Eur. J. Biochem. 2 4 8 1 -85 (1975)
Structure and Synthesis of a Lipid-Containing Bacteriophage A Polynucleotide-Dependent Polynucleotide-PyrophosphorylaseActivity in Bacteriophage PM2 Rolf SCHAFER and Richard M. FRANKLIN Ahtcilung fur Strukturbiologie,Biozentrum der Universitat Basel (Received February S/May 22,1975)
A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 "C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nucleic acids or polynucleotides as effectors. The product is not covalently linked to the effector.
The lipid-containing bacteriophage PM2 which replicates on the marine pseudomonad, Pseudomonas BAL-31 [l,21, has four distinct structural proteins [3]. A DNA-dependent RNA polymerase activity associated with the bacteriophage was first described by Datta and Franklin [4]. The polymerase activity was detected in the whole virus in the presence of a non-ionic detergent. Since the four structural proteins of the bacteriophage have been purified (R. Hinnen, H. Hitz, D. Schafer, R. Schafer, R. M. Franklin, and B. Wittmann, unpublished results), we were able to identify the protein responsible for the polymerising activity and analyze the nature of this reaction in vitro.
Assay of the Polymerisation Reaction Standard assays (0.5 mi) contained 25 mM Tris HCl, pH 8.1, 2.5 mM MnCl,, 20 mM 2-mercaptoethanol, 5 pg of viral protein, 50 pg RNA or DNA and 1 mM of a %I-labeled nucleotide triphosphate. The mixture was incubated at 28 "C for 2 h, diluted with 0.5 ml of ice water and precipitated with an equal volume of 10 % (w/v) trichloroacetic acid. The acidinsoluble material was collected, washed, and dried on nitrocellulose filters. The filters were placed in vials with 8 ml of toluene containing 3.6 g/1 PPO and 0.45 g/l dimethyl-POPOP. The radioactivity was determined in a Packard Tricarb liquid scintillation counter.
MATERIALS AND METHODS
Preparation of D N A
General
Superhelical (native) PM2 DNA was prepared as described by Espejo et al. [9]. Circular (nicked) PM2 DNA was prepared by the method of Richardson [lo] and T4 DNA was isolated according to Fuchs et al.
The preparation of highly purified bacteriophage PM2 was described by Hinnen et al. [6]. The purification of the proteins of the bacteriophage by electrophoresis on cellulose acetate strips in the presence of 8 M urea, by preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, or by gel filtration in the presence of 6 M guanidine hydrochloride will be described by Hinnen et al. in a future publication. Quantitative protein determinations were carried out as described by Lowry et al. [7] or by Heil and Zillig [8]. Sterile solutions were used for all of the procedures. This is paper number XX in the series. Enzyme. Polynucleotide pyrophosphorylase(EC 2.7.7.-).
Sources of Materials 3H-labeled deoxyribonucleoside and ribonucleoside triphosphates were purchased from the Radiochemical Centre (Amersham, England); PPO and POPOP, scintillation grade, were from Packard Instrument Company, Inc. (Illinois, U.S.A.); poly(U) and poly(G) were from Miles Laboratories Inc. (Illinois, U.S.A.); tRNA from Escherichia coli MRE 600 was purchased from Boehringer (Mannheim, Federal
82
Primed Pyrophosphorylase Activity in PM2
Republic of Germany); pronase E and other common reagents were from Merck (Darmstadt, Federal Republic of Germany).
RESULTS The four proteins of PM2 were separated into a mixture of proteins I and I1 (coat and spike proteins) and a mixture of proteins I11 and IV (the nucleocapsid core proteins) by Bligh and Dyer fractionation as modified for bacteriophage PM2 [12]. Both protein fractions were used for the standard assay of polymerase activity as described in Materials and Methods. No activity was measured in the mixture of proteins 1 and I1 ; the activity was associated with the mixture of proteins I11 and IV. Proteins I11 and IV were then separated from each other by three different methods, as will be described by Hinnen et al. later. The pure proteins were used in the standard assay. The polymerase activity was associated with protein IV, the protein which is combined with the DNA in the virion [6].Purification of the polymerase from bacteriophage PM2 is summarized in Table 1. The incorporation of ribonucleoside and deoxyribonucleoside triphosphates in the presence of PM2 DNA or poly(U) was linear up to 3 h. All of the assays described here were carried out for 2 h. All further assays were made in the presence of protein IV alone. Incorporation of nucleoside triphosphate took place in the presence of several different nucleic acids or synthetic polynucleotides (cJ Table 4). In the presence of poly(U), the synthesis of polyadenylate and polydeoxyadenylate were not very different and therefore poly(U) was used in the standard assay system to investigate the reaction. As described by Datta and Franklin [4], the enzymic reaction in the presence of bacteriophage PM2 was strongly dependent on the concentration of Mn2 ions, which were absolutely required, apparently as a cofactor. The optimum of Mn2+ions was 2.5 mM, the temperature optimum was around 25 "C and the pH optimum was at 8.1. Similar results were obtained in the present work in the presence of poly(U) and purified protein IV (Table 2). When protein IV was treated with sodium cyanate or p-phenylenediisocyanate which react with free amino groups of the N-termini and basic amino acid residues of the polypeptide, the synthesis of polyadenylate was depressed and the synthesis of polydeoxyadenylate was completely inhibited (Table 3). Sodium dodecyl sulfate in the assay depressed the enzymic reaction but did not inhibit it completely. The enzymic reaction was clearly protein-dependent since it was lost when protein IV was preincubated in the presence of the proteolytic enzyme pronase E. The synthesis of polyadenylate or polydeoxyadenylate +
Table 1. Purification of the polymerase activity associated with bocteriophoge PM2 The protein fractions were incubated under standard assay conditions (see Materials and Methods) in the presence of 30 vg of PM2 DNA (b, c); 0.2 % Nonidet P40 but no added DNA (cJ [4]) was in incubation mixture (a) Expt
a) PM2 virus b) Proteins 111, IV c) Protein IV
Protein
AMP incorporated
Enrichment
Pg
pmol/h
-fold
170 40 2
54 107 97.5
8.4 153.5
1
Table 2. Temperature, p H , and magnesium dependences of the polymerisotion reaction 50 pg of poly(U) was present in all incubation mixtures under standard assay conditions Temperature
AMP incorporatcd
"C
pmol h - ' (vgprotein IV)-'
22 28
104.8 168.7 128 77.1 12.9
31
48 58 PH
1.5 8 8.1 8.2 8.5 9
122.1 178.3 183.2 182.4 175.9 135.1
[Mn2+] rnM 0 1
2 2.5 3 5 10
22.1 79.4 132.8 194.2 185 163 157
was completely inhibited when protein IV was carboxymethylated. Incorporation was less when protein IV was heated at 98 "C in the presence of 2-mercaptoethanol for 30 min, but was equal to that of the control when oxygen was bubbled through the heated protein solution. Pyrophosphate added at the start of the incubation strongly inhibited the enzymic reaction. No inhibition was observed when pyrophosphate was added at the end of the incubation period. Neither T4 DNA nor poly(A), which was synthesized in the presence of T4 DNA, were hydrolyzed in a further
R. Schafer and R. M.Franklin
83
incubation period with excess pyrophosphate added to the incubation mixture. Magnesium ions did not replace manganese ions when native PM2 DNA (data not shown) or poly(U) were present in the
Table 3. Properties of protein IV in the pol.vmerisation reaction 50 pg of poly(U) was present in all incubation mixtures under standard assay conditions Treatment
Incorporated AMP
dAMP
pmol h-' (pg protein IV)-' Standard assay Protein treated with CNO(100 ng/ml) [17] Protein treated with p-phenylenediisocyanate (2 pg/ml) (181 0.5 % sodium dodecyl sulfate in the assay Protein heated for 30 min at 98 "C Protein treated with pronase E (1 pg) for 30 min at pH 7.4 and 40 "C Protein carboxymethylated [19] 40 mM pyrophosphate in the assay during the incubation period 40 mM pyrophosphate added to the assay at the end of the incubation 10 mM M 8 + , no Mnz+ in the assay 10 mM Mgz+ + 2.5 mM MnZ+ in the assay
0.15
188.5
207
133
2.3
108.9
1.9
155.3 112.7
176.5 120
4.8 3.9
5.2 3.2
61
36
185
213
Structure and Synthesis of a Lipid-Containing Bacteriophage A Polynucleotide-Dependent Polynucleotide-PyrophosphorylaseActivity in Bacteriophage PM2 Rolf SCHAFER and Richard M. FRANKLIN Ahtcilung fur Strukturbiologie,Biozentrum der Universitat Basel (Received February S/May 22,1975)
A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 "C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nucleic acids or polynucleotides as effectors. The product is not covalently linked to the effector.
The lipid-containing bacteriophage PM2 which replicates on the marine pseudomonad, Pseudomonas BAL-31 [l,21, has four distinct structural proteins [3]. A DNA-dependent RNA polymerase activity associated with the bacteriophage was first described by Datta and Franklin [4]. The polymerase activity was detected in the whole virus in the presence of a non-ionic detergent. Since the four structural proteins of the bacteriophage have been purified (R. Hinnen, H. Hitz, D. Schafer, R. Schafer, R. M. Franklin, and B. Wittmann, unpublished results), we were able to identify the protein responsible for the polymerising activity and analyze the nature of this reaction in vitro.
Assay of the Polymerisation Reaction Standard assays (0.5 mi) contained 25 mM Tris HCl, pH 8.1, 2.5 mM MnCl,, 20 mM 2-mercaptoethanol, 5 pg of viral protein, 50 pg RNA or DNA and 1 mM of a %I-labeled nucleotide triphosphate. The mixture was incubated at 28 "C for 2 h, diluted with 0.5 ml of ice water and precipitated with an equal volume of 10 % (w/v) trichloroacetic acid. The acidinsoluble material was collected, washed, and dried on nitrocellulose filters. The filters were placed in vials with 8 ml of toluene containing 3.6 g/1 PPO and 0.45 g/l dimethyl-POPOP. The radioactivity was determined in a Packard Tricarb liquid scintillation counter.
MATERIALS AND METHODS
Preparation of D N A
General
Superhelical (native) PM2 DNA was prepared as described by Espejo et al. [9]. Circular (nicked) PM2 DNA was prepared by the method of Richardson [lo] and T4 DNA was isolated according to Fuchs et al.
The preparation of highly purified bacteriophage PM2 was described by Hinnen et al. [6]. The purification of the proteins of the bacteriophage by electrophoresis on cellulose acetate strips in the presence of 8 M urea, by preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, or by gel filtration in the presence of 6 M guanidine hydrochloride will be described by Hinnen et al. in a future publication. Quantitative protein determinations were carried out as described by Lowry et al. [7] or by Heil and Zillig [8]. Sterile solutions were used for all of the procedures. This is paper number XX in the series. Enzyme. Polynucleotide pyrophosphorylase(EC 2.7.7.-).
Sources of Materials 3H-labeled deoxyribonucleoside and ribonucleoside triphosphates were purchased from the Radiochemical Centre (Amersham, England); PPO and POPOP, scintillation grade, were from Packard Instrument Company, Inc. (Illinois, U.S.A.); poly(U) and poly(G) were from Miles Laboratories Inc. (Illinois, U.S.A.); tRNA from Escherichia coli MRE 600 was purchased from Boehringer (Mannheim, Federal
82
Primed Pyrophosphorylase Activity in PM2
Republic of Germany); pronase E and other common reagents were from Merck (Darmstadt, Federal Republic of Germany).
RESULTS The four proteins of PM2 were separated into a mixture of proteins I and I1 (coat and spike proteins) and a mixture of proteins I11 and IV (the nucleocapsid core proteins) by Bligh and Dyer fractionation as modified for bacteriophage PM2 [12]. Both protein fractions were used for the standard assay of polymerase activity as described in Materials and Methods. No activity was measured in the mixture of proteins 1 and I1 ; the activity was associated with the mixture of proteins I11 and IV. Proteins I11 and IV were then separated from each other by three different methods, as will be described by Hinnen et al. later. The pure proteins were used in the standard assay. The polymerase activity was associated with protein IV, the protein which is combined with the DNA in the virion [6].Purification of the polymerase from bacteriophage PM2 is summarized in Table 1. The incorporation of ribonucleoside and deoxyribonucleoside triphosphates in the presence of PM2 DNA or poly(U) was linear up to 3 h. All of the assays described here were carried out for 2 h. All further assays were made in the presence of protein IV alone. Incorporation of nucleoside triphosphate took place in the presence of several different nucleic acids or synthetic polynucleotides (cJ Table 4). In the presence of poly(U), the synthesis of polyadenylate and polydeoxyadenylate were not very different and therefore poly(U) was used in the standard assay system to investigate the reaction. As described by Datta and Franklin [4], the enzymic reaction in the presence of bacteriophage PM2 was strongly dependent on the concentration of Mn2 ions, which were absolutely required, apparently as a cofactor. The optimum of Mn2+ions was 2.5 mM, the temperature optimum was around 25 "C and the pH optimum was at 8.1. Similar results were obtained in the present work in the presence of poly(U) and purified protein IV (Table 2). When protein IV was treated with sodium cyanate or p-phenylenediisocyanate which react with free amino groups of the N-termini and basic amino acid residues of the polypeptide, the synthesis of polyadenylate was depressed and the synthesis of polydeoxyadenylate was completely inhibited (Table 3). Sodium dodecyl sulfate in the assay depressed the enzymic reaction but did not inhibit it completely. The enzymic reaction was clearly protein-dependent since it was lost when protein IV was preincubated in the presence of the proteolytic enzyme pronase E. The synthesis of polyadenylate or polydeoxyadenylate +
Table 1. Purification of the polymerase activity associated with bocteriophoge PM2 The protein fractions were incubated under standard assay conditions (see Materials and Methods) in the presence of 30 vg of PM2 DNA (b, c); 0.2 % Nonidet P40 but no added DNA (cJ [4]) was in incubation mixture (a) Expt
a) PM2 virus b) Proteins 111, IV c) Protein IV
Protein
AMP incorporated
Enrichment
Pg
pmol/h
-fold
170 40 2
54 107 97.5
8.4 153.5
1
Table 2. Temperature, p H , and magnesium dependences of the polymerisotion reaction 50 pg of poly(U) was present in all incubation mixtures under standard assay conditions Temperature
AMP incorporatcd
"C
pmol h - ' (vgprotein IV)-'
22 28
104.8 168.7 128 77.1 12.9
31
48 58 PH
1.5 8 8.1 8.2 8.5 9
122.1 178.3 183.2 182.4 175.9 135.1
[Mn2+] rnM 0 1
2 2.5 3 5 10
22.1 79.4 132.8 194.2 185 163 157
was completely inhibited when protein IV was carboxymethylated. Incorporation was less when protein IV was heated at 98 "C in the presence of 2-mercaptoethanol for 30 min, but was equal to that of the control when oxygen was bubbled through the heated protein solution. Pyrophosphate added at the start of the incubation strongly inhibited the enzymic reaction. No inhibition was observed when pyrophosphate was added at the end of the incubation period. Neither T4 DNA nor poly(A), which was synthesized in the presence of T4 DNA, were hydrolyzed in a further
R. Schafer and R. M.Franklin
83
incubation period with excess pyrophosphate added to the incubation mixture. Magnesium ions did not replace manganese ions when native PM2 DNA (data not shown) or poly(U) were present in the
Table 3. Properties of protein IV in the pol.vmerisation reaction 50 pg of poly(U) was present in all incubation mixtures under standard assay conditions Treatment
Incorporated AMP
dAMP
pmol h-' (pg protein IV)-' Standard assay Protein treated with CNO(100 ng/ml) [17] Protein treated with p-phenylenediisocyanate (2 pg/ml) (181 0.5 % sodium dodecyl sulfate in the assay Protein heated for 30 min at 98 "C Protein treated with pronase E (1 pg) for 30 min at pH 7.4 and 40 "C Protein carboxymethylated [19] 40 mM pyrophosphate in the assay during the incubation period 40 mM pyrophosphate added to the assay at the end of the incubation 10 mM M 8 + , no Mnz+ in the assay 10 mM Mgz+ + 2.5 mM MnZ+ in the assay
0.15
188.5
207
133
2.3
108.9
1.9
155.3 112.7
176.5 120
4.8 3.9
5.2 3.2
61
36
185
213
