Further Characterization of HeLa DNA Polymerase ∊
1995; Elsevier BV; Volume: 270; Issue: 14 Linguagem: Inglês
10.1074/jbc.270.14.7799
ISSN1083-351X
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoDNA polymerase ∊ (pol ∊) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol α. 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 α and δ) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. 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 α, β, or δ. 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 ∊. DNA polymerase ∊ (pol ∊) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol α. 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 α and δ) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. 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 α, β, or δ. 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 ∊. INTRODUCTIONDNA polymerase ∊ (pol ∊) 1The abbreviations used are: polpolymerasePCNAproliferating cell nuclear antigenSSBsingle-stranded DNA-binding proteinHSSBhuman single-stranded DNA-binding proteinFPLCfast protein liquid chromatographyRFreplicating formRF-Areplication protein ARF-Creplication protein CA1activator 1 proteinGSTglutathione S-transferaseSpAS. aureus protein Abpbase pair(s)kbkilobase(s)kDakilodaltonsELISAenzyme-linked immunosorbent assayBSAbovine serum albuminFCSfetal calf serumDTTdithiothreitol. from HeLa cells, unlike yeast pol ∊(1Morrison A. Araki H. Clark A.B. Hamatake R.K. Sugino A. Cell. 1990; 62: 1143-1151Abstract Full Text PDF PubMed Scopus (276) Google Scholar), contains two apparent subunits of >200 and 55 kDa (2Syvaöja J. Linn S. J. Biol. Chem. 1989; 264: 2489-2497Abstract Full Text PDF PubMed Google Scholar) and appears to be required for long patch DNA repair in permeabilized cells(3Nishida C. Reinhard P. Linn S. J. Biol. Chem. 1988; 263: 501-510Abstract Full Text PDF PubMed Google Scholar). Unlike pol δ, 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)) 2RF-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. stimulate pol α and pol δ activities(4Tsurimoto T. Stillman B. EMBO J. 1989; 8: 3883-3889Crossref PubMed Scopus (187) Google Scholar, 5Kenny M.K. Lee S.H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9757-9761Crossref PubMed Scopus (164) Google Scholar). These replication factors are required for sequential initiation of lagging and leading strand DNA synthesis in reconstituted SV40 replication systems(6Tsurimoto T. Fairman M.P. Stillman B. Mol. Cell. Biol. 1989; 9: 3839-3849Crossref PubMed Scopus (61) Google Scholar, 7Tsurimoto T. Melendy T. Stillman B. Nature. 1990; 346: 534-539Crossref PubMed Scopus (318) Google Scholar). It has also been suggested that inhibition of HeLa pol ∊ by NaCl can be overcome by the presence of HSSB, A1, and PCNA(8Lee S.-H. Pan Z.Q. Kwong A.D. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1991; 266: 22707-22717Abstract Full Text PDF PubMed Google Scholar). 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 α prefers activated DNA as template, and pol δ prefers alternating poly(dA-dT) as template, whereas pol ∊ prefers long stretches of single-stranded regions in poly(dA)•oligo(dT) as template(9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar). 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 (1Morrison A. Araki H. Clark A.B. Hamatake R.K. Sugino A. Cell. 1990; 62: 1143-1151Abstract Full Text PDF PubMed Scopus (276) Google Scholar), 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 α, β, and δ. 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 PROCEDURESMaterials Nucleotides and Nucleic AcidsUnlabeled deoxy- and ribonucleotides were purchased from Pharmacia Biotech Inc.; [α-32P]dTTP and [α-32P]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(10Kaguni L.S. Rossignol J.-M. Conaway R.C. Banks G.R. Lehman I.R. J. Biol. Chem. 1983; 258: 9037-9039Abstract Full Text PDF PubMed Google Scholar, 11Campbell J. Annu. Rev. Biochem. 1986; 5: 733-773Crossref Google Scholar, 12Conaway R.C. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4585-4588Crossref PubMed Scopus (44) Google Scholar). 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 PlasmidsDNA pols α, δ, and ∊ and PCNA were purified from HeLa cells as described(9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar). Pol ∊ was purified further by Mono S FPLC column chromatography to remove residual pol α. HeLa pol β 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-β-D-galactopyranoside, and restriction enzymes were from Boehringer Mannheim.AntibodiesIgG-neutralizing monoclonal antibody against human DNA polymerase α (anti-pol α) 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 (13Nilsson B. Abrahmsen L. Uhlen M. EMBO J. 1985; 4: 1075-1080Crossref PubMed Scopus (262) Google Scholar) and maintained in E. coli RSB571 (derived from E. coli 697). This vector has both the lacq and the temperature-inducible λC1857 promoters. The glutathione S-transferase (GST) fusion vector, pGEX-1λT, was purchased from Pharmacia with the correct reading frame for the pol ∊ fragment and maintained in E. coli DH5α. 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) (14Kesti T. Frantti H. Syvaöja J.E. J. Biol. Chem. 1993; 268: 10238-10245Abstract Full Text PDF PubMed Google Scholar) contains the conserved region II of the α-class DNA polymerases(15Wong S.W. Wahl A.F. Yuan P.-M. Arai N. Pearson B.E. Arai K. Korn D. Hunkapiller M.W. Wang T.S. EMBO J. 1988; 7: 37-47Crossref PubMed Scopus (305) Google Scholar, 16Wang T. Annu. Rev. Biochem. 1991; 60: 513-552Crossref PubMed Scopus (451) Google Scholar).Monoclonal Antibody ProductionApproximately 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 (10−4M hypoxanthine, 10−6M aminopterin, 1.2 × 10−4M 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 × 10−4M hypoxanthine, 6 × 10−6M 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 TranscriptsThe probe for Northern analysis contained the conserved regions IV and II corresponding to nucleotides 1022-1835 of the cDNA for human pol ∊ (14Kesti T. Frantti H. Syvaöja J.E. J. Biol. Chem. 1993; 268: 10238-10245Abstract Full Text PDF PubMed Google Scholar). 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 α by Mono S ChromatographyHeLa DNA pol ∊ was purified as described (9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar) except that the final glycerol gradient separation was replaced by Mono S FPLC chromatography, which resulted in a complete separation of pol α from pol ∊. The pol ∊, hydroxylapatite fraction (9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar) 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 α activity eluted in 250 mM NaCl, and only the pol α 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 ExperimentA sequence-specific oligonucleotide was used to form a hairpin template as described by Insdorf and Bogenhagen(17Insdorf N.F. Bogenhagen D.F. J. Biol. Chem. 1989; 264: 21491-21497Abstract Full Text PDF PubMed Google Scholar). 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 [α-32P]dATP (3,000 Ci/mmol), 50 mM Hepes/KOH (pH 7.5), 5 mM DTT, 7.5 mM MgCl2, 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 AssaysStandard reaction mixtures (25 μl) for measuring pol ∊ activity contained 50 mM Hepes/KOH (pH 7.5), 15 mM MgCl2 (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 μM3H- or 32P-labeled dTTP, and 40 μM (dA)3000 and 4 μM oligo(dT)12−18 to give an interprimer distance of about 135 nucleotides. Pol α 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 MgCl2; 0.5 mM DTT; 0.2 mg/ml BSA; 50 μM dATP, dGTP, dCTP; and [3H]dTTP (203 cpm/pmol). Pol δ 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 MgCl2, 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(18Baker T. Kornberg A. DNA Replication. W. H. Freeman and Co., San Francisco1992Google Scholar).Processivity assays used either single primed (dA)3000•oligo(dT)12−18 or single primed M13mp18 DNA. Reactions were as above except that [α-32P]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(19Conaway R.C. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2523-2527Crossref PubMed Scopus (115) Google Scholar). 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 MgCl2, 1 mM ATP, 80 μM (dT)2500−5000, 20 μM [3H]dATP (200 cpm/pmol), and 0.5 unit of Klenow fragment. Incubations were for 30 min at 30°C.DNA Gel AnalysesTwo kinds of gels were used to analyze DNA products. Processivity studies utilized 8% polyacrylamide, 7 M urea slab gels prepared as described (20Cotterill S. Chui G. Lehman I.R. J. Biol. Chem. 1987; 262: 16100-16104Abstract Full Text PDF PubMed Google Scholar) 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 α, δ, and ∊ Preparations with Neutralizing Pol α AntibodyAnti-pol α antibody (SJK 132-20) was preincubated with HeLa DNA pol α 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 MgCl2. 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 MgCl2, 10 mM DTT, 40 μM (dA)3000, and 4 μM oligo(dT)12−18. Likewise, the preincubation buffer for pol δ 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 MgCl2, 10% (v/v) glycerol, 0.2 mg/ml BSA, 1 mM DTT, 40 μM (dA)3000, and 4 μM oligo(dT)12−18, 40 μM [3H]dTTP, and 1-2 ng/ml PCNA where indicated.Northern Blot AnalysisFragments 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 × 108 cpm/μg. Total RNA was prepared from 5-10 × 108 HeLa cells or 3-5 × 107 F65 normal fibroblasts by guanidium thiocyanate extraction followed by pelleting of the RNA through 5.7 M CsCl(21Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16620) Google Scholar). 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 × 108 mid-log phase HeLa cells were saved as the untreated control while 1-2 × 109 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/m2. 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 A260.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 × 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 32P-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 × SSC, 0.5% SDS for 1 h at room temperature and then twice in 0.1 × 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 × 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(9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar), 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 ProductionA 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 (14Kesti T. Frantti H. Syvaöja J.E. J. Biol. Chem. 1993; 268: 10238-10245Abstract Full Text PDF PubMed Google Scholar) 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-1λT, 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-β-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 A600 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 × 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.RESULTSCharacterization of the Pol ∊ PreparationIn our previous purification procedure of HeLa pol ∊(9Syvaöja J. Soumensaari S. Nishida C. Goldsmith J.S. Chui G.S.J. Jain S. Linn S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6664-6668Crossref PubMed Scopus (148) Google Scholar), pol α 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 α 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 α fractions. The purified pol α 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 α, confirmed the separation of HeLa pols α and ∊. One hundred to 200 ng of the IgG gave 50% inhibition for 0.06 unit of pol α, and additional antibody neutralized greater than 90% of the pol α 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%.To identify the catalytically active subunit(s) present in the preparation, the photolabeling procedure of Insdorf and Bogenhagen (17Insdorf N.F. Bogenhagen D.F. J. Biol. Chem. 1989; 264: 21491-21497Abstract Full Text PDF PubMed Google Scholar) was employed. This technique incorporates BrdUTP and [α-32P]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 (22Kesti T. Syvaöja J.E. J. Biol. Chem. 1991; 266: 6336-6341Abstract Full Text PDF PubMed Google Scholar) 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 OptimaTo 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 a
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