Vol

THE JOURNAL OF Bmm~~ca~ CHEMI8TnY 253, No. 3, Issue of February 10, pp. 758-764, Prmted III U S.A.

1978

Primase,
AN ENZYME

the dnaG Protein

WHICH STARTS DNA CHAINS*

of Escherichia

coZi
(Received for publication, August 3, 1977)

LEE

ROWEN

AND

ARTHUR

KORNBERG Stanford University School of Medicine, Stanford, California

From the Department 94305

of Biochemistr.y,

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and Cc) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the succeeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent K, for ribonucleoside triphosphates near 10 PM, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase be renamed primase.

enzymatic mechanisms of DNA replication in Escherichia coli can be analyzed (1, 2). Conversion of the single-stranded circle to the duplex replication form can be divided into three major stages: initiation, elongation, and termination. In all these phage DNA systems, chain elongation occurs by action of DNA polymerase III holoenzyme (3, 4). Given a primer, the holoenzyme can copy the circle to give the duplex replicative form.
* This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. This is the third paper in a series dealing with the replication of phage G4 DNA. The previous paper is Ref. 8. The costs of nublication of this article were defrayed iA part by the payment of page charges. This article must therefore be hereby marked uduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: holoenzyme, DNA polymerase III holoenzyme; albumin, bovine serum albumin; DBP, DNA-binding

The DNA chromosomes of the small bacteriophages G4, and +X174 have provided systems in which

M13,

Gaps in the replicative form can be filled by DNA polymerase I and sealed by DNA ligase to yield the closed circular duplex, RF I (5). The three phage DNA templates differ primarily in the mode of initiation of DNA replication. In each case DNAbinding protein (DBP) coats most of the DNA circle (6). Although this s&ices for priming of Ml3 DNA by RNA polymerase (7) and of G4 DNA by dnaG protein (8, 91, $X DNA, by contrast, must first be acted upon by additional proteins before priming by the dnaG protein can occur (2, lo12). A nucleoprotein intermediate is formed in the presence of ATP and Mg2+ in which proteins n (13) and i (13) act catalytically to put the dnaB protein (11, 13-15) and possibly the dnaC protein (13, 16) onto the DNA (10-12). Priming by dnuG protein on G4 DNA is by synthesis of a short RNA transcribed from a region about 5% of the genome length from the single cleavage site of EcoRl restriction endonuclease, the site determined as the origin of replication of G4 DNA (9). In this paper, a new purification procedure for the dnuG protein is described and evidence is given that the RNA polymerase activity co-purifies with the complementation activity for an extract of a dnuG mutant and with the priming activity for G4 DNA replication. All these activities are remarkably specific for G4 viral DNA coated by DNA-binding protein. In succeeding papers, the structure of the unique RNA transcript synthesized by the dnaG protein at the origin of G4 DNA replication and the utilization of deoxyribonucleoside triphosphates as well as ribonucleoside triphosphates by this enzyme are presented (17, 18). The action of the dnaG protein in the priming of +X DNA is discussed in a separate paper (11). Based on its role in priming DNA replication rather than in transcribing DNA regions for synthesis of messenger or other defined RNAs, and its capacity to use deoxynucleotides or ribonucleotides, we propose that the dnuG protein be designated primase.
MATERIALS AND METHODS

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Bacterial and Phage Strains - These were generously provided follows: Escherichia coli HMS83 (thy, lys, lac, rhu, &A, polB)

as by

protein; dNTPs, deoxyribonucleoside triphosphates; 4X, +X174; NMP, nucleoside monophosphate; RF, duplex circular replicative form of a phage DNA; RF I, covalently closed RF; rNTPs, ribonucleoside triphosphates; SDS, sodium dodecyl sulfate; SS, singlestranded.

758

dn.aG Gene Product

Dr. R. McMacken of Johns Hopkins University; E. coli PC3 (dnaG3, leu, thy, stp) by Dr. P. Carl of the University of Illinois, Urbana; phage G4 by Dr. G. N. Godson of Yale University. Growth of Cells and Phages-E. coli HMS83 was grown with high aeration in a New Brunswick loo-liter Fermacell to A,, = 8 to 10 in medium containing per liter: 10 g of K,HPO,; 1.85 g of KH,PO,; 10 g of Ardamine Z yeast extract; 10 mg of thiamin; 50 mg of thymine, 1% Cerelose; the pH was maintained between 7.1 and 7.3 by addition of NaOH. Cells were harvested in a Sharples continuous flow centrifuge. The cell paste was suspended in cold 50 mM Tris.Cl (pH 7.51, 10% sucrose to A,,, = 0.5 at a 1:400 dilution. The cell suspension, frozen by pouring it into liquid nitrogen, was stored at -20. E. coli PC3 (19) was grown as described (8). Phage G4 (20) was grown according to a delayed lysis procedure developed by Dr. G. N. Godson.* ChemicalsSources were as follows: rifampicin from Calbiochem; heparin from Sigma; streptolydigin and streptovaricin C from Upjohn; Actinomycin D from Merck, Sharp and Dohme; ammonium sulfate and sucrose from Schwarz/Mann, dithiothreitol from BioRad; imidazole from Baker or Eastman; Brij 58 from Pierce Co.; spermidine from Calbiochem; unlabeled rNTPs and dNTPs from PL Biochemicals. The dNTPs were further purified by chromatography on DEAE-Sephadex A-25 columns using a gradient of ammonium bicarbonate from 0.03 to 0.5 M. 3H- and 32P-labeled nucleoside triphosphates were from New England Nuclear or Amersham/ Searle. Enzymes - Bovine serum albumin was from Armour and lysozyme from Worthington. DBP (1.7 mg/ml) was electrophoretically pure (6). PC3 Fraction II was prepared as described previously for H560 (81, and heated for 10 min at 37 to reduce endogenous dnaG activity. Holoenzyme was purified (3) to give Fraction IV (8 x lo5 units/ml; 4.8 mg/ml) or Fraction VI (4 x 105 units/ml; 1.8 mg/ml). Purified RNA polymerase (1 mglml) was a gift of Dr. M. Chamberlin (University of California, Berkeley). DNA polymerase I was as described (21). DNA Templates - DNA from phage G4 was prepared by sedimentation of 5 x lOI plaque-forming units through a 5 to 20% sucrose gradient in 50 mM Tris.Cl (pH 7.51, 1 mM EDTA, 1 M NaCl for 3.5 h at 26,000 rpm, at 4 in the SW 27 rotor. The peak of infectivity was pooled, dialyzed against 50 mM sodium borate, adjusted to 1% SDS, and treated with 1 volume of distilled phenol equilibrated in 50 mM sodium borate. The extraction mixture was heated at 65 for 10 min; the aqueous phase was removed after centrifugation. The phenol phase was re-extracted with 0.5 volume of 50 mM sodium borate. Aqueous phases were combined, and the DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate (pH 5.51, and 2 volumes of isopropyl alcohol, followed by storage at -20 for 3 h. The DNA pellet obtained after centrifugation in a Sorvall centrifuge (18,000 rpm for 40 min at 0 in the SS 34 rotor) was resuspended in 200 ~1 of 10 mM Tris.Cl (pH 7.5), 1 mM EDTA, and dialyzed against the same buffer for 2 days at 4. As seen in the electron microscope with formamide spreading (221, 80% of the DNA molecules were circular. Sources of DNA were as follows: phage Ml3 (71, phage $X (23); phage Pf-1 a gift of Dr. D. Ray (University of California, Los Angeles), phage ST-1 (241, poly(dA) and poly(dC), Calbiochem; +X RF I and G4 RF I (25); phage A (26) and ColEl plasmid (27) respective gifts from T. White and G. Guild of this department; E. coli (28); trp attenuator (291, isolated from an HpaII restriction endonuclease digest of480pt190 was a gift of Frank Lee (Department of Biological Sciences, Stanford University). Resins - Bio-Rex-70 (100 to 200 mesh) was obtained from Bio-Rad, and equilibrated as follows: 4 pounds of resin in 4 liters of water were treated with 2.3 liters of 2 M HCl (final pH = 1). The resin was washed with water, by decantation, until the pH was 5. The resin was made 20% in glycerol (v/v), and kept for 1 h. The supernatant was replaced by 6 liters of 0.5 M imidazole base, 20% glycerol, and kept for 1 h. Finally, the resin was washed with and stored in Buffer I (pH 6.8) (see below). For reuse, the resin was treated with 0.5 M NaOH, washed, and then equilibrated as described above. DNA-cellulose (30) was prepared with calf thymus DNA (Calbiochem grade A (2618)). Upon mixing the DNA solution (2 mg/ml) with the cellulose, a thick paste was obtained (1 g of cellulose/3 ml of DNA). This paste was spread on sides of beakers and left to dry 2 G. N. Godson, personal communication.

of E. coli

759

at 37 for 2 weeks. It was then ground to a powder, and stored at -20. DNA-cellulose columns were stored in 2 M NaCl after each usage and were reused repeatedly. Valyl-Sepharose (31) was prepared by the cyanogen bromidecoupling procedure (32); the valine concentration in the resin was 22 rnM. Column BuffersThe following buffers were used: Buffer I, 20% v/v glycerol, 50 mM imidazole.Cl (pH adjusted at 50 mM, room temperature), 1 mM EDTA, 1 mM dithiothreitol; Buffer A, 20% glycerol, 50 mM Tris.Cl (diluted from 2 M Tris.Cl (pH 7.5), room temperature), 1 mM EDTA, 1 mM dithiothreitol; Buffer V, 1.24 M ammonium sulfate, 50 mM imidazole.Cl (pH 6.5), 1 mM EDTA, 1 mM dithiothreitol. Assays-RNA synthesis was assayed in a 25-~1 volume containing: 5 ~1 of Buffer P (pH 7.5) (50% glycerol, 250 mM Tris.Cl, 25 mM dithiothreitol, 0.5 mg/ml of albumin); 230 pmol of G4 DNA (nucleotide residues); 0.4 pg of DBP; 4 mM MgCl,; 20 PM each of ATP, CTP, GTP, and UTP, all labeled with 3H or 32P (3,000 to 20,000 cpm/ pmol); 4 pg/ml of rifampicin (if specified), and approximately 25 DNA replication units of primase. One unit of RNA synthesis is defined as 1 pmol of nucleotide incorporated/min at 30. The reaction was at 30 and was terminated by spotting the reaction mixture on a 1.5~cm, DE81 filter paper disc. The papers were washed four times in 0.3 M ammonium formate (pH 7.81, 10 mM sodium pyrophosphate for 5 min each time and rinsed between washes with water. They were then washed twice with 95% ethanol (no water rinse) and once with diethyl ether, dried, and counted. Recovery of a tetranucleotide with a 5-triphosphate end was 50%; longer oligonucleotides were recovered in greater or even full yield. DNA synthesis assays were as indicated for RNA synthesis, but with the addition of 100 PM ATP, 50 PM each of dATP, dCTP, and dGTP, 18 PM [3HldTTP (250 to 1000 cpmlpmolf, and approximately 25 units of holoenzyme. Incubation was at 30 and terminated either as described for RNA synthesis, or by addition of 50 ~1 of 0.1 M sodium pyrophosphate and 1 ml of 10% trichloroacetic acid and filtered (8). A unit is defined as 1 pmol of nucleotide incorporated into DNA per min at 30. Complementation assays differed from DNA synthesis assays in that 1 ~1 of PC3 Fraction II (15 pg of protein containing 14 units of endogenous DNA synthetic activity dependent upon addition of dnaG protein) was used in place of DBP and holoenzyme; DNA replication was dependent upon addition of dnoG protein, and was not stimulated by DBP, holoenzyme, or both. In assays of all primase activities, the value obtained without primase was subtracted from the experimental value; this value never exceeded 2 residues per input DNA circle for RNA synthesis, or 5 pmol of nucleotide for DNA synthesis. DNA polymerase I was assayed with activated calf thymus DNA (33). Other Methods - Conductivities were read in Radiometer conductivity meter at 0 after a loo-fold dilution of the sample in water. SDS-polyacrylamide gel electrophoresis was performed using the Tris/glycine system (34). Glycerol gradient centrifugation was performed as described (35). Protein determinations were as described

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(36).
RESULTS

of Primase - Activity was extracted (Table I) by lysozyme lysis in the presence of a nonionic detergent, Brij 58; spermidine was added to facilitate removal of cell debris and DNA by low speed centrifugation. The clear lysate contained 3,000 to 6,000 units/g of cell paste. Addition of solid ammonium sulfate to 40% saturation precipitated about 15% of the protein including several replication proteins (dnaC
Purification protein, protein n, and rep), as well as primase. These proteins

were resolved on Bio-I&x-70, a polyacrylic acid resin. Primase was eluted first near 0.24 M NaCl (pH 6.81. At lower pH, the proteins required higher salt concentration for elution. Successive fractionations on DNA-cellulose and valyl-Sepharose yielded a primase fraction 50 to 80% pure. Final resolution was achieved by chromatography on DEAE-cellulose. This fraction was stable on ice for up to 2 months and to freezing and storage in liquid nitrogen for at least 1 year. Replication

760

dnaG Gene Product of E. coli

TABLE

Purification
All operations were carried out near 4. Fraction I (15,720 ml) was prepared from 3.2 kg of HMS83 cell paste lysed as follows: frozen cell paste (see Materials and Methods) was thawed and diluted with 0.5 volume of 50 mM Tris.Cl (pH 7.5), 10% sucrose to A 595 = 0.30 to 0.35 cl:400 dilution). 4 M KC1 was added to 0.15 M, 0.4 M dithiothreitol to 1 mM, 1 M spermidinecl to 20 mM, 0.5 M EDTA to 20 mM, and 10% Brij 58 to 0.1%. The pH was adjusted to 8.5 with solid Tris base. Lysozyme was then added to 0.2 mg/ml. After 20 min at 2 the lysate was centrifuged at 18,000 rpm at 0 for 45 min in the Sorvall SS34 rotor. The supernatant is Fraction I. Ammonium sulfate (0.24 g/ml) was added to Fraction I. The precipitate was backwashed with a volume of Buffer A (containing 0.1 M NaCl and 0.24 g/ml of ammonium sulfate1 equivalent to one-tenth of Fraction I. The precipitate was resuspended in a minimal volume of Buffer I (pH 6.81, 200 mM KCl, and frozen in liquid nitrogen (Fraction II). Fraction II (350 ml) was dialyzed for 2 h against Buffer I (pH 6.8), 200 mM KCl, then diluted 20-fold into 20% glycerol, 1 mru dithiothreitol, 1 mM EDTA (to a conductivity of 50 pmho), and applied to a Bio-Rex-70 column (1.7 liters, 15 x 9 cm) at 30 mg of protein/ml of resin. The column was successively washed with 2.5 column volumes of Buffer I containing first 240 mM KCl, then 400 mM KCl, finally 700 mM KCl. Three fractions were collected for each wash and to each was added 0.35 g/ml of ammonium sulfate. Pellets were resuspended in minimal volumes of Buffer I (pH 6.8) and frozen in liquid nitrogen. Fractions with primase activity were thawed, pooled, and dialyzed 16 h against Buffer I (pH 6.5) (Fraction 1111. Fraction Protein Activity
w units X 10m5

I of primuse Dialyzed Fraction III (15 ml, representing one-half the total) was clarified (15,000 rpm, 10 min in a Sorvall SS34 rotor, 0) and diluted to conductivity <43 pmho with Buffer I (pH 6.5) and applied to an 80-ml (3.8 x 7.0 cm) DNA-cellulose column (15 mg of protein/ml of resin) equilibrated in Buffer I (pH 6.51. The column was washed with 0.5 column volume of Buffer I; 2 column volumes of Buffer I, 100 mM NaCl (conductivity 53 pmhol and 2 column volumes of Buffer I, 200 mM NaCl. Primase (31 ml), found in the Buffer I, 200 rnM wash, was precipitated with 0.35 g/ml of ammonium sulfate. The pellet was resuspended in 15 ml of Buffer V. Insoluble material was removed by centrifugation (see above); the supematant was retained (Fraction IVa). Fraction IVa (30 mg of protein) was applied to a valyl-Sepharose column (6 ml, 1.5 x 3.5 cm) equilibrated in Buffer V. The column was washed at flow rate of 6 column volumes/ h with 15 column volumes of Buffer V; activity was eluted with a lo-column-volume gradient from Buffer V to Buffer I (pH 6.5). Primase activity (Fraction V, 14 ml) was dialyzed (less than 16 hl against Buffer A. The sample was diluted (conductivity <35 pmho) with Buffer A and applied to a 2-ml DEAE-cellulose (DE52) column equilibrated in Buffer A. The column was washed with 2 column volumes of Buffer A and the activity was eluted with a lo-columnvolume gradient from 0 to 200 mM NaCl in Buffer A. The sample was stored in liquid nitrogen; it also was stable on ice (Fraction VI). DNA synthesis assays were for 10 min at 30 (see Materials and Methods). Specific activity
(unitimgi x 10m3

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Yield
5%

PllIi&XtiOIl -fold

I. II. III. IV. IVa. V. VI.

Extract Ammonium sulfate Bio-Rex-70 DNA-cellulose Ammonium sulfate Valyl-Sepharose DEAE-cellulose

283,000 46,900 3,960 124 60 8.7 0.64

(11,500)0 11,500 8,800b 6,600 6,200 3,700 7ooc

(0.0411 0.25 2.2 53 103 430 1,090

(100) 100 77 57 54 32 6 sulfate fraction

(1.0) 6.0 54 1,290 2,500 10,500 26,600 is cited here as a

a Inasmuch as assays could not be performed on Fraction I, the activity recovered in the ammonium minimal value. b From this stage on, only half of Fraction III was used and values are corrected accordingly. c Activity in eluted Fractions 11 to 17 were determined for each fraction (see Fig. 1).

of G4 DNA in vitro is dependent upon addition of purified primase (Table II). Molecular Weight of PrimaseAn SDS-polyacrylamide gel electrophoretic profile of the most purified fraction showed it to be 80 to 90% of the Coomassie blue-staining material with a molecular weight of 60,000 (Fig. 1). In a glycerol gradient (Fig. 21, the primase activities co-sedimented with hemoglobin, indicating a native molecular weight near the 60,000 value observed on electrophoresis under denaturing conditions (Fig. 1). Thus, the isolated primase (9, 38) is a single subunit enzyme. Co-purification of Three Primase Activities: Priming, Complementation, and RNA Synthesis- Ratios of these three activities remained fairly constant throughout the last four steps of puriiication (Table III). Assay of RNA synthesis activity in Fraction II was complicated by the presence of another rifampicin-resistant RNA synthetic activity, possibly that described by Ohasa and Tsugita (39). Co-migration of the three primase activities was observed on valyl-Sepharose and DEAE-cellulose columns (Fig. 3). Thus these several activities appear to reside in the same protein. Factors Influencing RNA Polymerase Action of Primase: Substrate Concentration, Mg+, Temperature, pH, and SaltPrimase, in the presence of DBP, Mg2+, and the four rNTPs,

TABLE

II

of G4 SS to RF DNA synthesis assays were performed in 25 ~1 containing (when complete) 230 pmol of G4 DNA; 0.4 pg of DBP; 4 mM MgCl,; 10 PM each of CTP, GTP, UTP; 100 PM ATP, 50 PM dNTPs (138 cpmlpmol of nucleotide), 0.1 pg of rifampicin, 0.25 ~1 of primase (DEAE, Fraction 13, see Fig. 1) and 40 units of holoensyme (Fraction IV). Incubation was for 6 min at 30. DNA synthesis Component omitted pl?d 78 None G4 DNA 0.3 DBP 0.6 Mg2+ 0.2 Rifampicin 65 23 CTP, GTP, UTP ATP 3.2 Primase 1.6 Holoenzyme 0.2

Requirements for conversion

synthesizes a unique RNA transcript (26 to 29 nucleotides long) at the origin of replication of G4 DNA (17). The apparent K, for the NTPs is in the neighborhood of 10 PM (Fig. 4). RNA synthesis occurs within a rather narrow range of Mg2+ concentration (2 to 8 mru) (Fig. 5); this amount of MgZ+

dnaG Gene Product

DEAE CELLULOSE FRACTION

of E. coli
TABLE III

761

Ratios of primate activities during purification

, 17 Purification of primase from 2.7 kg of cells, following the procedure described in Table I, yielded fractions with activities (106 units) and specific activities (10s tmits/mg) as follows: II, 16.6, 0.24; III, 15.0, 6.8; IV, 7.6, 108; IVa, 4.9, 153; V, 3.3, 362; VI, 0.92, 765. Overall yield was 6%. Activities of the primase fractions in the complementation assay (103 units/ml) were, III, 365; IV, 26; IVa, 14.5; v, 7.7; VI, 15. RNA synthesis Fraction purification Priming activity
475

, 11

, 12

, 13

, 14

, 15

, 16

Complementation activity
228 302 315

46,300

35,800

;, :. ,.

.Y

.,

<

..

,) : I ,\ : ,.: (, . : :~ :,\,:Q ,: ._ ., ,

:. . ., ,.. : i_,Y ~1: :..

III. IV. IVa. V. VI.

Bio-Rex-70 DNA-cellulose Ammonium sulfate Valyl-Sepharose DEAE-cellulose

140
2,200 3,000 7,200

1,279
460 731 590

189
245

15,300 is defined as 1 pmol

o One unit of activity sixed/min at 30.

of nucleotide

synthe-

2.4

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1.6 m :

FIG. 1. SDS-polyacrylamide gel electrophoresis of puritied primase. DEAE-cellulose column peak fractions (40 ~1, see Table I) were applied to a 10% acrylamide, 0.25% bisacrylamide slab gel. Electrophoresis was at 25 mA until the bromphenol blue dye marker reached the bottom of the gel (4 h). The protein concentrations (micrograms per ml) of Fractions 11 to 17 were, respectively: 17, 63, 108, 120, 100, 80, and 57. Their primase activities (10s units/ml) were, respectively: 40,87,135,140,110,53, and 35. Protein standards were: 10 fig of albumin (M, = 68,0001,12 ~lg of+X proteins (gene F, 46,300, gene H, 35,800; gene G, 19,000 (37)).
3.0 . 2.5 ,I SEDIMENTATION I I .Suul

0.8

0 (81 DEAECELLULOSE 100 so COMPLEMENTATION RNA SYNTHESIS so 40 0.5 0.4 1 o.3 2 0.2

poll

Hb

cytC +

-E
E cl ti E z f w 9 LT 0.5 2.0 RNA SYNTHESIS

26

0.1

1.5 COMPLEMENTATION 1.0

00

12 FRACTION

16 NUMBER

20

24

26

OO

16 FRACTION

24 NUMBER

32

FIG. 2. Glycerol gradient sedimentation litem (550 ng) of Fraction VI primase (see a 20 to 40% glycerol gradient containing 6.81, 10 mM dithiothreitol, 1 mu EDTA, sulfate. Sedimentation, in an SW 60 rotor h is shown from right to left. Standards gradients were DNA polymerase I @oZ I), 64,ooO, and cytochrome c (cyt C), 12,800.

of primase. Fifty microTable III) were applied to 60 mM imidaxole . Cl (pH and 0.1 M ammonium at 58,000 rpm at 4 for 19 sedimented in parallel 109,ooO; hemoglobin (ZZbf,

FIG. 3. Co-migration of primase activities on valyl-Sepharose and DEAE-cellulose. A, valyl-Sepharose: Fraction IVa primase (690,000 units, 4.5 mg of protein) (Table III), were applied to a l-ml valyl-Sepharose column. The column was washed with 15 ml of Buffer V, followed by a 20-ml gradient from Buifer V to Buffer I (10 ml each). Volume of Fractions 1 to 5 was 5 ml; of Fractions 6 to 20, 1.7 ml. B, DEAE-cellulose: Fractions 10 and 11 from the valylSepharose column (470,000 units, 1.3 mg) were dialyzed against Buffer A, and applied to a DEAE-cellulose column (1 ml). The column was washed with 1 ml of Buffer A, and developed with a loml gradient from 0 to 150 nnu NaCl in Buffer A.

is far in excess of that needed to bind the substrates (80 PM) or DNA (10 PM). At 30 and 37, both the rate and extent of RNA synthesis were about the same, but at 20 the rate was reduced to 50%, and at 45, only 20% of the full extent of RNA synthesis was

observed (probably due to inactivation of prima&. No RNA synthesis was observed at O, even though binding of primase to DBP-coated G-4 DNA does occt~.~ RNA synthesis was observed between pH values of 6.5 and 9 with the fastest rate occurring near pH 7.5. The rate at pH 6.5 was 45% that at pH 7.5 in imidazole buffer; at pH 9 (glycine buffer) the rate was 26% that at pH 7.5. RNA synthetic activity of primase was affected by ionic
s D. L. Bates and A. Komberg, unpublished results.

dnaG

Gene Product

of E. coli

strength. Inhibition of 50% occurred when 50 mM NaCl was added to the buffers in the assay components, estimated to be near 20 mM in NaCl in conductivity value. RNA synthesis by primase is sensitive to phosphate (40% inhibition at 40 PM). Stoichiometry of Primase and TemplateThe rate and extent of RNA synthesis on G4 DNA was influenced by the amount of primase present (Fig. 6). It appears that at least one molecule of primase is required per circle to attain the synthesis of a complete primer. That no more than one copy of primer is synthesized per circle is suggested by the limit of incorporation of rNTPs at 30 residues per circle. Additional primase increases the rate at which RNA priming of all the circles is completed. When either primase or DNA was in excess (1.5, or 0.2 primase molecules/circle, respectively) only a full length RNA product was observed (by polyacrylamide gel electrophoresis (17)) throughout the course of the reaction; products of intermediate size were not found. Thus, the ratelimiting step appears to be initiation of RNA synthesis rather than elongation of the RNA chain. Inhibitors of RNA Synthesis Activity-Primase was not inhibited by several of the well known inhibitors which bind Y P g P t I IA) 24 I I I

E. coli RNA polymer-ax (Table IV) further indicating that contamination by the latter enzyme is not responsible for the observed RNA synthesis. Actinomycin D, which binds G-C pairs in DNA (40, 411, inhibits both enzymes. E. coli DNAbinding protein, in an amount sufficient to cover the G4 DNA, was essential for primase action, but almost completely inhibited transcription by RNA polymerase. Influence of Template on RNA Polymerase Action-l%?mase in the presence of DBP, was highly specific for G4 DNA (Table V). DNA of the closely related phage ST-1 was the only other template which showed significant activity; polyacrylamide gel electrophoretic analysis indicated that the RNA product was the same size as that for G4 DNA (data not shown). Lack of transcriptional activity by primase in the case of Ml3 DNA and 4X DNA is probably due to the
I
40 d-G

I
PROTEIN/G4

I
CIRCLE:

32

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24

16 20 -

0 0 10

-.----.-m--L 20

30 TIME (MINI

40

50

60

ATP

CTP, GTP, UTP

FIG. 4. Influence of ribonucleoside triphosphate concentrations on primase activity. RNA synthesis assays were performed as described under Materials and Methods, except that in A, ATP was added to the indicated concentrations in the presence of 20 pM each of CTP, GTP, and UTP (all 3H-labeled, 3000 cpm/pmol nucleotide), and in B, the concentration of the 3H-labeled CTP, GTP, UTP mixture was varied, and ATP was 20 PM. Primase was DEAEFraction 13 (Fig. l), 1 ~1, 108 ng. Incubations were for 3.5 min at 30.
I I I I I

FIG. 6. RNA synthesis at different levels of primase. RNA synthesis (see Materials and Methods) in 25 ~1 containing 2300 pmol of G4 DNA, 8.5 pg of DBP, and primasa (DEAE-Fraction 15, 100 pglml) (Fig. 1) at levels of 0, 0.25, 0.5, 1, 2.5, and 5 ~1. Incubation was at 30. At indicated times, 2 ~1 (containing 184 pmol of DNA) were removed. The number of primase molecules per circle is based on a purity of 25%. The preparation, originally 80% pure, had decayed to 32% of its original activity (110,000 to 35,000 units/ml). One microliter of Fraction 15 (containing an estimated 25 ng or 2.5 x 10 molecules of active primase) is sufficient to prime 2300 pmol of DNA (2.6 x 10 single-stranded circles) at a level of one monomer per circle.
TABLE IV Effect of RNA polymerase inhibitors on primase action RNA synthesis by primase (see Materials and Methods) was in 25 ~1 containing 460 pmol of G4 DNA, 0.85 pg of DBP, and 1 ~1 of primase (DEAE-Fraction 15, see Fig. 1). RNA synthesis by RNA polymerase was in 25 ~1 containing 230 pmol of G4 DNA and 1 ~1 of RNA polymerase holoenzyme (Fraction PC 10, 1 mg/ml, 22,200 units/mg). Inhibitors were added before enzyme as indicated. Incubation was for 20 min at 30. Control values (NMP/circle in absence of inhibitor) were: 26.3 for nrimass and 555 for RNA aolvmerase. Inhibition of RNA synthesis Inhibitor Concentration RNA poIymerPrimase as! % pglml Rifampicin 40 99 0 Heparin 40 99 29 Streptolydigin 40 72 0 Streptovaricin C 40 96 10 Actinomycin D 40 94 99 DBP 16 99 0

24 -

16 -

40 0 A 2 I 4 I 6 Mg2+, mM I 8 I 10 , 12

FIG. Source 30.

5. Effect of MgZ+ on RNA polymerase of primase was as in Fig. 4. Incubations

action of primase. were for 10 min at

dnaG Gene Product

TABLE V of templates on primuse and RNA polymerase actions in presence of DNA-binding protein RNA synthesis (see Materials and Methods) was in 50 ~1 containing DNA and DBP as indicated, and 4 ~1 of primase (DEAEFraction 17 (Fig. 1)) or 1 ~1 of RNA polymerase; (see Table IV). Incubation was for 30 min at 30. Reactions with G4 SS DNA, Ml3

of E. coli

763

Influence

adequate for physical and functional analysis an important objective. Using the dependence of G4 DNA replication upon primer formation as an assay for primase, we have purified the enzyme 26,000-fold to near-homogeneity. From the specific
activity of the purified enzyme and the number of units in the

SS DNA, $X SS DNA, pf-1 SS DNA, poly(dA),

and poly(dC),

contained 920 pmol of nucleotide residues and 3.2 pg of DBP. Reactions with G4 RF I DNA, I$X RF I DNA, T4 DNA, ColEl DNA, A DNA, and Escherichia coli DNA, contained 1840 pmol of nucleotide residues and 3.2 pg of DBP. The reaction with the trp attenuator DNA contained 185 pmol of nucleotide and 3.2 pg of DBP. Values of NMP/molecule template were based on template size of 5.5 kb (kb = 1000 residues for SS DNA or 1000 base pairs for duplex DNA) for C4, M13, $X, and pf-1 DNAs (421, 6.5 kb for ColEl DNA (271, 46 kb for A DNA (43), 144 kb for T4 DNA (44), and 0.56 kb for trp attenuator DNA.4 For E. coli, poly(dA) and poly(dC) DNA templates, the number of residues synthesized per 5.5 kb of input template is given. These numbers represent 47%, 0.178, and 1.3% transcription of the template, respectively. A value for NMP/molecule template of zero indicates incorporation of radioactivity less than 30% above the background value obtained in absence of enzyme. RNA synthesis
DNA

most active cell extract, there appear to be only 50 to 100 molecules of primase per cel1.j In confirmation of an earlier report (9) the native protein is a polypeptide of 60,000 daltons. It is unlike the large, multisubunit E. coli RNA polymerase
not only in size and organization but also in its insensitivity

to rifampicin and other inhibitors that bind and inactivate the p subunit of the large polymerase. The RNA-synthesizing activity of primase coincides
throughout tion activity its priming the purification for an extract capacity for procedure with its complementaof a dnaG mutant, as well as with DNA replication. This is a strong

template

G4 ss ST-l SS Ml3 SS c#ax ss Pf-1 ss

Frimase NMPlmokcule 21.8 4.0 0 0 0

RNA

polymerase

oftemplate 4.0 417 353 40 100

G4RFI $XRFI
ColEl A T4 E. coli trp attenuator

0 0
1.4 0 0 0 0.8

11,000 11,200
4,020 10,700 33,400 2,600 44

Poly(dA)
Poly(dC)

0
0

11
79

inability of the protein to bind to these DNAs3 RNA transcribed by RNA polymerase on trp attenuator DNA (29) contains a hairpin region with seven G-C base pairs4 resembling the RNA primer synthesized from G4 DNA (17); however primase did not transcribe trp attenuator DNA to a significant extent under the conditions tested. The duplex DNA templates tested were all inert for primase action (~0.03% of the template transcribed) with the possible exception of ColEl DNA, the small amount of RNA synthesized in this case has
not been further characterized.
DISCUSSION

indication that all these activities reside in the primase molecule and, along with other distinctions, eliminates any possibility that the potent, rifampicin-sensitive RNA polymerase contaminates our preparations. Perhaps the most remarkable feature of primase is the great transcriptional specificity it displays for certain templates. Among single-stranded DNA circles coated with DNAbinding protein, only phage G4 (or ST-l) DNA can serve and it can sustain the synthesis of a unique 29-residue, transcript (17). The DNA-binding protein may promote formation of a particular secondary structure in the DNA or it may prevent nonspecific, unproductive binding of primase to the DNA. For the 4X DNA circle to be utilized by primase, masking by DBP does not suffice. Instead, a replication intermediate containing one dnaB protein molecule in a complex with the DNA (11) (produced through the action of dnaC protein, proteins i and n, and ATP) is essential (12). Both the G4 and 4X systems afford attractive opportunities for understanding the actions of primase, the dnaB protein and other replication proteins responsible for initiation events in replication of the E. coli chromosome. The succeeding paper (17) describes the structure of the 29residue RNA transcript synthesized by primase on G4 DNA when deoxynucleoside triphosphates are excluded. When the ribo- and deoxynucleoside triphosphates are all present, the enzyme synthesizes a shorter, hybrid transcript, which is even more abbreviated when priming DNA synthesis (18).
Acknowuledgments - We would like to acknowledge Dr. Joel Weiner and John Scott for developing the early stages of the primase purification procedure, and Janey Beuchel for preparation of G4 phage. Note Added in Proof-J. Sims, D. Hourcade, and D. Dressler (personal communication) have found a DNA sequence complementary to the primer RNA at a region identified, through in viuo studies, as the origin of G4 negative strand synthesis. REFERENCES 1. Schekman, R., Weiner, A., and Kornberg, A. (1974) Science

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The product of the dnaG gene is needed continuously during replication of the E. coli chromosome (45). Studies with lysates of temperature-sensitive dnaG mutants suggested a function in the initiation of nascent (Okazaki) fragments (46). Previous enzymatic studies of the dnaG protein showed it to be an RNA polymerase whose product served as a primer for DNA replication (8). The enzyme has been renamed primase because we recognize its role in forming primers for DNA replication at very special loci. The importance of this protein and its remarkable properties made its isolation in quantities
4 F. Lee and C. Yanofsky, personal communication.

5 The calculated number of molecules/cell is based upon assumptions of 5 X 10 cells/g; a specific activity of pure primase of 1.3 X 106 units/me: a molecular weight of nrimase of 60,000 almol; and that the activity in FrII repres&ts the total activity in the cell, i.e. 3600 units/g for the preparation described in Table I and 6100 units/ g for that described in Table III.

764

dnaG

Gene Product

of E. coli

186, 987-993 2. Wickner, S., and Hunvitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4120-4124 3. McHenry, C., and Komberg, A. (1977) J. Biol. Chem. 252,64786484 4. Wickner, S. (1976)Proc. Natl. Acad. Sci. U. S. A. 73,3X1-3515 5. Westergaard, O., Bmtlag, D., and Komberg, A. (1973) J. Biol. Chem. 248, 1361-1364 6. Weiner, J. H., Bertsch, L. L., and Komberg, A. (1975) J. Biol. Chem. 250, 1972-1980 7. Geider, K., and Komberg, A. (1974) J. Biol. Chem. 249, 39994005 8. Bouche, J.-P., Zechel, K., and Komberg, A. (1975) J. Biol. Chem. 250, 5995-6001 9. Zechel, K., Bouche, J.-P., and Komberg, A. (1975) J. Biol. Chem. 250, 4684-4689 10. McMacken. R.. Bouche. J.-P., Rowen, S. L., Weiner, J. H., Ueda, K:, Thelander; L., h&Henry, C., and Kornberg, A. (1977) in Nucleic Acid-Protein Recognition (Vogel, H. J., ed) 15-29 11. McMacken, R., Ueda, K., and Komberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4190-4194 12. Weiner, J. H., McMacken, R., and Komberg, A. (19761 Proc. Natl. Acad. Sci. U. S. A. 73, 752-756 13. Schekman, R., Weiner, J. H., Weiner, A., and Komberg, A. (1975) J. Biol. Chem. 250, 5859-5865 14. Wickner, S. H., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787 15. Wrieht. M.. Wickner. S. H.. and Hurwitz. J. (1973) Proc. Natl. Acad: Sci. U. S. A. 70, 3120-3124 16. Wickner, S. H., Berkoner, I., Wright, M., and Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2369-2373 17 Bouche, J.-P., Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 765-769 18 Rowen, L., and Komberg, A. (1978) J. Biol. Chem. 263, 770774 19. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122 20. Godson, N. G. (1974) Virol0g.y 58, 272-289 21. Jovin, T. M., Englund, P. I?, and Bertsch, L. L. (1969) J. Biol. Chem. 244, 2996-3008 22. Davis, R. W., Simon, M., and Davidson, N. D. (1972) Methods Enzymol. 21D, 413-428 23. Iwaya, M., Eisenberg, S., Bartok, K., and Denhardt, D. T.

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