Purification and characterization of PRD1 DNA polymerase

ELSEVIER

Biochimica et Biophysica Acta 1219 (1994) 267-276

etBiochi~ic~a BiophysicaA~ta

Purification and characterization of PRD1 D N A polymerase Weiguo Zhu, Junetsu Ito

*

Department of Microbiology and Immunology, College of Medicine, The University of Arizona, Tucson, AZ 85724, USA Received 13 October 1993; revised manuscript received 23 February 1994

Abstract

A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of the PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in Escherichia coli cells. We have developed a simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing the recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for the PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme. Keywords: Purification; DNA polymerase; DNA replication; Bacteriophage; Guanidine hydrochloride denaturation; (E. coli)

1. Introduction

Bacteriophage PRD1 is a lipid-containing phage infecting Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium which harbor plasmids of the P, N, or W incompatibility types [1,2]. The genome of PRD1 is a linear double stranded D N A of 14 925 bp [3]. A 28 kDa terminal protein is covalently linked to the 5' ends of the genome. The linkage between the terminal protein and genomic D N A is a phosphodiester bond between the tyrosine residue at position 190 and the terminal deoxynucleotide d G M P of the genome [4]. The PRD1 D N A contains perfect inverted terminal repeats (ITRs) of 110 to 111 bp [5,6]. The phage PRD1 genome is replicated by a protein-primed mechanism similar to those of adenovirus [7,8] and ~b29 [9,10]. Thus, PRD1 D N A replication occurs via a protein-primed strand displacement mechanism where D N A synthesis begins at either end of the terminal redundant D N A molecule with the covalent linkage of d G M P to the tyrosine-190 of the terminal protein. The subsequent D N A chain elonga-

* Corresponding author. Fax: + 1 (602) 6262100. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 0 7 3 - C

tion displaces one parental D N A strand. The terminal 20 bp of the ITRs are the minimal origin of replication and are required for in vitro replication [11]. Sitespecific mutagenesis and in vitro DNA replication analyses show that the sequence of the origin of replication is highly specific. This implies that the interactions between the replication origin and the D N A polymerase-terminal protein complex are highly specific. It has also been demonstrated that the minimum requirement for phage encoded proteins needed to synthesize genome length replication products in vitro are the D N A polymerase and the terminal protein [12,13]. The PRD1 D N A polymerase (EC 2.7.7.7) is a multifunctional enzyme. It has at least three enzymatic activities: (1) it catalyzes the formation of terminal protein-dGMP complex; (2) a D N A chain elongation activity; and (3) a 3' to 5' exonuclease activity. This protein primed D N A polymerase consists of 553 amino acid residues with a calculated molecular mass of 63.3 kDa [14,15]. Thus, it is the smallest known D N A polymerase which contains 3' to 5' exonuclease. Based on amino acid sequence comparison, the PRD1 D N A polymerase has been classified as being a family B D N A polymerase which shares the sequence

268

W. Zhu, J. lto /Biochimica et Biophysica Acta 1219 (1994) 267-270

homology with major eukaryotic replicative DNA polymerases: DNA polymerase a, 3 and e [14,16-20] as well as archaebacterial DNA polymerase [21]. Therefore, the PRD1 DNA polymerase can serve as a model system with which to study the structure-function relationships of DNA polymerase molecules, especially family B DNA polymerases. In order to understand the molecular basis of the protein-primed DNA replication and to study the structure-function relationships of the PRD1 DNA polymerase, we have cloned the PRD1 DNA polymerase gene into a phagemid expression vector pEMBLex3 [22]. Attempts to purify large amount of the PRD1 DNA polymerase from this clone were unsuccessful because most of the overproduced recombinant PRD1 DNA polymerase aggregated into an insoluble form. Similar results have been described by Savilahti et al. [13]. To develop a simple and rapid purification procedure for large amount of the PRD1 DNA polymerase, we applied a guanidine hydrochloride denaturation and renaturation procedure permitting the purification of the enzyme to near homogeneity. In this paper, we first describe a procedure for purification of the overexpressed PRD1 DNA polymerase and a detailed comparative analyses of the enzymatic properties of both soluble and insoluble forms. Further characterizations of the PRD1 DNA polymerase are reported. The PRD1 DNA polymerase is a highly versatile enzyme.

2. Materials and methods

2.1. Bacteria, phage and plasmids Escherichia coli JL 2443 (A(lac-pro AB), thi, hsd D5, Sup E, F'(tra D36, pro AB, lac I q lac Z AM15)), obtained from Dr. J.W. Little of the University of Arizona, was used as the host strain for overproduction of the PRD1 DNA polymerase. Plasmid pEJG containing the PRD1 DNA polymerase gene was derived from phagemid pEMBLex3, obtained from Dr. G. Cesaren of the European Molecular Biology laboratory [23], which carries a strong lambda phage pR promoter and a temperature sensitive repressor ci857 gene [22]. Bacteriophage PRD1 was kindly provided by both R. Olsen of the University of Michigan and L. Mindich of the Public Health Research Institute of the City of New York. Bacteriophage PRD1 was grown in Salmonella typhimurium strain LT2 carrying the drug resistance plasmid pLM2. Phage particles were prepared by 12% P E G / 0 . 4 M NaCI precipitation and 5-20% sucrose gradient centrifugation. The PRD1 DNA-terminal protein complex was prepared according to the method of Sharp et al. [24], which involves sucrose gradient centrifugation in GuHC1. The PRD1 DNA was prepared

from purified phage particles as described [25]. The purified PRD1 terminal protein was provided by Dr. Shih-Yi S. Chang.

2.2. Nucleotides, column supports and other chemicals Deoxyribonucleoside 5'-triphosphates, poly(dC)olig°(dG)12 15 and Sephacryl S-200 were purchased from Pharmacia LKB. DEAE-cellulose DE52 was from Whatman. Hydroxylapatite was from Bio-Rad. Heparin-agarose, aphidicolin and N'-ethylmaleimide were from Sigma. NEN DuPont was the supplier for all radioactive labeled deoxyribonucleoside 5'-triphosphates. M13mpl8 ssDNA and the universal sequencing primer (17mer) were from USB.

2.3. Expression and purification of PRD1 DNA polymerase Purification of the soluble form of PRD1 DNA polymerase

50 1 of E. coli JL2443 (pEJG) was grown in LB-broth containing ampicillin (100/zg/ml) at 30°C until A590 = 0.3. The culture temperature was shifted up to 42°C. After 2 h of heat induction, the cells were collected by centrifugation (Sorvall GSA, 10 min, 5000 rpm at 4°C) and lysed according to the procedure of Burgess and Jendrisak [26]. The cell lysing buffer contained 50 mM Tris-HC1 (pH 8.0), 2 mM EDTA, 0.1 mM Dq~F, 1 mM /3-mercaptoethanol. Lysozyme was added to 300 # g / m l and phenylmethylsulfonyl fluoride (PMSF) was added to 1 mM. After 30 rain of vigorous stirring at 25°C, sodium deoxycholate was added to 0.05%, and PMSF was added again to a final concentration of 1.5 raM. The lysate was stirred at 25°C for 10 min, and then placed on ice for another 20 min. The lysate was then sonicated at 4°C for 5 min (Branson sonifier, set 4) and centrifuged (Sorvall SS-34, 2 hours, 15 000 rpm at 4°C). The supernatant was brought up to 35% ammonium sulfate saturation by adding of powdered ammonium sulfate with constant stirring. The precipitable material was removed by centrifugation and the supernatant was bought up to 70% ammonium sulfate saturation and centrifuged. The pellet was disolved in 2000 ml DEAE buffer (20 mM Tris-HC1 (pH 7.6), 2 mM /3mercaptoethanol, 1 mM EDTA, 10% glycerol and 0.1 M NaCI) and dialyzed twice against 20 1 of the same buffer. DEAE cellulose chromatography: the dialyzed sample was applied to a DE52 column (5 × 20 cm, 100 m l / h ) preequilibrated with DEAE buffer. The column was washed with 1000 ml of DEAE buffer and the bound proteins were eluted with a 2000 ml linear gradient from 0.1 to 1.0 M NaC1 in the same buffer. Fractions of 10 ml were collected. Both protein-priming and DNA polymerase activities were measured and positive fractions were pooled (fractions #60-#110

W. Zhu, J. lto / Biochimica et Biophysica Acta 1219 (1994) 267-276

eluting from 0.43 to 0.59 M NaCI). These pooled fractions were then dialyzed aganist 4 1 of heparinagarose buffer (50 mM Tris-HCl (pH 7.6), 2 mM /3mercaptoethanol, 0.5 mM EDTA, 10% glycerol, 0.1 M NaCI, 5 mM MgCI2). Heparin-agarose chromatography: the DE52 pool was applied to a heparin-agarose column (1.5 × 6 cm, 10 m l / h ) preequilibrated with the heparin-agarose buffer. The column was washed with 100 ml heparinagarose buffer and eluted with a 100 ml linear gradient from 0.1 M to 1.5 M NaCI in the same buffer. Fractions of 2 ml were collected. The assay positive fractions were pooled (fractions #11-#25 eluting from 0.4 to 0.8 M NaCI) and dialyzed against 1 liter of hydroxylapatite buffer (10 mM sodium phosphate (pH 6.8), 2 mM/3-mercaptoethanol, 0.5 mM EDTA, 10% glycerol, 0.1 M NaCI). Hydroxylapatite column chromatography: the sample was applied to 5 ml hydroxylapatite column (1.1 × 5 cm, 10 m l / h ) preequilibrated with the hydroxylapatite buffer. The column was washed with 50 ml of 0.5 M NaC1 in the same buffer and eluted with a linear gradient from 10 to 400 mM of sodium phosphate in the same buffer. Fractions of 1 ml were collected and the assay positive fractions were pooled (fractions # 8 #17 eluting from 75 to 145 mM sodium phosphate). Sephacryl S-200 chromatography: the hydroxyapatite pool was concentrated to 3 ml by Centriprep Concentrator 30 (Amicon) and loaded to Sephacryl S-200 (1.6 x 100, 15 ml/h) preequilibrated with hydroxylapatite buffer. The column was eluted with 200 ml of the same buffer. Fractions of 1 ml were collected and the assay positive fractions were analyzed in a 10% SDS-PAGE with Coomassie blue staining. Purified DNA polymerase fractions were dialyzed to enzyme storaging buffer (40 mM potassium phosphate (pH 7.0), 2 mM DTI', 0.5 mM EDTA, 50% glycerol, 0.1 M NaCI) and stored at -20°C.

Purification of the insoluble form of PRD1 DNA polymerase 250 ml of E. coli JL 2443 (pEJG) was grown and lysed as described above. The lysate was centrifuged and the pellet was washed three times with cell lysing buffer. Then the pellet was resuspended in 10 ml of denaturing buffer (50 mM Tris-HCl (pH 8.0), 5 mM DTT, 6 M GuHC1) and stirred at 4°C for 30 min. The suspension was then centrifuged (Sorvall SS-34, 30 min, 10000 rpm at 4°C). The supernatant was saved and pumped (at a speed of 5 m l / h ) to 200 ml of heparin-agarose buffer which was under constant stirring. The flocculant precipitate was removed by centrifugation. The supernatant was dialyzed against 2 1 of heparin-agarose buffer and then subjected to heparinagarose column chromatography and subsequently to hydroxylapatite column chromatography. The proce-

269

dures for heparin-agarose and hydroxylapatite column chromatography were described above. After these two column chromatographic steps, the enzyme was found to be near homogeneity and the purified fractions were stored under the same conditions as that of the soluble preparation.

2.4. Assay conditions DNA polymerase assay: The standard assay for DNA polymerase was performed as described previously [9]. The reaction mixture of 40 ~1 contained 50 mM TrisHCI (pH 7.6), 5 mM MgCI2, 1 mM DTT, 0.5 mg/ml BSA, 5% glycerol, 0.3/xM of poly(dC)-oligo(dG)12_18, 30/zM [3H]dGTP (500 cpm/pmol). The K m values for primer terminus and dGTP were determined as described [27], using a homopolymer substrate poly(dC)oligo(dG)lz_18 at an equal molar ratio of template to primer. A series of six concentrations of dGTP or poly(dC)-oligo(dG)12_18 were used, chosen so as to bracket the expected K m values. Triplate reactions were carried out for each substrate concentration. The reaction rate (v) measured at a series of substrate concentrations (S) were plotted using the LineweaverBurk plot (1/v plotted against 1/S), allowing the determination of K m and Vm~x. For each K m, 10 nM of purified DNA polymerase was added so that the amount of template-primer or dGTP substrate was sufficient to saturate the enzyme. The inhibition studies were performed using the standard DNA polymerase assay except that the dGTP concentration was reduced to 10 ~M and 5 nM DNA polyrnerase was added. Inhibition curves were plotted and data were also presented as concentration of each inhibitor that causes 50% inhibition. All the DNA polymerase assays were carried out at 30°C. One unit of DNA polymerase activity was defined as the amount of DNA polymerase that incorporates 1 nmol of labeled dGMP into acid-insoluble DNA at 30°C in 30 min under the standard assay conditions. Exonuclease assay: the 50/zl reaction mixture contained 50 mM Tris-HCl (pH 7.6), 10 mM MgCI 2, 1 mM DTT, 0.5 mg/ml BSA, 5% glycerol. The labeled substrate was prepared by incubating poly(dC)oligo(dG)12_18 with Sequenase version 2.0 (Exo- T7 DNA polymerase, from USB) and [3H]dGTP. The substrate was added to the reaction mixture at 10 /zg/ml (30000 cpm total). The double stranded DNA was heat denatured for the exonuclease assay on single stranded DNA. After incubating the reaction mixture at 30°C for 30 min, the ethanol soluble materials were measured for their radioactivity. One unit of exonuclease was defined as the amount of DNA polymerase required to catalyze the release of 1 nmol of labeled dGMP to ethanol soluble form in 30 min at 30°C under the standard assay conditions.

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144 Zhu, J. Ito / Biochimica et Biophysica Acta 1219 (1994) 267-276

The D N A polymerase strand-displacement assay was carried out as described by Blanco et al. [28]. A 25 ~1 sample of the reaction mixture contained 50 mM TrisHCI (pH 7.6), 10 mM MgC12, 1 mM DTF, 0.5 m g / m l BSA, 5% glycerol, 150 /zM of each dATP, dCTP and TTP, 30 /xM of [ot-32p]dGTP (5 /xCi), 0.5 /zg primedM13mpl8 ssDNA and 100 ng of purified D N A polymerase. After incubation for the indicated times at 30°C, the reaction was stopped by adding E D T A to 20 raM. The samples were ethanol precipitated and subjected to electrophoresis in a 0.7% alkaline agarose gel along with a-32p-labeled D N A length markers and then the labeled D N A was visualized by autoradiography. In vitro replication of PRD1 D N A with purified components was carried out as described [12]. The reaction mixture (200 /zl) contained 50 mM Tris-HC1 (pH 7.6), 7.5 mM MgCI 2, 1 mM DTT, 0.5 m g / m l BSA, 10 mM (NH4)2804, 100 /zM of each dATP, dCTP and TTP, 10 /zM of [a-32p]dGTP (5 /xCi), 1 /xg of the purified PRD1 terminal protein, 100 ng of PRD1 D N A polymerase and 1 /zg of the PRD1 DNA-terminal protein complex as template. The reaction was carried out at 37°C and samples of 25 /zl were withdrawn at the indicated times. The samples were treated with proteinase K ( 2 0 0 / z g / m l , 37°C, 30 min), extracted with phenol and analyzed by alkaline agarose gel electrophoresis. Other assays and procedures: the conditions for PRD1 terminal protein-dGMP complex formation were the same as described [4]. D N A polymerase filling-in assay was carried out according to Bernad et al. [29]. The reaction mixture (25 izl) was the same as that of the strand-displacement assay except that 0.5/zg of the B a n I digested PRD1 D N A was used as the template. D N A polymerase exchange-replacement reaction: the

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reaction mixture (25 #1) contained 100 mM Tris-HC1 (pH 7.6), 20 mM MgCI 2, 1 mM DTT, 0.5 m g / m l BSA, 5% glycerol, 0.5/zg of the D r a I digested PRD1 DNA, 1 0 0 / z M of each dATP, dGTP, dCTP and 0.25/zM of [a-32p]TTP (5 /xCi), 100 ng of the PRD1 D N A polymerase. The reaction mixture was incubated at 30°C for 20 min and stopped by adding 20 mM EDTA. The samples were processed and counted as that of the filling-in assay. Two-dimensional gel electrophoresis was carried out according to O'Farrell [30] with 3 / 1 0 Biolyte (Bio-Rad) in the first dimension and 10% polyacryamide uniform gel in the second dimension. Isoelectric focusing was run at 1000 V for 4 h. The slab gel electrophoresis was run at 20 mA. The gels were stained with Coomassie Brillant Blue R250 (0.05% in 50% m e t h a n o l / 1 0 % acetic acid) and destained with several changes of a 5% m e t h a n o l / 7 % acetic acid solution. Protein concentration was determined according to Bradford [31]. The NH2-terminal amino acid sequence analysis was carried out by the amino acid sequencer facility of the University of California, San Diego.

3. Results 3.1. Expression and solubility o f recombinant PRD1 D N A polymerase in E. coli cells

To analyze the levels of expression and solubility of the PRD1 D N A polymerase, an E. coli strain carrying plasmid p E J G was grown at 28°C to mid-log phase and then transfered to 42°C to derepress the p R promoter. Aliquots were removed at different time intervals and total cell proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Electrophoretic patterns of insoluble and soluble proteins are shown in Fig. 1A

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~ii~,i ~ iiiiiiii;~¸ ~i!i!;¸ ii,i~i i~iii~i i~ii~ ~ i d Fig. 1. Time course and solubility of PRD1 DNA polymerase expression. The E. coli JL 2443 ceils harboring recombinant plasmid pEJG were cultured and samples were taken at intervals of 15 min after heat induction at 42°C. The cells were lysed as described under Materials and methods. The total cell lysate was centrifuged for 2 h at 15000 rpm in a Sorvall SS-34 centrifuge at 4°C. The soluble supernatant as well as the insoluluble pellet were separated and disolved in 2 × SDS-PAGE sample loading buffer. Equal amount of samples were loaded onto 10% SDS-PAGE followed by 5 min at 100°C.The gels were stained with Coomassie Brilliant Blue. In panel A and B, lanes 1 to 10 represent the times of heat induction from 0, 15, 30, 45, 60, 75, 90, 105, 120 to 135 min, respectively. Panel A and B represent the insoluble and the soluble fractions of cellular proteins respectively. The position of PRD1 DNA polymerase is indicated by arrows.

W. Zhu, J. Ito / Biochimica et Biophysica Acta 1219 (1994) 267-276

and B. A protein band corresponding to the PRD1 DNA polymerase could be observed after 60 min of heat induction (Fig. 1A). The PRD1 DNA polymerase was estimated to comprise approximately 30% of the total insoluble proteins after 120 min at 42°C. On the other hand, only a small amount of the soluble PRD1 DNA polymerase can be detected after 45 min at 42°C and this reached a maximum level of about 1% of total soluble fraction of cellular proteins after 60 min of heat induction (Fig. 1B). The amount of the soluble form PRD1 DNA polymerase decreased after 90 rain at 42°C, due probably to aggregation as the concentration of the protein increased following the induction. In an attempt to overcome the aggregation problem, several methods were tried. These include: induction at 38°C instead of 42°C, subcloning the PRD1 DNA polymerase gene into other expression vectors such as pTZ18u, pKK223-3 and pUC19. However, none of these approaches helped to solve the problem. Attempts to extract the PRD1 DNA polymerase in the precipitate with various salts or by using detergents such as Tween 20, Briji 58, Sarkosyl or Triton X-100 were also unsuccessful.

3.2. Purification of PRD1 DNA polymerase The foregoing results indicated that a large portion of the PRD1 DNA polymerase was in the cell pellet. A preliminary experiment suggested that the insoluble PRD1 DNA polymerase could be extracted from the pellet by protein-denaturing agents, such as 6 M guanidine hydrochloride or 8 M urea and that the denatured protein could be renatured by dilution to form an active enzyme. We, therefore, developed a simple, rapid procedure for purification of the PRD1 DNA polymerase based on 6 M GuHCI denaturation and renaturation. The detailed procedure was described under Materials and methods. Briefly, an E. coli JL 2443 (pEJG) was grown in 250 ml LB at 28°C to mid-log phase and the cell cultures were transfered to 42°C and shaken for 120 min. The cells were collected and lysed. The insoluble fraction of the cell lysate was washed three times with cell lysing buffer. Judging from SDS-polyacryamide gel electrophoresis, the washed material contained the PRD1 DNA polymerase that is approximately 50% pure, although inactive. The washed pellet was resuspended in 6 M GuHCI solution containing Tris-HC1 buffer at pH 7.6 and the suspension was centrifuged in a Sorvall at 4°C to remove the insoluble materials. The supernatant was diluted with renaturation buffer containing Tris-HCl (pH 7.6), 5 mM MgCI z and 0.1 M NaC1. At this stage, the PRD1 DNA polymerase was found to be active in both protein-primed initiation reaction and DNA polymerization. The active PRD1 DNA polymerase was then purified to near homogeneity using heparin-agarose and hydroxyl-

271

apatite column chromatography as described under Materials and methods. To confirm the identity of overexpressed recombinant PRD1 DNA polymerase, the NH2-terminal amino acid sequence of the purified enzyme was analyzed. The NH2-terminal amino acid sequence is Pro-ArgArg-Ser-Arg-Lys. Although the first amino acid methionine is absent, which is not uncommon for the Table 1 Comparison of soluble and insoluble preparations of PRD1 D N A polymerase Activities a,b K m (dNTP),/zM gca t, s - 1 g c a t / K m, s -1/zM -1 K m (primer terminus) c, nM 5' ~ 3' Polymerase activity: Initiation-complex forming activity, (U/mg) Elongation-filling-in activity a, (U/mg) 3' --, 5' Exonuclease activity: on single-stranded DNA, (U/mg) on double-stranded DNA, (U/mg) Replacement reaction e, ( U / m g )

Soluble Insoluble preparation preparation 1.3 2.5 2.0 11.0

1.8 4.0 2.2 31.0

38.2

41.9

3.98

118.5 69.9 3.6

DNA replication on different templates f: poly(dC)- oligo(dG)12_ls, 970.0 (U/mg) primed M13 single-stranded DNA, 225.1 (U/mg) activated calf thymus DNA, 175.4 (U/mg) PRD1 DNA-TP complex, 482.9 (U/mg) Inhibitions: KCI, (15o, mM) NaC1, (I5o, mM) NH4CI, (I50, mM) Aphidicolin g, (I50 , # M ) N-Ethylmaleimide g, (I50, mM) Dideoxy-GTP g, (150,/xM)

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a The standand reactions were performed using poly(dC)oligo(dG)iz_18 (1 : 1 ratio) as template in 50 mM Tris-HCl (pH 7.6), 5 mM MgCI2, 1 mM D'I"F, 0,5 m g / m l BSA, 3 0 / z M [3H]dGTP (500 cpm/pmol) and 0.3/xM of template. b All kinetic parameters were calculated from Lineweaver-Burk plots by the method of least squares. c The molar concentration of the primer terminus was calculated based on the average molecular mass of 100 kDa for poly(dC)oligo(dG)12_18. d The template was BanI digested PRD1 DNA which contains 3'-CPuPyG-5' as 5'protruding ends. e The template was DraI digested PRD1 DNA which contains 3'-TIT-5' blunt ends. f The template concentrations in the reactions were 0.3 ~tM for poly(dC)-oligo(dG)12_ls, 2 5 / z g / m l for primed M13, 250/~g/ml for activated calf thymus DNA and 100 /zg/ml for PRD1 DNA-TP complex. The PRD1 D N A polymerase was added to 5 nM which ensured that the templates were at a saturating concentration. g The dGTP concentration was decreased to 10/zM.

W. Zhu, J. Ito /Biochimica et Biophysica Acta 1219 (1994) 267-276

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recombinant proteins expressed in E. coli, the rest of the sequence matched perfectly to the predicted sequence deduced from the nucleotide sequence of the PRD1 D N A polymerase gene [14]. Since the other characteristics of the purified enzyme such as apparent molecular weight and p I value are all in agreement with that of the native PRD1 D N A polymerase, we conclude that the purified recombinant enzyme is the PRD1 DNA polymerase.

soluble and insoluble preparations are indistinguishable as determined by two dimensional gel electrophoresis. The apparent molecular masses of both proteins were 63 kDa, in good agreement with the predicted molecular mass of 63336 Da from the sequencing data [14,15]. The isoelectric points of both proteins were 6.55 which were also similar to the predicted p I value of 6.68 [21]. Next, various kinetic parameters and enzymological properties were compared between the two DNA polymerase preparations. The results are summarized in Table 1. The steady-state kinetic parameters, K m for dNTP, Kcat for the DNA polymerase reaction and K m for the primer terminus were measured for both DNA polymerase p r e p a r a t i o n s using a h o m o p o l y m e r poly(dC)-oligo(dG) as the DNA substrate. Although the gcat and K m for the primer terminus of the insoluble PRD1 DNA polymerase were somewhat higher than that of the soluble enzyme, the values were of the same order of magnitude. Since the protein-primed initiation of DNA synthesis involves highly specific protein-protein and prot e i n - D N A interactions, it is expected to provide a sensitive assay for detecting structural and conformational changes of the protein. There is no appreciable difference in the formation of covalent linkage of a d G M P to the terminal protein between the two DNA

3.3. Comparison between soluble and insoluble preparations of PRD1 DNA polymerase In order to characterize the PRD1 D N A polymerase purified from the insoluble material, we used the soluble PRD1 D N A polymerase as a reference protein. The soluble PRD1 D N A polymerase was purified from a large amount of material using several columns for chromatography as described under Materials and methods. First, the purity of the PRD1 D N A polymerase was determined by SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining. The purity of both PRD1 D N A polymerase preparations was over 95%. The insoluble PRD1 D N A polymerase preparation was somewhat purer than the reference soluble enzyme preparation (data not shown). Both

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Iv.. Zhu, J. Ito / Biochimica et Biophysica Acta 1219 (1994) 267-276

was ruled out by a sensitive gel assay (unpublished data). The PRD1 D N A polymerase is also capable of performing 'replacement reaction', analogous to the T4 D N A polymerase [32]. This activity is due to two catalytic activities, the 3' to 5' exonuclease and the 5' to 3' D N A polymerase activity. This reaction consists of cycles of removal and replacement of the 3' terminal nucleotides from the blunt ended D N A substrate. The template-primer preference was also compared. As shown in Table 1, the PRD1 D N A polymerase is more active on poly(dC)-oligo(dG) than the native template of the PRD1 genome with terminal protein. Specific activities of the enzyme prepared by denaturationrenaturation procedure are usually higher than the soluble PRD1 D N A polymerase. This is probably due to the fact that the insoluble preparation is actually purer than the soluble preparation. The sensitivities of two PRD1 D N A polymerase preparations to various inhibitors and salt concentra-

polymerase preparations (Table 1). Likewise, filling-in activities are the same between these enzyme preparations. This evidence implies that the structure of the PRD1 D N A polymerase after denaturation-renaturation is virtually identical to that of the soluble D N A polymerase. Like many family B D N A polymerases, the PRD1 D N A polymerase contains an intrinsic 3' to 5' exonuclease activity which is believed to increase the accuracy of D N A replication by removing misincorporated nucleotides [13]. The PRD1 D N A polymerase can hydrolyze both single- and double-stranded DNAs. However, as is the case of a proofreading 3' to 5' exonuclease, single-stranded D N A is the optimal substrate for PRD1 D N A polymerase. The comparative studies have shown that the insoluble preparation has somewhat higher exonuclease activity than the soluble preparation (Table 1). The possibility of 5' to 3' exonuclease activity and endonuclease activity associated with PRD1 D N A polymerase under the employed assay conditions

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Fig. 3. In vitro replication of PRD1 genome with purified DNA polymerase and terminal protein. The reaction mixture was as described under Materials and methods. After incubation at 37°C for the times indicated, the samples were withdrawn and treated with proteinase K. Alkaline agarose gel electrophoresiswas performed as described [12]. Both 32P-labeledPRD1 DNA marker and DNA length markers are also shown. For the 45 min samples, half amount of the total samples were loaded.

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a b e d e f g h

i

j

k

I b) DP

1.13

116 ~3

i57

~22 127

Fig. 4. Strand-displacement synthesis of primed M13 D N A by P R D I D N A polymerase. The reaction mixture was as described under Materials and m e t h o d s which contained 100 ng of the purified PRD1 D N A polymerase, either the soluble preparation or the insoluble preparation, or 5 units of the Klenow enzyme (from New EngLand Biolab, Inc.). The reactions were carried out at 30°C for 5, 10, 15, 30 and 60 rain and stopped by adding E D T A . Samples were processed and analyzed by alkaline agarose gel electrophoresis along with D N A length markers. Lane A: Klenow enzyme incubated for 30 min. Lanes b to f represent the soluble preparation and lanes g to k represent the insoluble preparation of the P R D I D N A polymerase. Lanes b and g, lanes c and h, lanes d and i, lanes e and j, lanes f and k correspond to the time of 5, 10, 15, 30, 60 min. respectively. The size of D N A length and M13 D N A are shown on the right side by arrows.

Template/DNA

Pol

Soluble preparation I v 1:4 1:2 I:i 2=I 4:1 8:1

Insoluble

preparation

r

1:4

i

1:2

1:1

2:1

4:1

8~1

TOP-

(Kb) 23.13-

9.4166.577-

4.361-

2.322-

Fig. 5. Processive synthesis of primed M13 D N A by the PRD1 D N A polymerase. The processivity assay was carried out under the same conditions as the strand-displacement assay described under Materials and methods. T h e template-primer to D N A polymerase ratios are indicated. T h e reactions were carried out at 30°C for 30 rain. The samples were processed and analyzed by alkaline agarose gel electrophoresis.

w. Zhu, J. lto / Biochim&a et Biophysica Acta 1219 (1994) 267-276

tions are compared (Fig. 2 and Table 1). As shown in Fig. 2A, B and C, KCI, NaC1 and NH4C1 have dual effects on PRD1 DNA polymerase activity. At lower concentration (< 150 mM), these salts stimulate PRD1 DNA polymerase activity. However, at higher concentration (> 200 mM), they inhibit the activity. Under the same conditions, these salts have little effect on the activity of the Klenow fragment of E. coli DNA polymerase I. All the family B DNA polymerases so far tested, are sensitive to aphidicolin, although the degree of sensitivity varies [33]. It has been shown that in vitro PRD1 DNA replication was inhibited by aphidicolin [12]. In agreement with the in vitro DNA synthesis, the PRD1 DNA polymerase is sensitive to aphidicolin (Fig. 2D). Under the same conditions, the Klenow fragment of E. coli DNA polymerase I is insensitive. These inhibition studies are consistent with evidence that PRD1 DNA polymerase prepared by denaturation-renaturation is very similar to that of the soluble enzyme. The efficiency of the protein-primed genome DNA replication was next compared between two DNA polymerase preparations. Fig. 3 demonstrates that both preparations can efficiently synthesize the unit length of PRD1 DNA. For both preparations, the unit length PRD1 DNA was reached after 7.5 rain incubation and plateaued during further incubation at 37°C. This rate is nearly comparable to that obtained in the cell-free replication system for the PRD1 genome [12]. Proteinprimed DNA replication initiates at one end of the genome and proceeds in the 5' to 3' direction with concomitant displacement of the parental 5' strand [34]. Blanco et al. [28] have shown that the ~b29 DNA polymerase is also capable of catalyzing strand-displacement DNA synthesis without any other protein factors. Having confirmed that the purified PRD1 DNA polymerase is capable of performing strand-displacement synthesis, we compared two PRD1 DNA polymerase preparations as to their ability to catalyze strand-displacement DNA synthesis without any assistance of other proteins. Fig. 4 shows that both preparations are quite capable of performing strand-displacement synthesis. Under the same conditions, the Klenow fragment of E. coli DNA polymerase I could not. Finally, the processivity of the PRD1 DNA polymerase was compared between the two preparations. For the processivity assay, we used primed M13 ssDNA as template and DNA synthesis was measured by increasing the ratio of primer-template to DNA polymerase. Fig. 5 illustrates that the molar ratio of primer-template to DNA polymerase ranges from 1 : 4 to 8:1. Under these conditions, a distributive DNA polymerase would give a decreased size of replication products. Conversely, the size of replication products should remain the same for a processive DNA polymerase. As can be seen from Fig. 5, the size of the

275

replication products does not decrease as the ratio of primer-template to DNA polymerase increases.

4. Discussion

The PRD1 DNA polymerase is the smallest known member of the family B DNA polymerases [20]. The family B DNA polymerases include: T4, 4)29, eukaryotic replicative DNA polymerases (a, ~ and E), archaebacterial DNA polymerases and viral DNA polymerases. Because of its small size and because of the amenability to genetic and biochemical manipulations, the PRD1 DNA polymerase provides a useful model system with which to study structure-function relationships of the DNA polymerase molecules. The overexpression and purification of the PRD1 DNA polymerase has been hampered by the insolubility of the enzyme in an overexpressed system [13,22]. We report here a rapid and efficient purification procedure for the PRD1 DNA polymerase. This procedure is based on the property of the PRD1 DNA polymerase that it is highly sensitive to the denaturing agent, but becomes fully active by dilution of the denaturing agent. We have used 6 M guanidine hydrochloride to solublize the insoluble PRD1 DNA polymerase and renatured the enzymatic activity by dilution. This method has been used successfully with a variety of enzymes including ofactor of E. coli RNA polymerase [35], p factor [36], Bacillus subtilis or factor [37,38], /3-galactosidase and alkaline phosphatase [36]. To determine that the PRD1 DNA polyrnerase after denaturation and renaturation is similar to the native soluble enzyme, we have compared two preparation extensively. These include: kinetic parameters, various enzymatic activities such as 3' to 5' exonuclease, repair type DNA polymerase activity, exchange replacement activity, strand-displacement activity and sensitivities to various salts and inhibitors. All properties compared are indistinguishable between two preparations of the PRD1 DNA polymerase. Perhaps, the most significant test for conformational changes of the two forms of PRD1 DNA polymerase is the protein-primed DNA replication test. Since protein-primed DNA synthesis requires specific interactions between DNA polymerase, terminal protein, template DNA and substrate nucleotides, it provides a highly sensitive assay for comparing the two preparations. Our results showed that the rate of protein-primed D N A synthesis of both enzyme preparations in the presence of purified PRD1 terminal protein and intact PRD1 DNA-terminal protein complex as template, are virtually identical (24-26 nucleotides per second at 37°C). These results are very similar to the other protein-primed DNA replication systems. The rate of the protein-primed ~b29 DNA syntesis was about 10 nucleotides per second at 30°C

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W. Zhu, J. Ito / Biochimica et Biophysica Acta 1219 (1994) 267-276

[39]. In the case of adenovirus DNA replication in vitro, the value of 10-20 nucleotides per second at 30°C was described [40]. Bodnar and Pearson [41] reported that the rate of adenovirus DNA replication in infected ceils is 1600 + 170 nucleotides per minute at 32°C. This value would be equivalent to 26 nucleotides per second. All of these results indicate that the PRD1 DNA polymerase prepared by the denaturation-renaturation procedure is indistinguishable from the soluble PRD1 DNA polymerase. The studies presented here identify several unique properties of the PRD1 DNA polymerase which make it suited for its role in protein-primed, strand-displacement DNA replication. The PRD1 DNA polymerase is highly processive and is capable of performing stranddisplacement DNA synthesis without any other protein factors. These properties are reminiscent of the DNA polymerase encoded by Bacillus phage ~b29 [28]. The availability of a simple purification procedure for the PRD1 DNA polymerase, in addition to its amenability to genetic manipulation, should facilitate detailed biochemical and structural studies of the mechanism of protein-primed, strand-displacement DNA synthesis.

Acknowledgments We thank Dr. Mark C. Leavitt for his help in the initial stage of this research. We also thank Dr. Stella S.S. Chang for supplying the purified PRD1 terminal protein. We are grateful to Dr. Harris Bernstein and Ms. Vivian Gage for their critical reading of this manuscript. This research was supported in part by grant from Takara Shuzo Co. Ltd.

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