v) Triton X-100 for each liter of culture. Suspensions were sonicated 10 times for 20 s at 0 °C with a Branson sonicator and then centrifuged at 15,000 times g for 5 min. The induced fusion proteins were soluble. The SpA-pol fusion protein was purified on an IgG-Sepharose 6 FF column according to the procedure from Pharmacia. The protein had a molecular mass of about 46 kDa, consistent with the combination of the 31-kDa SpA and the 15-kDa polypeptide expected from the 435-bp pol DNA fragment. The GST fusion protein was purified with glutathione-Sepharose 4B purchased from Pharmacia. The protein had a molecular mass of about 41 kDa as expected for a combination of the 26-kDa GST protein and the 15-kDa pol fragment. Both fusion proteins appeared to be more than 90% homogeneous by denaturing gel analysis.

Monoclonal Antibody Production against Native HeLa DNA Pol

Forty µg of the Mono S FPLC-purified HeLa pol was injected four times over a period of 1 month into a mouse to stimulate antibody production. Antiserum was collected by tail bleeding and then tested for antibody response by either ELISA, Western immunoblot, or DNA polymerase neutralization assays. Preimmune serum was saved as a negative control.

To obtain monoclonal antibodies, the mouse with the best antigenic response was sacrificed. Its spleen was removed, minced gently, and then the spleen cells were fused with myeloma cells by electrofusion at the ratio of five spleen cells to one myeloma cell. Immediately following the fusion, the cells were grown in Iscove's modified Dulbecco's medium with 20% FCS and 10% macrophage-conditioned medium. After 24 h, the unfused cells were selected against using HAT medium, and then the fused hybridoma cells were maintained in the same medium with half-strength HT concentrations. Fused hybridoma cells began to form colonies in about 10 days to 2 weeks. Aliquots of supernatant from wells with a single colony about one-third of the size of the well were screened for antibody response by ELISA. High titer samples were tested further by polymerase neutralization assays and Western immunoblots. Based on the above results, interesting cell lines were subcloned by serial dilution and dispensed into five 96-well plates such that each well would contain only one cell. Hybridomas with positive responses, producing monoclonal antibodies with the ability to recognize pol subunits or to neutralize pol activity, were then selected and scaled up for IgG production. Cell lines were placed in Iscove's modified Dulbecco's medium with 10% dimethyl sulfoxide and 30% FCS and frozen in liquid nitrogen for long term storage.


RESULTS

Characterization of the Pol Preparation

In our previous purification procedure of HeLa pol (9) , pol alpha and pol were not completely separated at the final glycerol gradient step. However, if the penultimate hydroxylapatite fraction is chromatographed on a Mono S column as described under ``Experimental Procedures,'' pol and pol alpha activities are well resolved, eluting at 130 mM NaCl and 250 mM NaCl, respectively. Separation was confirmed by the different template preferences for the two peaks and the intrinsic primase activity that was present only in the pol alpha fractions. The purified pol alpha and pol fractions were characterized further by SDS-polyacrylamide gel electrophoresis, which showed them to be separated and pol to be nearly homogeneous (Fig. 1). Finally, purified IgG from SJK 132-20, a monoclonal cell line producing neutralizing antibody against KB cell pol alpha, confirmed the separation of HeLa pols alpha and . One hundred to 200 ng of the IgG gave 50% inhibition for 0.06 unit of pol alpha, and additional antibody neutralized greater than 90% of the pol alpha activity. However, 2 µg of antibody slightly neutralized the hydroxylapatite pol fraction but actually stimulated an equivalent amount of Mono S-purified pol fraction by 15%.


Figure 1: SDS-polyacrylamide gel analysis of HeLa DNA pol purified by Mono S FPLC. The Mono S fraction of HeLa DNA pol (0.1 mg) was electrophoresed on a 7% SDS-polyacrylamide gel and stained with silver. The positions of molecular mass markers are shown on the left in kDa.



To identify the catalytically active subunit(s) present in the preparation, the photolabeling procedure of Insdorf and Bogenhagen (17) was employed. This technique incorporates BrdUTP and [alpha-P]dATP into a synthetic hairpin-primed template. The polymerase stalls at a site where the complimentary dNTP is absent, and then UV-irradiation is used to cross-link the pol to the hairpin template via BrdU. SDS-polyacrylamide gel electrophoresis is then employed to resolve the pol heterodimer, leaving the pol catalytic subunit covalently attached to the radioactive template. Autoradiography revealed two major bands of >200 and 122 kDa (data not shown). Kesti and Syvaöja (22) identified a 258-kDa polypeptide in a similar DNA polymerase trap experiment which, when cleaved by trypsin, generated an active 122-kDa fragment. Thus the Mono S fraction had both forms of the pol catalytic subunit.

Salt Optima

To optimize reaction conditions for the studies below, salt effects upon pol activity were investigated. The addition of KCl up to 50 mM, or potassium glutamate at 100 mM, increased pol activity by as much as 2-fold. In contrast, 100 mM KCl, NaCl, (NH(4))(2)SO(4), or K(2)SO(4) completely inhibited pol . Pol alpha was also stimulated about 2-fold by 100 mM potassium glutamate, although pol was inhibited.

When assayed with (dA)bulletoligo(dT)12-18, pol alpha had maximal activity between 1 and 15 mM MgCl(2), whereas pol had the highest activity, between 10 and 15 mM MgCl(2). Thus the two pols can be distinguished by their different MgCl(2) optima when assayed on poly(dA)bulletoligo(dT). However, when assayed on primed M13 DNA, both pols had a maximal activity at 1 mM MgCl(2).

Activity of Pols alpha, , and on RNA-primed Templates

As a possible clue to the function of pol , its activity on both DNA-primed and RNA-primed templates was compared with those of pols alpha and (Table 1). Pol alpha was especially active with poly(dT)bulletoligo(rA) and had considerable activity with RNA-primed M13 DNA. Pol also demonstrated activity upon RNA-primed templates, and it is noteworthy that PCNA had no strong effect on this activity. Like pol alpha, pol also was especially active upon poly(dT)bulletoligo(rA) and had activity with RNA-primed M13 DNA. In conclusion, all three enzymes efficiently extended RNA primers with deoxyribonucleotides.



Effect of Replication Factors on Pol Activities

The effects of RF-A (HSSB^2), RF-C (A1), and PCNA on pol activity and processivity were studied. For comparison, experiments were performed using either 1 mM MgCl(2) or 10 mM MgCl(2). When assayed on single primed M13 DNA, RF-A (and HSSB) had no effect on pol activity, RF-C (and A1) stimulated pol activity slightly, and T4 gene 32 protein inhibited pol activity (Table 2, Experiments I and II). Pol has higher activity with 1 mM MgCl(2) on this substrate, so levels of enzyme were adjusted to give similar levels of incorporation (in the absence of added factors).



As seen in Fig. 2A, the factors also had little effect upon processivity of pol in these same reactions; the enzyme was extremely processive in the presence or absence of these factors. In fact, pol was able to copy the entire M13mp18 genome in the absence of factors, and PCNA had no effect on pol activity or the DNA product size (Fig. 3).


Figure 2: Effect of replication factors on the processivity of pol with single primed M13 DNA. Panel A, reactions are those of Table 2, Experiment I. The pause site noted near nucleotide 120 is the synthetic polylinker region of M13mp18 DNA. Panel B, reactions are those of Table 2, Experiment II, with 1 mM MgCl(2), either in the presence or absence of 100 mM NaCl. DNA products were analyzed on 8% polyacrylamide, 7 M urea gels.




Figure 3: Effect of PCNA on the processivity of pol with single primed M13 DNA. Assays (25 µl) contained 1 mM MgCl(2), 2.5 pmol of primer termini, and 80 ng of HeLa PCNA from various phenyl-Sepharose fractions and were as described under ``Experimental Procedures.'' DNA products were isolated and analyzed on a 0.8% alkaline agarose gel. Lane M, DNA size markers with their apparent lengths (in nucleotides) shown on the left. Reactions for lanes 1-4 contained no PCNA and fractions 80, 78, and 84 of PCNA, respectively. Incorporation for the four reactions was 6.6, 5.3, 7.5, and 7.7 pmol of dNMP, respectively.



Pol activity was strongly inhibited by the presence of 100 mM NaCl, and the various replication factors alone had very little effect on this salt inhibition, although this inhibition could be slightly overcome when all three replication factors were present (Table 2; Fig. 2B).

When the assays were repeated with single primed (dA)bulletoligo(dT), there was also no major effect on pol either by PCNA or by all three factors, although RF-A and RF-C individually stimulated pol activity slightly in either 1 or 15 mM MgCl(2) (see Table 3, Experiment I; Fig. 4A). When 60 mM NaCl was added, activity and processivity were both greatly reduced, and the individual factors had little effect upon pol . However, when RF-A, RF-C, and PCNA were all present, pol activity was enhanced almost 2-fold and a somewhat longer DNA product was observed (Table 3, Experiment II; Fig. 4B). Similar experiments were performed with 100 mM NaCl present (Table 3, Experiment III; Fig. 4C), and again none of the factors individually or together was able to overcome the severe inhibition of activity or product length by the salt.




Figure 4: Effect of replication factors on the processivity of pol with single primed poly(dA)bulletoligo(dT). Reactions were as described in Table 3, Experiments I, II, and III. DNA products were analyzed on 8% polyacrylamide, 7 M urea gels. The spot located on the top of the rightmost track of panel C is spurious radioactivity. Longer exposures of the gel in panel C showed no material in the gel, only enhanced spots at the tops of the tracks.



When the effects of the replication factors on pol activity with poly(dA)bulletoligo(dT) in the presence of 175 mM potassium glutamate were determined, none of the factors had any significant effect (Table 3, Experiment IV). In effect, the pol activity was inhibited by 90% when 175 mM potassium glutamate was present, but this salt inhibition was not overcome by either HSSB, A1, PCNA, or the three in combination, although PCNA and A1 showed weak enhancement. These results are in contrast to those reported by Lee et al.(8) in which under the same conditions the presence of the various replication factors (HSSB, A1, and PCNA) overcame the salt inhibition.

Comparison of Levels of Pol Activity and mRNA in Cycling, Noncycling, and UV-irradiated Cells

Using an 814-bp probe (nucleotides 1022-1835) cloned from a HeLa pol cDNA library, Northern blot analysis of total RNA detected a single, 7.7- kb transcript (Fig. 5). The transcript size was calculated by using either commercial markers or the 5.1- and 1.9-kb ribosomal RNAs stained with ethidium bromide as internal markers. The effect of serum stimulation 30 h after quiescence on the abundance of the transcript was measured (Fig. 5A), and the transcript amount increased 5-fold. As an internal control, the levels of actin transcript present in the same preparations were also measured and found not to differ significantly between the preparations. When the level of pol transcript was measured during a time course after removal of a serum block (Fig. 5B), the amount of pol transcript peaked 18 h after the serum addition, rising 16-fold, and then dropped off. Since the division time for HeLa cells is 24-26 h, this result suggests that maximal pol transcript occurs somewhat prior to cell division.


Figure 5: Northern blot analyses for the transcript of pol catalytic subunit in quiescent versus serum-stimulated cells. Panel A, 30 µg of total RNA was present in each lane. Preparations of RNA, blotting, and quantitation were as described under ``Experimental Procedures.'' Quiescent HeLa cells were mid-log phase cells that were resuspended in medium containing only 0.1% FCS and held for 96 h. Half of these serum-starved cells were then fed medium containing 10% FCS for 30 h to obtain proliferating cells. As a control, a 800-bp chicken actin cDNA fragment was used as a probe. The levels in all samples were determined by gel scanning. Panel B, procedures were as in panel A, except that cells were held in quiescence for 48 h and then fed medium containing 10% FCS. Total RNA was prepared at the times indicated for Northern analyses.



The level of transcript 4 h after UV irradiation in total RNA of HeLa cells or diploid normal fibroblasts was also studied (Fig. 6, A and B, respectively), and no effects from the UV radiation were seen. Similar results were obtained when purified mRNA was probed. Hence the UV radiation does not appear to alter expression of the gene.


Figure 6: Northern blot analyses for the transcript of pol catalytic subunit after UV irradiation of HeLa cells and diploid fibroblasts. Thirty µg of total RNA obtained 4 h after irradiation for 7 s at 2 J/m^2/s was used in each lane, and all cells were at mid-log phase during treatment. The preparation for total RNA, blotting procedures, and irradiation conditions are described under ``Experimental Procedures.'' As a control, a 800-bp chicken actin cDNA fragment was used as a probe. The levels in all samples were determined by gel scanning. Panel A, HeLa cells; panel B, normal diploid human fibroblasts.



Pol activities were measured in parallel to mRNA levels. In the presence of the pol alpha antibodies, greater than 95% of the pol alpha activity was depleted, allowing pol activity to be measured specifically. Pol alpha activities were also monitored for comparison and did not change appreciably (Table 4). When DEAE fractions (9) from quiescent and proliferating cells (18 h after quiescence) were compared, pol activities (as normalized by the total cell number) increased by roughly 40%, and pol alpha activities increased by 62% (Table 4) as calculated from incorporation with their preferred templates. Likewise, when the levels of catalytic subunit in DEAE fractions from quiescent and proliferating cells were compared by immunoblotting with pol monoclonal antibody, they contained a similar level of antigen per unit of activity (Fig. 7). Similar constancy was observed when assays were done with unfractionated crude extracts (not shown). Hence in contrast to the approximately 6-16-fold increase of mRNA, the amount of the catalytic subunit of pol as determined by activity or by antigenicity changed little. This result recalls similar results with pol beta wherein mRNA levels changed without corresponding changes in activity(23) .




Figure 7: Immunoblotting of pol catalytic subunit from DEAE-Sephacel fractions from quiescent and proliferating HeLa cells. Proteins in each lane were the pooled fractions from DEAE chromatography from extracts of either quiescent or proliferating cells. Various amounts of the above proteins were resolved by electrophoresis on a 7% SDS-polyacrylamide gel and probed with mouse monoclonal IgG 3C5.1 at 1 µg/ml as described under ``Experimental Procedures.'' The secondary antibody was horseradish peroxidase-conjugated sheep anti-mouse IgG at a 1:5,000 dilution. Lanes 1-6 were from quiescent cells. Lanes 7-12 were from proliferating cells. Polymerase activities were: lanes 1 and 7, 0.5 unit; lanes 2 and 8, 0.33 unit; lanes 3 and 9, 0.17 unit; lanes 4 and 10, 0.07 unit; lanes 5 and 11, 0.033 unit; lanes 6 and 12, 0.017 unit. Lane 13 contained prestained protein standards with their apparent molecular masses shown on the right in kDa.



When pol activities from 30-50% ammonium sulfate fractions of UV irradiation were compared, it was seen that the UV irradiation also produced no significant change in the activity of pol or alpha with HeLa or normal fibroblast cells (Table 4).

Antibodies to Pol

Polyclonal antibodies were raised against a 46-kDa fusion protein, of staphylococcal protein A, and amino acids 1611-2045 of the catalytic subunit of pol (SpA-pol ) which includes the region II conserved domains of the alpha-like polymerases (see ``Experimental Procedures''). A second fusion protein, GST-pol , contained the same pol fragment fused to glutathione S-transferase from Schistosoma japonicum(24) and was used as a comparison antigen. Antisera from four immunized mice bound both fusion proteins as well as purified HeLa pol in ELISAs. Immunoblots showed that the pol large (>200-kDa) subunit from various fractions during the purification was recognized but not the pol alpha catalytic (180-kDa) subunit, HeLa pol beta, nor HeLa pol (Fig. 8). However, IgG from this serum neither neutralized nor immunodepleted pol activity. Since this antibody was raised against a small portion of the pol catalytic polypeptide, it was possible that this antibody could not recognize the epitope of the catalytic site. Therefore, intact, purified pol was used to make monoclonal antibodies. Antisera from two mice specifically recognized HeLa pol by ELISA and immunoblot assays. Neutralization assays (Fig. 9) showed that this serum neutralized HeLa pol activity by >95%.


Figure 8: Immunoblotting of various fractions of DNA pols alpha, beta, , or with polyclonal antibody against the SpA-pol fusion protein. The antibody was a purified IgG (987.1) at a concentration of 0.3 µg/ml from ascitic fluid of a mouse that was immunized with a protein A-pol fusion protein (SpA-pol ) as described under ``Experimental Procedures.'' Purified HeLa pols were resolved by electrophoresis on a 7% SDS-polyacrylamide gel then probed with the antibody. Lanes from left to right contained 0.12 unit of the Mono S fraction of pol alpha, 0.15 unit of the phosphocellulose fraction of pol beta, 0.11 unit of the glycerol gradient fraction of pol , 0.03 unit of the hydroxylapatite fraction of pol , and 0.15 unit of the Mono S fraction of pol . The positions of protein markers are indicated on the left in kDa. The catalytic subunits of pols alpha, beta, and migrate with M(r) values of 180,000, 40,000, and 125,000, respectively. The hydroxylapatite fraction of pol did not appear because of the low level of enzyme used. It does show when the same amount of the same fraction is probed with a monoclonal antibody as in Fig. 10B.




Figure 9: Neutralization of pol with polyclonal antibody against native HeLa DNA pol . Assays were as described under ``Experimental Procedures.'' Hydroxylapatite fraction (0.05 unit) of pol was used for each reaction. The relative activity of 1.0 was equal to 26.7 pmol of dTMP incorporated.




Figure 10: Immunoblotting with a purified IgG directed against the pol catalytic subunit. The antibody, obtained after immunization of a mouse with HeLa pol , was a monoclonal purified IgG (3C5.1) (see ``Experimental Procedures''). It was used at 1 µg/ml. Panel A, 3-µl samples of fractions from DEAE-Sephacel chromatography were resolved on a 7% SDS-polyacrylamide gel and probed with antibody. The numbers at the top represent the fraction numbers. Panel B, purified HeLa pols alpha, beta, , and were resolved by electrophoresis on a 7% SDS-polyacrylamide gel. Lanes 1 and 7, 0.12 unit of a Mono S fraction of pol alpha; lanes 2 and 8, 0.15 unit of a phosphocellulose fraction of pol beta; lanes 3 and 9, 0.11 unit of a glycerol gradient fraction of pol ; lane 4, 0.03 unit of hydroxylapatite fraction of pol ; lane 5, 0.15 unit of a Mono S fraction of pol ; lane 10, 0.15 unit of a hydroxylapatite fraction of pol ; lane 6 contained rainbow marker protein standards whose apparent molecular masses and positions are shown on the left in kDa. The catalytic subunits of alpha, beta, and migrate with M(r) values of 180,000, 40,000, and 125,000, respectively.



Monoclonal lines of hybridoma cells were then prepared by fusion of mouse spleen cells with myeloma cells, and we have obtained several monoclonal antibodies that recognize the catalytic subunit of pol . Using purified IgG from one of these (3C5.1), fractions across the DEAE-Sephacel gradient were probed, and the intensity of the 220-kDa subunit on an immunoblot correlated with the pol activity (Fig. 10A). Moreover, the antibody did not recognize pol alpha, beta, or (Fig. 10B).


DISCUSSION

Pol from HeLa cells was initially identified in our laboratory as a DNA repair factor for permeabilized human fibroblasts(3) . Unlike pol , pol alpha was not easily resolved from pol , but the utilization of Mono S FPLC chromatography has now allowed complete separation of the two pols, albeit still with a somewhat low yield of pol activity. Homogeneous HeLa pol appears by protein staining with silver to consist of two subunits: >200 kDa and 55 kDa, unlike yeast pol , which has a large (>200-kDa) catalytic subunit and three smaller subunits(25) . At present, it is not known whether homologs of the small yeast subunits exist in human cells and interact with human pol . Pols alpha, , and are each considerably less sensitive to inhibition by potassium glutamate than by NaCl, KCl, ammonium sulfate, and K(2)SO(4). Richy et al.(26) have demonstrated that potassium and glutamate are major intracellular ionic osmolytes, and Griep and McHenry found that E. coli DNA polymerase III holoenzyme could tolerate a much higher concentration of glutamate than chloride(27) . Moreover, specific protein-DNA interactions also tolerate much higher glutamate than chloride concentrations(28, 29) . Therefore, potassium glutamate should probably be more commonly utilized as an in vitro buffer and salt.

Neither the activity nor the processivity of our preparations of HeLa pol is greatly affected by HSSB (RF-A), A1 (RF-C), or PCNA, individually or in combination. There is a major pause site for pol within the M13mp18 DNA template which results in a band at position 120 bp and maps to the vicinity of the polylinker (see Fig. 2). The addition of replication factors did not affect this pausing.

Strong inhibition by salt of pol was observed, but this inhibition could not be completely overcome by any of the above replication factors, although when all three replication factors were present, both pol activity and processivity were enhanced roughly 2-fold. These results contrast to those of Lee et al.(8) in which the three replication factors (HSSB, A1, and PCNA) together could overcome the salt inhibition of pol under identical conditions used here. Different forms of pol have been reported in various studies: a large form of pol has a polypeptide that is greater than 200 kDa; but smaller forms of catalytic subunits between 125 and 170 kDa have been observed(1, 22, 30, 31, 32) , which appear to be related by proteolytic cleavage(22) . Our preparation seemed to contain both forms, so the origin of the differing results of this study and those of Lee et al.(8) cannot be attributable to proteolysis. Whatever the differences are due to, they are worthy of further study, however, since PCNA has been observed to affect DNA repair synthesis (33) , which could be attributable to pol .

Only a 7.7-kb HeLa pol transcript has been identified, so there is no reason to believe that shorter forms of the active subunit are controlled at the transcriptional level. Although the levels of pol activity and the catalytic subunit antigenicity increased only slightly after serum stimulation of quiescent cells, the pol transcript level increased up to 16-fold. After UV irradiation, however, neither the mRNA nor activity levels increased. In yeast, mRNA levels of DNA ligase and DNA pol beta increased after DNA damage, yet their enzymatic activities did not change(23) . In none of these cases is it clear what the significance is of the mRNA increase without a concomitant protein increase. It does not appear, however, that pol synthesis is increased, but the protein produced is less active because the antibody studies showed roughly equal antigen:activity ratios.

A comparison of pols alpha, , and for their primer-template preferences shows that all three pols can efficiently use ribonucleotide-primed templates, so no clue to the functions of pols and in replication were found in this regard. It is curious that pols and can utilize RNA primers, since one expects pol alpha to add DNA to its primase products. One wonders, therefore, why pol alpha contains DNA polymerase or, conversely, why pol alpha products appear to be extended by pols or .


FOOTNOTES

*
This research was supported in part by Grants GM30415 and P30ES011896 from the National Institutes of Health. 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.

§
Supported in part by Training Grant T32 ES07075 from the National Institutes of Health. Present address: Dept. of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, CA 94305-5307.

To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-7583; Fax: 510-643-5035.

(^1)
The abbreviations used are: pol, polymerase; PCNA, proliferating cell nuclear antigen; SSB, single-stranded DNA-binding protein; HSSB, human single-stranded DNA-binding protein; FPLC, fast protein liquid chromatography; RF, replicating form; RF-A, replication protein A; RF-C, replication protein C; A1, activator 1 protein; GST, glutathione S-transferase; SpA, S. aureus protein A; bp, base pair(s); kb, kilobase(s); kDa, kilodaltons; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; FCS, fetal calf serum; DTT, dithiothreitol.

(^2)
RF-A and RF-C refer to factors supplied by Bruce Stillman's laboratory. Human SSB and Activator-1 refer to the corresponding factors, respectively, obtained from the laboratory of Jerard Hurwitz.


ACKNOWLEDGEMENTS

We thank Ann Fischer for expert tissue culture work and Dr. Alex Karu and Karen Marcus for the expert assistance and advice in making polyclonal and monoclonal antibodies.


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