Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius : Purification, characterization and immunological properties

  • Published on
    30-Sep-2016

  • View
    212

  • Download
    0

Transcript

Eur. J. Biochern. 178,619-626 (1989) 0 FEBS 1YXY Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius Purification, characterization and immunological properties Christiane ELIE, Anne Marie DE RECONDO and Patrick FORTERRE Biologie Molkculaire de la RCplication, Groupe de Biologie et Gknktique Molkculaires, Equipe de Recherche 272 du Centre National de la Recherche Scientifique, Villejuif (Received May 18/August 23, 1988) - EJB 88 0571 We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S , the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2,3-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80C and is active between 55C and 85C in the presence of activated calf-thymus DNA. Cellular organisms require DNA polymerases to replicate and repair their genome. In spite of common mechanistic features, eukaryotic and eubacterial DNA polymerases differ in many properties such as their size, ability to interact with the accessory proteins and inhibitor sensitivity [l, 21. Members of a third cell lineage, archaebacteria, exhibit a mixture of eukaryotic and eubacterial features at the molecular level, as well as specific archaebacterial traits [3]. Consequently, comparative analysis of archaebacterial DNA polymerases should yield information about putative evolutionary re- lationships between DNA polymerases from the three king- doms, as was shown in the case of RNA polymerases [4]. Indeed, we have previously reported the inhibition of DNA replication in vivo in halophilic archaebacteria by aphidicolin [5], an inhibitor of eukaryotic DNA polymerases CI and 6 and of DNA polymerases from some animal viruses and bacterio- phages [6]. In vitro, aphidicolin also inhibits DNA polymerase activities from several archaebacteria [7 - 91. These results suggest that eukaryotic and some viral and archaebacterial replicases share common features. A meaningful comparison between DNA polymerases from different cell lineages would require the comparison of their amino acid sequences. A prerequisite to this goal is the purification of archaebacterial DNA polymerases and the cloning of the genes which encode these enzymes. In addition to information about DNA-polymerase evolution, studies of these enzymes from sulfur-dependent archaebacteria, the most thermophilic organisms known today, should lead to a better understanding of DNA synthesis at very high temperatures. Correspondence to P. Forterre, Institut de Microbiologie, Universite de Paris-Sud, Batiment 409, F-91405 Orsay Cedex, France Abbreviations. PhMeS02F, phenylmethylsulfonyl fluoride; Me2S0, dimethyl sulfoxide; ddTTP, 2,3-dideoxyribosylthymine triphosphate. Enzyme. DNA polymerase or deoxynucleoside-triphos- phate : DNA deoxynucleotidyl transferase (EC 2.7.7.7). We are interested in DNA replication of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium which grows optimally at 80C [lo]. In a preliminary communi- cation, we described the association between S. acidocaldarius DNA polymerase activity and a 40-kDa polypeptide [l l] ; the same result has been reported by Prangishvili [12]. Neverthe- less, in later work, we have found that the purified 40 kDa polypeptide was devoid of DNA polymerase activity and that this activity co-migrated with a 100-kDa polypeptide exclus- ively. We describe here the purification and characterization of the 100-kDa DNA polymerase of S. ucidoculdarius. Our procedure allows rapid purification of an enzyme which is stable and exhibits a high specific activity. Klimczak et al. have previously reported a 100-kDa DNA polymerase in the same Sulfolobus strain [13] but, in contrast to these authors, we did not detect 3 - 5-exonuclease activity associated with this DNA polymerase. In addition, we describe a method for obtaining immune serum against this enzyme, which specifi- cally inhibits its activity and recognizes the 100-kDa polypep- tide by immunoblotting. We also present studies on the thermophilicity and the thermostability of this DNA poly- merase. MATERIALS AND METHODS Chemicals PhMeS02F and N-ethylmaleimide were purchased from Sigma; leupeptin, pepstatin and unlabeled dNTPs from Boehringer ; [p3P]ATP from New England Nuclear; [3H]TTP and 251-labeled protein A from Amersham, polyacrylamide from Bio-Rad and Freunds complete and incomplete adjuvant from Difco. Aphidicolin was a gift from Dr A. H. Todd (Imperial Chemical industry, GB) and was prepared at 10 mg/ml in 100% MezSO; this stock solution was diluted in distilled water just before use. Phosphocellulose 620 P31 was purchased from Whatman, heparin-Sepharose CL- 6B and blue-Sepharose CL-6B were from Pharmacia. Enzymes Escherichia coli DNA polymerase I and Klenow fragment were purchased from Pharmacia, calf-intestinal alkaline phos- phatase and calf-thymus terminal deoxynucleotidyltrans- ferase from Boehringer, T4 polynucleotide kinase from New England Nuclear. Thermoplasrna acidophilum DNA poly- merase was purified in our laboratory using a procedure that will be published elsewhere. Nucleic acids Calf-thymus DNA (Sigma) was treated with pancreatic DNase until 10% was rendered acid-soluble [14]. Poly(rA) * (dT), was purchased from Boehringer, poly(dA) was from Pharmacia, M13 single-stranded DNA and SV40 [3H]DNA were kindly provided by S. Salhi and Dr 0. Jean- Jean from this laboratory, respectively. SV40 [3H]DNA (3 x lo4 cpm/pg) was linearized by PvuII in order to obtain blunt-ended double-stranded DNA. Poly(dA) ( z 500 residues) was 5- and 3-end-labeled accord- ing to the following procedures. For 5-end labeling, poly(dA) was first treated with calf-intestinal alkaline phosphatase, phenol/chloroform-extracted (twice), ethanol-precipitated and labeled with T4 polynucleotide kinase and [Y-~~PIATP (3000 Ci/mmol) ; the specific activity of 54abeled poly(dA) was 2 x lo7 cpm/pg. For 3-end labeling, poly(dA) was treated with terminal deoxynucleotidyl transferase and [3H]dTTP (30 Ci/mmol) and applied on a G-50 column in order to eliminate the free nucleotides; the specific activity of 34abeled poly(dA) was 8 x lo4 cpm/pg. Enzymatic assays All reaction mixtures contained 50 mM Tris/HCI, pH 7.5, 5 mM 2-mercaptoethanol, 4 mM MgCI2, 10 mM NH4C1 and 400 pg/ml bovine serum albumin in a final volume of 50 pl. DNA polymerase assay. Reaction mixtures contained 100pM of each of the four dNTPs, 0.66 pM [3H]dTTP (1 pCi), 5 pg activated calf-thymus DNA and 0.3 unit DNA polymerase. The mixtures were incubated at 70C for 30 min and the acid-insoluble material determined. One unit of DNA polymerase activity is defined as the amount of enzyme re- quired to convert 1 nmol dNMPs into acid-insoluble product, under these conditions. Primasr assay. Reaction mixtures contained 50 pM (each) GTP, CTP, UTP, 1 mM ATP, 100 pM of each of the four dNTPs, 0.66 pM [3H]dTTP (1 pCi), 1.26 pg M13 DNA and 0.8 - 12 units DNA polymerase. Incubations were performed for 30 min at 70C or 37C. When it was performed at 37C, 0.4 unit E. coli DNA polymerase I was added to the reaction mixture. Endonuclease assay. DNA polymerase (0.8 - 12 units) was incubated with 0.5 pg supercoiled pBR322 in 20 p1 reaction mixture (final volume). Incubations were performed for 30 min at 37C or 70C and the DNA was analyzed on 1 YO agarose gel. Exonuclease assay. For double-stranded-DNA exonucle- ase assay, 0.8 - 12 units DNA polymerase were incubated with 1.5 x lo4 cpm linearized SV40 [3H]DNA. For single-stranded- DNA exonuclease assay, 0.8 - 12 units DNA polymerase were incubated with either 7 x lo3 cpm 3-labeled poly(dA), for 3-5-exonuclease assay, or with 12 x lo5 cpm of 5-labeled poly(dA), for 5 - 3-exonuclease assay. All assays were performed in the presence or absence of the four dNTPs for 30 min at 37 C or 70 C. After trichloroacetic acid precipi- tation in the presence of 10 pg calf-thymus DNA as carrier, the acid-insoluble material was determined. Protein determination with bovine serum albumin as standard. Protein was determined by the method of Bradford [I51 Sodium dodecyl suljiite/polyacrylamide gel electrophoresis Denaturing gel electrophoresis was performed according to Laemmli [16]. Protein bands were revealed by silver stain- ing [17]. S. acidocaldarius cultures S . acidocaldarius DSM 639 was obtained from Dr W. Zillig (Max Plank Institut). The cells were grown at 75C in 300-1 fermenters with aeration of 1 vvm (1 air volume . 1- medium volume . min-) at a pressure of lo5 Pa (Laboratoire dExtraction et de Fermentation, CNRS, Gif-sur-Yvette). The medium used contains 1.3 g (NH4)2S04, 0.3 g KH2P04, 0.3 g MgS04, 0.1 g CaC12, 1 g yeast extract, 2 g sucrose and 1 g casaminoacids in 1 1. The pH was adjusted to 3 with H2S04. The cells were harvested during the exponential phase and frozen at -70C. Poly (ethyleneimine) preparation The poly(ethy1eneimine) (polymin P, Sigma) used for nucleic acid precipitation was first diluted in 50 mM Tris/ HC1, pH 7.5, 1.2 M NH4C1. The pH was adjusted to 7.5 with HCl and the solution was then dialyzed against 11 dilution buffer overnight at 4C. Purification of the DNA polymerase All the procedures were carried out at 4C. Crude extract. 150 g S . acidocaldarius DSM 639 cells were thawed in 600 ml50 mM Tris/HCl, pH 7.5,l mM dithiothrei- tol, 0.5 mM EGTA, 1.2 M NH4CI, 1 mM PhMeSOzF con- taining 1 pg/ml leupeptin and pepstatin. The cells were then disrupted in a homogeniser (Manton Gaulin) under pressure (400 kg/cm2) for 4 min. The nucleic acids were precipitated by addition of polymin P, under gentle agitation, to a final concentration of 0.37%. After centrifugation (15000 x g, 1 h), the supernatant was collected (fraction I). Ammonium sulfate precipitation. 330.4 g solid (NH&S04 were slowly added to fraction I, under gentle agitation, at 0C [70% saturation (NH4)2S04]. After 1 h of additional stirring and centrifugation (1 5 000 x g, 1 h), the pellet was resuspended in 120ml buffer A (50mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 0.5 mM EGTA). The suspension was then dialyzed for one day against 3 1 buffer A containing 40 mM NH4CI, with three changes of buffer (fraction 11). Phosphocellulose chromatography. Fraction I1 was loaded onto a phosphocellulose column (2.7 x 36 cm), equilibrated with buffer A containing 40 mM NH4Cl. After washing the column with 2.4 1 equilibrating buffer, the activity was eluted as a single peak, with buffer A containing 250 mM NH4CI. Active fractions were pooled as fraction 111. 62 1 Heparin-Sepharose chromatography. Fraction I11 was di- luted in buffer A to an ionic strength equivalent to that of 100 mM NH4C1 and loaded onto a heparin-Sepharose CL- 6B column (2.7 x 6 cm) equilibrated at the same ionic strength. The column was first washed with 20 vol buffer A containing 200 mM NH4Cl and eluted with 11 vol of a linear gradient of 0.2 - 1 M NH4Cl in buffer A. The activity eluted in the range 400 - 500 mM NH4Cl (fraction IV). Blue-Sepharose chromatography. Fraction IV was diluted in buffer B (buffer A containing 10 mM MgC12 and 0.02% Triton X-100) to an ionic strength equivalent to that of 40 mM NH4Cl and loaded onto a blue-Sepharose CL-6B column (1.6 x 7 cm) equilibrated with buffer B containing 40 mM NH4C1. The column was washed with 1Ovol equilibrating buffer and eluted with 12 vols of a linear gradient of 40- 700 mM NH4Cl in buffer B. Active fractions eluted in the range 100 - 200 mM NH4Cl and were pooled as fraction V. Sucrose-gradient centrifugation. Fraction V was concen- trated tenfold with a Centricon-30 and loaded onto a linear gradient of 5-20% sucrose in buffer B containing 300 mM NH4Cl. The gradients were centrifuged for 40 h at 38000 rpm in a SW41 Beckman rotor. Fractions of 0.5 ml were collected from the bottom of the gradients and the active fractions were pooled as fraction VI. Production of anti-(DNA polymerase) serum Fraction V (blue-Sepharose) was resolved on a 5% pre- parative SDS/polyacrylamide gel and polypeptide bands were revealed by KCI staining [18]. The 100-kDa polypeptide was removed and electroeluted overnight at room temperature into a membrane trap (Biotrap Schleicher & Schull) in SDS/ Tris/glycine buffer. 80 pg of the electroeluted 100-kDa poly- peptide (referred as DNA polymerase antigen) was emulsi- fied with complete Freunds adjuvant and injected into mul- tiple subcutaneous sites on the back of a rabbit. Six weeks later, new injections were performed with 60 pg DNA- polymerase antigen emulsified with incomplete Freunds adju- vant. Ten days later, blood was removed and serum aliquoted. Pre-immune serum was obtained just prior to immunization. Western blotting was performed essentially by the method of Burnette [19], except that nitrocellulose sheets (BA 83- 0.2 pm, Schleicher & Schull) were saturated with 5% non-fat milk instead of bovine serum albumin and washing buffers contained 0.1 %V Tween (Merck) instead of Nonidet P-40. RESULTS Purification ofthe S . acidocaldarius DNA polymerase S . acidocaldarius cells were lyzed in a high-ionic-strength buffer to prevent protease action and to decrease protein/ nucleic acid interactions. A thermophilic DNA-polymerase activity was detected in the crude extract and after precipi- tation of the nucleic acids with polymin P. To determine if this activity was due to one, or several, enzyme(s), a polymin P supernatant was fractionated with (NH4),S04 by successive backwashes of a first precipitate obtained with 80% (NH4)2S04 (decreases of 10% for each step). DNA poly- merase activity was assayed in each fraction and its sensitivity to salts, N-ethylmaleimide, ddTTP and aphidicolin was deter- mined. When using these characterizations, a unique type of DNA polymerase activity emerged in the range 70 - 35% (NH4)2S04 and this activity was resistant to aphidicolin. This DNA polymerase was purified by successive chromatographies on phosphocellulose, heparin-Sepharose and blue-Sepharose and by centrifugation through a sucrose gradient (Table 1). A single peak of activity was eluted at each chromatographic step and following fractionation of the sucrose gradient. To avoid a large loss of activity, due either to adsorption or instability of the enzyme at low protein concentration, it was necessary to silanize all the laboratory ware and to include 0.02% Triton X-100 in the buffers of the two last steps of the purification. In contrast to Triton X-100, glycerol or poly- (ethylene glycol) did not protect the DNA-polymerase ac- tivity. The recovery of DNA polymerase activity after sucrose gradients, performed with and without Triton X-100, were 95% and 5%, respectively. Electrophoretic analysis in SDS/polyacrylamide gels of the different fractions of the purification is shown in Fig. 1. The purified fraction contains a major polypeptide of 100 kDa which co-migrated with the DNA polymerase activity on su- crose gradient, together with two minor polypeptides of 33 kDa and 45 kDa (Fig. 2). The densitometric analysis of the gel revealed that the percentage of the lOO-kDa, 45-kDa and 33-kDa polypeptides were 83%, 6% and lo%, respectively. The DNA polymerase has been purified 5500-fold with a yield of 13% and a specific activity of 600000 units/mg protein was obtained. The purified DNA polymerase was stable for more than 12 months when the fraction was stored at 4C (0.05% sodium azide included) or at -70C. Native molecular mass and subunit composition The sedimentation of the DNA polymerase on sucrose gradients in high- or low-ionic-strength buffers (50 mM, 300 mM or 600 mM NH4Cl) gave a sedimentation-coefficient value of 6 S (Fig. 2). By gel filtration on HPLC TSK 3000 (LKB), we have determined the Stokes radius of the enzyme to be 4.2 nm (Fig. 3). Using the equation of Siege1 and Monty [20], we calculated the native molecular mass of the DNA polymerase to be 109 kDa. These results suggest that the 100-kDa polypeptide of purified fractions, detected by SDS/ PAGE, is the single subunit of the DNA polymerase. Antibodies raised against the DNA polymerase The 100-kDa polypeptide electroeluted from preparative polyacrylamide gels was injected into a rabbit (see Materials and Methods). This electroeluted polypeptide (100-kDa anti- gen) was free of contaminating polypeptides as shown by silver staining of the gel (Fig. 4A). To determine the specificity of the immune serum, immunoblot experiments were performed; enzymatically active fractions, V and VI (blue- Sepharose and sucrose gradient), and the 100-kDa antigen were resolved by SDS/PAGE and probed with pre-immune and immune serum at equal dilutions (Fig. 4B). Whereas there is no response with pre-immune serum (Fig. 4B, lanes, 4, 5 and 6), the immune serum contains antibodies that react with the 100-kDa antigen and the 100-kDa polypeptide of fractions V and VI (Fig. 4B, lanes 1, 2 and 3). The immune serum also recognizes specifically the 45-kDa and 33-kDa polypeptides in fractions V and VI, suggesting that these two polypeptides are degradative products of the 100-kDa polypeptide. Fig. 5 shows that the immune serum raised against the 100-kDa antigen specifically neutralized the activity of S. acidocaldarius DNA polymerase and had no effect on the activity of Thermoplasma acidophilum DNA polymerase, 622 Table 1. Purification scheme of the S. acidocaldarius DNA polymerase Fraction Volume Protein 1 O - j x activity Yield Specific activity Purification ml mg units % units/mg -fold Polymine P (I) 700 15400 1663.2 100 108 - Ammonium sulfate (11) 300 7 200 1612.8 97 224 2 Heparin-Sepharosc (IV) 92 50.6 599.5 36 11 848 110 Sucrose gradient (VI) 13 0.38 226.8 13 600 000 5555 Phosphocellulose (111) 200 340 1380.4 83 4060 38 Blue-Sepharose (V) 42 1.26 252.0 15 200 000 1852 Fig. 1 . SDS/polyacrylamide gel electrophoresis of the enzymatic frac- tions of the purification. The fractions (100 units of each stcp) were run on a 7.5% acrylamide slab gel as described under Materials and Methods. Lane 1 , phosphocellulose; lane 2, heparin-Sepharose; lane 3, blue-Sepharose; lane 4, sucrose gradient. Marker proteins indicated by their molecular masses in kDa were: phosphorylase b (98 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa) and a-chymo- trypsinogen (25 kDa) 50 - 40 k - 0 W 5 30 E 0 0 a a z a u *O 5 10 U 0 5 A c - 1 2 3 4 *\ , */; , , *.* I I I I 1 3 5 7 9 11 13 15 17 19 21 23 FRACTION NUMBER another thermoacidophilic archaebacterium. This confirms that DNA-polymerase activity is associated with the 100-kDa polypeptide. Reaction requirements The DNA-polymerase activity, in the presence of activated calf-thymus DNA as substrate, absolutely requires the four deoxyribonucleoside triphosphates and a divalent metal ca- tion; maximum activity was observed with 4 m M MgC12 whereas only 30% activity was obtained with MnC12, at its optimum concentration of 0.2 mM. In 50 mM Tris/HCl buffer, the optimum pH was 7.5 and 70% activity was obtained at pH 6.5 and pH 9. The activity is inhibited by monovalent salts; 50% inhibition was observed with 150 mM NH4Cl. In the absence of bovine serum albumin, the same rate of DNA synthesis was obtained when poly(ethy1ene gly- col) 6000 or 10000 was included in the reaction mixture; the effect of poly(ethy1ene glycol) occurs in a narrow range of polymer concentration, i.e. 17-27%, with a maximum at 20% poly(ethy1ene glycol) 6000. In addition, the inclusion of poly(ethy1ene glycol) in the reaction mixture allows the DNA polymerase to be active at higher ionic concentration; the rate of DNA synthesis in the presence of 20% poly(ethy1ene glycol) 6000 was the same in the range 0- 150 mM NH4Cl and 50% inhibition was observed with 400 mM NH4C1. Glycerol and sucrose inhibit the DNA-polymerase activity; 50% inhibition was obtained with 15% glycerol or 4% sucrose. Fig. 2. Sedimentation profile of the D N A polymerase on sucrose gradi- ent. An aliquot of blue-Sepharose fraction (2800 units) was centri- fuged on a 5 -20% linear sucrose gradient as described under Ma- terials and Methods. (A) Aliquots (4 pl, diluted 50-fold) of sucrose gradient fractions were assayed for DNA-polymerase activity under standard conditions. The arrows indicate the position of protein markers run in parallel gradients: 1 , yeast alcohol dehydrogenase (7.6 S); 2, E. coli DNA polymerase I(5.5 S); 3, bovine serum albumin (4.3 S); 4, ovalbumin (3.6 S) and 5 , cytochrome c (1.7 S). (B) Aliquots (45 pl) of sucrose gradient fractions were run on a 7.5% SDS/ polyacrylamide slab gel as described under Materials and Methods. The lane number indicates the fraction number. Marker proteins indicated by their molecular masses in kDa were: phosphorylase b (98 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa) and a-chymotrypsinogen (25 kDa) 623 40 - 0 30 - B 2 20 8 2 10 !i 4 K z 0 FRACTION NUMBER Fig. 3. Gelfiltration of the S. acidocaldarius DNA polymerase. Fraction V was run on a TSK 3000 gel filtration column in buffer containing 50 mM Tris/HCI pH 7.5, I mM dithiothreitol, 0.5 mM EGTA, 300 mM NH,CI and 0.02% Triton X-100; 0.2-ml fractions were collected and assayed for DNA polymerase activity. Standard proteins run with the same procedure were: 1, cytochrome c (1.7 nm); 2, chymotrypsinogen (2.25 nm); 3, bovine serum albumin (3.5 nm) and 4, aldolase (4.4 nm) Fig. 4. Specificity of the ant i - (DNA polymerase) serum. (A) SDS/ polyacrylamide gel electrophoresis of the electroeluted 100-kDa poly- peptide used for immunization of the rabbit (the position of phos- phorylase b, 98 kDa, is indicated by the arrow). (B) Polypeptides were separated by SDS/polyacrylamide gel electrophoresis, transferred to a nitrocellulose sheet which was then cut in two parts, incubated for 1 h with the rabbit pre-immune or immune serum at the same dilution (1/900) and then with '251-labeled protein A as described under Ma- terials and Methods. The nitrocellulose sheets were autoradiographed for 1 5 h at -70C. Lanes 1 and 4,60 units sucrose gradient fraction; lanes 2 and 5,60 units blue-Sepharose fraction; lanes 3 and 6,0 .4 pg of the electroeluted 100-kDa polypeptide. Lanes 1, 2 and 3 were incubated with the immune serum and lanes 4, 5 and 6 with the pre- immune serum. I4C-labeled markers indicated by their molecular masses in kDa were: phosphorylase b (98 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa) and lactoglobulin A (18 kDa) Inhibitors The activity is resistant to 20 pg/ml aphidicolin (we have verified that the aphidicolin solution used inhibited the DNA polymerase activity of calf-thymus DNA polymerase a). The DNA-polymerase activity is inhibited by ddTTP; 17% and 100 n t z k v 50 Y 0 -o-o----.o-o-o T: acidophilum Sacidocaldarius 1 2 3 4 5 6 7 8 SERUM (%) Fig. 5 . Neutralization of the S. acidocaldarius DNA-polymerase ac- tivity by the immune serum raised against the 100-kDa polypeptide. 0.3 unit S. acidocaldarius (fraction V) or T. acidophilum DNA poly- merases were preincubated with equal amounts of pre-immune or immune serum (expressed as a percentage of the final volume of the reaction mixture) for 1 h at 37C in incomplete reaction mixture. The four dNTPs, [3H]dTTP and the DNA substrate were then added and the samples were incubated for 30 min at 70C. The percentage activity is the ratio between the activity determined in the presence of the immune serum and the activity determined in the presence of the pre-immune serum at the same dilution. The 100% value represents the incorporation of 60 pmol dTMP for the two DNA polymerases 33% inhibition were observed with ddTTP/dTTP ratios of 1 : 1 and 5 : 1, respectively. Preincubation of the DNA poly- merase for 15 min at 70C with 2 mM and 5 mM N-ethyl- maleimide inhibited the activity by 35% and 66%, respective- 624 40 50 60 70 80 90 TEMPERATURE ('c) Fig. 6. Dependence of the S . acidocaldarius DNA polymerase activity on the incubation temperature. The D N A polymerase (0.2 unit, frac- tion VI) was assaycd in standard reaction mixture for 30 min at the indicated temperature ly, whereas no inhibition was observed when the preincu- bation was performed at 0C. Template primer specificity The DNA polymerase was not able to use a poly- ribonucleotide poly(rA) . (dT)12 as a template primer either at 37C or 70C. In addition, no primase activity has been detected when the enzyme was assayed on single-stranded circular DNA in the presence of the four deoxy- and ribonu- cleotides at 70C or at 37C. In this last case, since S. acidocal- darius DNA polymerase is not active at 37"C, 0.4 unit E. coli DNA polymerase I was added to the reaction mixture, in order to detect a putative primase activity at this temperature. Nuclease activities An endonuclease activity was detected up to the blue- Sepharose chromatography step but was completely removed following sucrose-gradient centrifugation. Neither 5' - 3', nor 3' - 5'-exonuclease activity has been detected on double- or single-stranded substrates either at 37 "C, 60 "C or 70 "C. In the same assays at 37C 0.2 unit E. coli Klenow fragment released 95% of the acid-insoluble radioactivity from 3'- labeled poly(dA) and 0.2 unit E. coli exonuclease I11 degraded 50% of the acid-insoluble radioactivity from linearized SV40 [3H]DNA. Thermophilicity und thermostability The nucleotide incorporation obtained with the DNA polymerase assayed on activated calf-thymus DNA for 30 rnin at different temperatures is shown in Fig. 6 ; the incorporation increased up to 70C and rapidly decreased at higher tempera- tures. The kinetics were different below and above 70C (Fig. 7); first-order kinetics were observed with increasing velocity up to 7 0 T , whereas the nucleotide incorporation stopped after the first 10 min of the reaction at higher tem- 40 - 0 E 0 3 0 2- G : 20 00 f 10 k a a z 70y0 o/ 0 10 20 30 TIME (min) Fig. 7. Kinetics of nucleotide incorporation at different temperatures. The D N A polymerasc (0.2 unit, fraction VI) was assayed in a standard reaction mixture at 55"C, 70"C, 80C and 85C for the indicated time peratures. The initial velocity (during the first 8 min) was identical at 70C and 80C. The arrest of the polymerization process could be due to thermal inactivation of the enzyme and/or to the effect of high temperature on the DNA sub- strate. To test these two hypotheses, we determined the effect of preincubation of either the DNA polymerase or the DNA substrate at 70C or 80C. After preincubation, the DNA polymerase activity was assayed under standard conditions (30 rnin at 70C). Preliminary experiments have shown that the thermostability of the enzyme was dependent upon protein concentration; thus, the preincubations were performed di- rectly in the reaction mixture which contained 400 pg/rnl bo- vine serum albumin, in the absence of the dNTPs. The result, illustrated in Fig. SA, shows that the DNA-polymerase ac- tivity was resistant to preincubation for 40 min, both at 70C and 80C (more than 80% of residual activity). In contrast, the activated calf-thymus DNA was still an efficient template after preincubation at 70C but not at 80C (Fig. 8B); only 50% and 20% residual activity was obtained with the DNA substrate preincubated at 80 "C for 2 min and 40 min, respec- tively. Our results indicate that the enzyme is stable in our in vitro conditions at the temperatures which are optimal for growth of S. acidocaldarius cells (70-80C). This is not the case above 80C; the DNA polymerase is heat-inactivated and 50% residual activity was obtained when the enzyme was preincubated for 15 rnin at 87 "C. DISCUSSION We have purified to near homogeneity a thermophilic DNA polymerase from the archaebacterium S. aciduculdarius. Several experimental results indicate that this DNA poly- merase is a monomer of 100 kDa: (a) SDS/polyacryIamide gel electrophoresis of the purified fraction shows a polypeptide of 100 kDa which co-purifies with the DNA polymerase activity throughout the purification procedure; (b) combination of gel-filtration and sucrose-gradient centrifugation data indi- cate that the enzyme has an apparent native molecular mass 625 b o 2 4 100 50 0 10 20 30 40 TIME OF PREINCUBATION (rnin) Fig. 8. Effect o f the oreincubation of the S . acidocaldarius DNA pojymera.& or the DNA substrate atd70"C and 80C on the DNA- polymerase activity. The DNA polymerase (0.2 unit, fraction VI) in the absence of the DNA substrate (A) or the DNA substrate in the absence of the DNA polymerase (B) were preincubated for different time at 70C or 80C in the reaction mixture which did not contain the four dNTPs and [3H]dTTP (45 pl, final volume). The preincu- bations were stopped by addition of the four dNTPs, [3H]dTTP and the DNA substrate or the DNA polymerase; the samples were then incubated for 30 min at 70C. The final reaction mixture (50 pl) was identical to that used in the standard polymerase assay of 109 kDa; (c) the immune serum raised against the 100-kDa polypeptide inhibits specifically the DNA-polymerase activity and recognizes a 100-kDa polypeptide in active fractions in immunoblot experiments. Two minor polypeptides of 45 kDa and 33 kDa are re- vealed by SDS/polyacrylamide gel electrophoresis of the most purified fraction in addition to that of 100 kDa. We have shown that these polypeptides were immunologically related to the 100-kDa polypeptide and so that they appear as degradative products of this polypeptide. Klimczack et al. [13] found that a single enzyme is respon- sible for the DNA polymerase activity detected in crude extracts of S. acidocaldarius. In contrast, Prangishvili [ 121 reported that this activity was also due to an abundant multimeric DNA polymerase composed of four identical 40- kDa subunits. During our first purification procedures, we noticed that an abundant 40-kDa polypeptide co-purified with the DNA-polymerase activity on all the chromatographic columns used : DEAE-Sephacel, phosphocellulose, heparin- Sepharose, single-stranded DNA ultrogel, hydroxyapatite and blue-Sepharose. The 100-kDa polypeptide, which is much less abundant, was not always detected and the DNA- polymerase activity could be ascribed to the 40-kDa polypep- tide. The inclusion of 0.02% Triton X-100 in the sucrose- gradient buffer allowed us to stabilize the DNA-polymerase activity and to separate the 100-kDa and 40-kDa polypep- tides. The 100-kDa polypeptide (as well as the 45-kDa and 33-kDa polypeptides already described) co-migrated with the DNA-polymerase activity (sedimentation coefficient of 6 S) whereas the 40-kDa polypeptide migrated as a tetrameric protein (sedimentation coefficient of 7.5 S) devoid of DNA polymerase activity (not shown). This 40-kDa polypeptide is not immunologically related to that of 100 kDa. When Triton X-100 is included during blue-Sepharose chromatography, which is the case in the purification scheme described in this article, the 40-kDa polypeptide is removed at this step. The abundance of the 40-kDa polypeptide compared to the 100- kDa protein, the difficulty encountered in separating it from the DNA-polymerase activity and its sedimentation coef- ficient suggest that this polypeptide could correspond to the subunit of the putative tetrameric DNA polymerase described by Prangishvili. In conclusion, our results suggest that this tetrameric protein is not a DNA polymerase and that a single enzyme (100 kDa) is responsible for the DNA polymerase activity detected in crude extracts of S. acidocaldarius. This data confirms the results of Klimczak et al. We have further characterized the purified enzyme and, in agreement with Klimczak et al., we found that S. acidocalda- rius DNA polymerase is resistant to aphidicolin and inhibited by ddTTP, N-ethylmaleimide and monovalent salts. In ad- dition, we showed that the DNA-polymerase activity is stimu- lated by inclusion or poly(ethy1ene glycol) in the reaction mixture and that poly(ethy1ene glycol) allows the enzyme to be active at higher ionic strength; the same phenomenon has been observed by Zimmerman and Harrison [21] with B. coli DNA polymerase I and T4 DNA polymerase. We also showed that sensitivity to N-ethylmaleimide is greatly increased by preincubation of the DNA polymerase at 70C. This indicates that S . acidocaldarius DNA polymerase should undergo a conformational change between 4C and 70 "C (the same phe- nomenon was observed with T. acidophilum DNA poly- merase; unpublished result). The DNA polymerase activity assayed on activated calf- thymus DNA is optimal at 70 "C. Nevertheless, we show here that the highly purified enzyme is stable up to 80 "C, which is the optimum growth temperature of S. acidocaldarius and that the decrease of activity observed between 70C and 80C is due to the temperature effect on the DNA substrate. This indicates that the DNA polymerase does not require specific stabilizing cofactors at 80C in vitro. In vivo, DNA protec- tion against thermal denaturation could be performed by S. acidocaldarius DNA-binding proteins such as those re- cently described by Reddy et al. [22]; these proteins can stabilize DNA up to 90C. A major discrepancy between our results and those of Klimczack et al. [13] is their report of a 3'-5'-exonuclease activity associated with the 100-kDa polypeptide. In our case, we have never detected such activity in purified enzyme frac- tions on single- or double-stranded DNA. Either an exo- nuclease activity is associated with the 100-kDa polypeptide and our purified enzyme had lost this activity, or this activity is associated with another polypeptide which co-purifies in low amounts with the 100-kDa protein during the purification procedure described by Klimczak et al. The 100-kDa DNA polymerase of S. ucidocaldarius did not exhibit typical eukaryotic or eubacterial features and its biological function is unknown. Its monomeric structure and molecular mass are similar to those of the major eubactcrial DNA polymerases (DNA polymerase I) and eukaryotic fl-like DNA polymerases. However, it should be remembered that a DNA replicase could be isolated as a monomeric enzyme (the catalytic subunit alone). The enzyme is resistant to aphidicolin; in contrast, Rossi et al. [9] reported that the main DNA-polymerase activity detected in S. solfataricus, another Sulfolobale, is aphidicolin-sensitive. The same situation exists among methanogenic archaebacteria since the DNA polymerase of Methanococcus vaniellii is sensitive to aphidicolin [7], whereas the enzyme from Methanobacterium thermoautotrophicum is resistant [23]. It is also possible that aphidicolin-resistant and aphidicolin-sensitive DNA poly- merases coexist in the same archaebacterium. For instance, Prangishvili [12] detected a weak DNA polymerase activity, which is aphidicolin-sensitive, in S. acidocaldarius. Neverthe- less, this is a preliminary result since no polypeptide has been ascribed to this activity. Furthermore, it should be re- membered that the growth of S. acidocaldarius is not sensitive to aphidicolin [5]. All these results emphasize the importance of isolating the gene coding for S. acidocaldarius DNA polymerase in order to compare its sequence with those of other DNA poly- merases ; we have undertaken this task using the polyclonal antibodies described in this paper. The sensitivity of the enzyme to ddNTPs allows use of S . acidocaldarius DNA polymerase for DNA sequencing at 70 "C, by the method of Sanger (S. Salhi, personal communi- cation). This could avoid some problems arising from hairpin formation at 37 "C when mesophilic DNA polymerases are used. Another recent interest in thermophilic DNA poly- merases is that these enzymes improve the performance of amplification tests using the polymerase chain reaction method [24]. The capacity of S. acidocaldarius DNA poly- merase to perform this reaction is currently being tested in our laboratory. We thank A. Escaut and D. Mane for S. acidocaldarius cultures, S . Salhi, J. M. Rossignol, M. Nadal and M. Duguet for helpful discussions, and R. H. Elder for his critical reading of the manuscript. This work was supported by funds from the Association de la Re- cherche contre le Cancer and C.E. was supported by a fellowship from the Ligue Nationale Francaise contre le Cancer. REFERENCES 1. Kornberg, A. (1980) DNA replication W. H. Freeman, San Francisco. 2. Kornberg, A. (1988) J . Biol. Chem. 263, 1-4. 3. Woese, C. R. (1987) Microbiol. Rev. 51,221 -271. 4. Huet, J . , Schnabel, R., Sentenac, A. & Zillig, W. (1983) EMBO 5. Forterre, P., Elie, C. & Kohiyama, M. (1984) J . Bacteriol. 159, 6. Bernad, A., Zaballos, A., Salas, M. & Blanco, L. (1987) EMBO 7. Zabel, H. P., Holler, E. &Winter, J. (1987) Eur. J . Biochem. 165, 8. Nakayama, M. & Kohiyama, M. (1985) Eur. J . Biochem. 152, 9. Rossi, M., Rella, R., Pensa, M., Bartolucci, S . , De Rosa, M., Gambacorta, A., Raia, C. A. & Dell'Aversano Orabona, N. (1 986) Sysi. Appl. Microbiol. 7, 337 - 341. 10. Brock, T. D., Brock, K. M., Belly, R. T. & Weiss, R. L. (1972) Arch. Mikrobiol. 84, 54-68. 11. Forterre, P., Nadal, M., Elie, C., Mirambeau, G., Jaxel, C. & Duguet, M. (1986) Syst. Appl. Microbiol. 7, 67-71. 12. Prangishvili, D. A. (1986) Mol. Biol. 20, 380-389. 13. Klimczak, L. J., Grummt, F. &Burger, K. J. (1985) Nucleic Acids 14. Aposhian, H. V. & Kornberg, A. (1962) J . Biol. Chem. 237,519- 15. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 16. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 17. Switzer, R. C., Merril, C. R. & Schifrin, S. (1979) Anal. Biochem. 18. Hager, D. A. & Burgess, R. R. (1980) Anal. Biochem. 109, 19. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. 20. Siegel, L. M. & Monty, K. J . (1966) Biochim. Biophys. Acta 112, 21. Zimmerman, S. B. & Harrison, B. (1987) Proc. Natl Acad. Sci. 22. Reddy, T . R. & Suryamarayana, T. (1988) Biochim. Biophys. Acta 23. Klimczak, L. J., Grummt, F. & Burger, K. J. (1986) Biochemistry 24. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Erllich, H. A. (1988) Science (Wash. D C ) 239,487-491. J . 2, 1291 - 1294. 800 - 802. J. 6,4219-4225. 171-175. 293 - 297. Res. 13, 5269 - 5282. 525. 98,231 -237. 76 - 86. 346 - 362. USA 84,1871 -1875. 949,87 - 96. 25,4850-4855.

Recommended

View more >