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THE J OURNAL OF Bmm~~c a ~ CHEM8TnYVol 253, No. 3, Issue of February 10, pp. 758-764, 1978

Prmted I I I U S.A.

Primase, the dnaG Protein of Escherichia coZiAN ENZYME WHICH STARTS DNA CHAINS*

(Received for publication, August 3, 1977)

LEE ROWEN AND ARTHUR KORNBERGFrom the Department of Biochemistr.y , Stanford University School of Medicine, Stanford, California94305

Conversion of the viral DNA of phage G4 to the duplexform provided an opportunity to isolate and determine thefunct ion of the dnaG protein, the product of a gene knownto be essential for replication of the Escherichia coli chro-mosome. This stage of G4 DNA replication requires actionof three proteins: the E. coli DNA-binding protein, thednaG protein, and the DNA polymerase III holoenzyme.The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains asingle polypeptide of 60,000 daltons. It has been assayed forits act ivi ty on G4 DNA in three ways: (a) RNA synthesis,(b) complementation for replication of an extract of atemperature-sensitive dnaG mutant, and Cc) priming of DNAreplication by DNA polymerase III holoenzyme. The dnaGprotein is highly specific for G4 DNA and synthesizes aunique 29-residue RNA primer to be described in the suc-ceeding paper. Other single-stranded and duplex DNA tem-plates are inactive. RNA primer synthesis by the dnaGprotein has an apparent K, for ribonucleoside triphosphatesnear 10 PM, and a narrow optimum for Mg2+.The sharp specific ity of the dnaG protein in choice oftemplate and the utilization of either deoxyribonucleotidesor ribonucleotides to produce a hybrid piece only a fewresidues long (as described in a succeeding paper) suggeststhat the dnaG protein previously named RNA polymerasebe renamed primase.

The DNA chromosom es of the sma ll bacteriophages M13,G4, and +X174 have provided systems in which enzymaticmechanisms of DNA replication in Escherichia coli can beanalyzed (1, 2). Conversion of the single-stranded circle to theduplex replication form can be divided into three major stages:initiation, elongation, and termination. In all these phageDNA systems, chain elongation occurs by action of DNApolymerase III holoenzyme (3, 4). Given a primer, the holoen-zyme can copy the circle to give the duplex replicative form.

* This work was supported in part by grants from the NationalInstitutes of Health and the National Scien ce Foundation. This isthe third paper in a series dealing with the replication of phage G4DNA. The previous paper is Ref. 8. The costs of nublication of thisarticle were defrayed iA part by the payment of page charges. Thisarticle must therefore be hereby marked uduertisement in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: holoenzyme, DNA polymerase IIIholoenzyme; albumin, bovine serum albumin; DBP, DNA-binding

Gaps in the replicative form can be filled by DNA polymeraseI and sealed by DNA ligase to yield the closed circular duplex,RF I (5).The three phage DNA templates differ primarily in themode of initiation of DNA replication. In each case DNA-binding protein (DBP) coats most of the DNA circle (6).Although this s&ices for priming of Ml3 DNA by RNApolymerase (7) and of G4 DNA by dnaG protein (8, 91, $XDNA, by contrast, must f irs t be acted upon by additionalproteins before priming by the dnaG protein can occur (2, lo-12). A nucleoprotein intermediate is formed in the presence ofATP and Mg2+ in which proteins n (13) and i (13) act cata lyti -cal ly to put the dnaB protein (11, 13-15) and possibly thednaC protein (13, 16) onto the DNA (10-12).Priming by dnuG protein on G4 DNA is by synthesis of ashort RNA transcribed from a region about 5% of the genomelength from the single cleavage site of EcoRl restrictionendonuclease, the site determined as the origin of replicationof G4 DNA (9).In this paper, a new purification procedure for the dnuGprotein is described and evidence is given that the RNApolymerase activity co-purifies with the complementation ac-tivi ty for an extract of a dnuG mutant and with the primingact ivi ty for G4 DNA replication. All these activities areremarkably specific for G4 viral DNA coated by DNA-bindingprotein. In succeeding papers, the structure of the uniqueRNA transcript synthesized by the dnaG protein at the originof G4 DNA replication and the utilization of deoxyribonucleo-side triphosphates as well as ribonucleoside triphosphates bythis enzyme are presented (17, 18). The action of the dnaGprotein in the priming of +X DNA is discussed in a separatepaper (11).Based on its role in priming DNA replication rather thanin transcribing DNA regions for synthesis of messenger orother defined RNAs, and its capacity to use deoxynucleotidesor ribonucleotides, we propose that the dnuG protein bedesignated primase.

MATERIALS AND METHODSBacterial and Phage Strains - Thes e were generously provided as

follows: Esche richia coli HMS83 (thy, lys, lac, rhu, &A, polB) byprotein; dNTPs , deoxyribonucleoside triphosphates; 4X, +X174;NMP, nucleo side monopho sphate; RF, duplex circular replicativeform of a phage DNA; RF I, covalently closed RF; rNTPs, ribonucleo-side triphosphates; SDS, sodium do decyl su lfate; SS, single-stranded.

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dn.aG Gene Product of E. coli 759Dr. R. McMacken of Johns Hopkins University; E. coli PC3 (dnaG3,leu, thy, stp) by Dr. P. Carl o f 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 highaeration in a New Brunsw ick loo-liter Fermacell to A,, = 8 to 10 inmedium containing per liter: 10 g of K,HPO,; 1.85 g of KH,PO,; 10 gof 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 additionof NaOH. Cells were harvested in a Sharples continuo us flowcentrifuge. The cel l paste was suspended in cold 50 mM Tris.Cl(pH 7.51, 10% sucrose to A,,, = 0.5 at a 1:400 dilution. The cellsuspe nsion, 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 proceduredeveloped by Dr. G. N. Godson.*Chem icals- Sources were as follows: rifamp icin from Calbiochem ;heparin from Sigma; streptolydigin and streptovaricin C from Up-john; Actinomyc in D from Merck, Sharp and Dohme; ammon iumsulfate and sucrose from Schwarz/Mann, dithiothreitol from Bio-Rad; imidazole from Baker or Eastman; Brij 58 from Pierce Co.;spermidine from Calbiochem ; unlabeled rNTPs and dNTP s from P-L Bioch emica ls. The dNTP s were further purified by chromatogra-phy on DEAE-Sephadex A-25 column s using a gradient of ammo-nium bicarbonate from 0.03 to 0.5 M. 3H- and 32P-labeled nucleo sidetriphosphates were from New England Nuclear or Amersham/Searle.Enzymes - Bovine serum albu min was from Armour and lysozymefrom 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 endogeno us dnaGactivity. Holoenzyme was purified (3) to give Fraction IV (8 x lo5units/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 asdescribed (21).

DNA Temp lates - DNA from phage G4 was prepared by sedimen-tation of 5 x lOI plaque-forming units through a 5 to 20% sucrosegradient in 50 mM Tris.Cl (pH 7.51, 1 mM EDTA, 1 M NaCl for 3.5 hat 26,000 rpm, at 4 in the SW 27 rotor. The peak of infectivity waspooled, dialyzed against 50 mM sodium borate, adjusted to 1% SDS,and treated with 1 volume of distilled phenol equilibrated in 50 mMsodium borate. The extraction mixture was heated at 65 for 10 min;the aqueous p hase was removed after centrifugation. The phenolphase was re-extracted with 0.5 volume of 50 mM sodium borate.Aqueou s phase s were combine d, and the DNA was precipitated byaddition of 0.1 volume of 3 M sodium acetate (pH 5.51, and 2volumes of isopropyl alcoho l, 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 resuspen ded in200 ~1 of 10 mM Tris.Cl (pH 7.5), 1 mM EDTA, and dialyzed againstthe same buffer for 2 days at 4. As seen in the electron microsco pewith formamide spreading (221, 80% of the DNA mole cules werecircular.Sources of DNA were as follows: phage Ml3 (71, phage $X (23);phage Pf-1 a gift of Dr. D. Ray (University of California, LosAngeles), phage ST-1 (241, poly(dA) and poly(dC), Calbiochem ; +XRF I and G4 RF I (25); phage A (26) and C olEl plasm id (27)respective gifts from T. White and G. Guild of this department; E.coli (28); trp attenuator (291, isolated from an HpaII restrictionendon uclease digest o f480pt190 was a gift of Frank Lee (Departmentof Biolog ical Scien ces, 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 waterwere treated with 2.3 liters of 2 M HCl (final pH = 1). The resin waswashed with water, by decantation, until the pH was 5. The resinwas made 20% in glycerol (v/v), and kept for 1 h. The supernatantwas replaced by 6 liters of 0.5 M imidazole base, 20% glycerol, andkept for 1 h. Finally, the resin was washed with and stored inBuffer I (pH 6.8) (see below). For reuse, the resin was treated with0.5 M NaOH, washed, and then equilibrated as described above.DNA-ce llulose (30) was prepared with calf thymus DNA (Calbi-ochem grade A (2618)). Upon mixing the DNA s olution (2 mg/ml)with the cellulos e, a thick paste was obtained (1 g of cellulose /3 mlof DNA). This paste w as spread on side s of beakers and left to dry

2 G. N. Godson, personal comm unication.

at 37 for 2 weeks. It was then ground to a powder, and stored at-20. DNA-cellulose colum ns were stored in 2 M NaCl after eachusage and were reused repeatedly.Valyl-Sepharose (31) was prepared by the cyanogen bromide-coup ling procedure (32); the valine concen tration in the resin was22 rnM.Column Buffers- The following buffers were used: Buffer I, 20%v/v glycerol, 50 mM imidazole.Cl (pH adjusted at 50 mM, roomtemperature), 1 mM EDTA, 1 mM dithiothreitol; Buffer A, 20%glycerol, 50 mM Tris.Cl (diluted from 2 M Tris.Cl (pH 7.5), roomtemperature), 1 mM EDTA , 1 mM dithiothreitol; Buffer V, 1.24 Mammo nium sulfate, 50 mM imidazole.Cl (pH 6.5), 1 mM EDTA, 1mM dithiothreitol.Assays-RNA synthes is was assayed in a 25-~1 volume contain-ing: 5 ~1 of Buffer P (pH 7.5) (50% glycerol, 250 mM Tris.Cl, 25 mMdithiothreitol, 0.5 mg/ml of albumin); 230 pmol of G4 DNA (nucleo-tide 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 25DNA replication units of primase. One unit of RNA synthesis isdefined as 1 pmol of nucleotide incorporated/min at 30. The reactionwas at 30 and was terminated by spotting the reaction mixture ona 1.5~cm, D E81 filter paper d isc. The papers were washed four timesin 0.3 M ammon ium formate (pH 7.81, 10 mM sodium pyrophosphatefor 5 min each time and rinsed between washes with water. Theywere then washed twice with 95% ethanol (no water rinse) and oncewith diethyl ether, dried, and counted. Recovery of a tetranucleotidewith a 5-triphospha te end was 50%; longer oligonu cleotides wererecovered in greater or even full yield.DNA synthesis assays were as indicated for RNA synthesis, butwith the addition of 100 PM ATP, 50 PM each of dATP, dCTP, anddGTP, 18 PM [3HldTT P (250 to 1000 cpmlpm olf, and approximately25 units of holoenzyme. Incubation was at 30 and terminated eitheras described for RNA synthes is, or by addition of 50 ~1 of 0.1 Msodium pyrophosphate and 1 ml of 10% trichloroacetic acid andfiltered (8). A unit is defined as 1 pmol of nucleotide incorporatedinto DNA per min at 30.Compleme ntation assays differed from DNA synthes is as says inthat 1 ~1 of PC3 Fraction II (15 pg of protein containing 14 units ofendogen ous DNA synthetic activity depen dent upon addition ofdnaG p rotein) was used in place of DBP and holoenzyme; DNAreplication was dependent upon addition of dnoG protein, and wasnot stimulated by DBP, holoenzyme, or both.In assays of all primase activities, the value ob tained withoutprimase was subtracted from the experimental value; this valuenever exceeded 2 residues per input DNA circle for RNA synthesis,or 5 pmol of nucleotide for DNA synthes is.DNA polymerase I was assayed with activated calf thymus DNA(33).Other Methods - Condu ctivities were read in Radiometer conduc-tivity meter at 0 after a loo-fold dilution of the samp le in water.SDS-polyacrylamide gel electrophoresis was performed u sing theTris/glycine system (34). Glycerol gradient cen trifugation was per-formed as described (35). Protein determinations were as described(36).

RESULTSPurification of Primase - Activity was extracted (Table I)by lysozyme lysis in the presence of a nonion ic detergent, Brij

58; spermidine was added to facilitate removal of cell debrisand DNA by low speed centrifugation. The clear lysate con-tained 3,000 to 6,000 units/g of cell paste. Addition of solidammonium sulfate to 40% saturation precipitated about 15%of the protein including several replication proteins (dnaCprotein, protein n, and rep), as well as primase. Thes e proteinswere resolved on Bio-I&x-70, a polyacrylic acid resin. Primasewas eluted fir st near 0.24 M NaCl (pH 6.81. At lower pH, theproteins required higher salt concentration for elution. Succes-sive fractionations on DNA-cellulose and valyl-Sepharoseyielded a primase fraction 50 to 80% pure. Final resolutionwas achieved by chromatography on DEAE-cellulose. Thisfraction was stable on ice for up to 2 months and to freezingand storage in liquid nitrogen for at least 1 year. Replication

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760 dnaG Gene Product of E. coliTABLE I

Purification of primuseAll 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 c ell paste (see Materials and Methods) was thawed anddiluted with 0.5 volume of 50 mM Tris.Cl (pH 7.5), 10% sucrose toA 595 = 0.30 to 0.35 cl:40 0 d ilution). 4 M KC1 was added to 0.15 M, 0.4M dithiothreitol to 1 mM, 1 M sperm idinecl to 20 mM, 0.5 M EDTAto 20 mM, and 10% Brij 58 to 0.1%. The pH was adjus ted to 8.5 withsolid Tris base. Lysozyme was then added to 0.2 mg/ml. After 20min at 2 the lysate was centrifuged at 18,000 rpm at 0 for 45 minin the Sorvall SS34 rotor. The supernatant is Fraction I. Ammon iumsulfate (0.24 g/ml) was added to Fraction I. The precipitate wasbackwashed with a volume of Buffer A (containing 0.1 M NaCl and0.24 g/ml of ammon ium sulfate1 equivalent to one-tenth of FractionI. The precipitate was resuspended in a minima l 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 dithiothre-itol, 1 mM EDTA (to a conductivity of 50 pmho), and applied to aBio-Rex-70 column (1.7 liters, 15 x 9 cm) at 30 mg of protein/ml ofresin. The column was successively washed with 2.5 column volumesof Buffer I containing first 240 mM KCl, then 400 mM KCl, finally700 mM KCl. Three fractions were collecte d for each wash and toeach was added 0.35 g/ml of ammonium sulfate. Pellets wereresuspended in minima l volumes of Buffer I (pH 6.8) and frozen inliquid nitrogen. Fractions with primase activity were thawed,pooled, and dialyzed 16 h against Buffer I (pH 6.5) (Fraction 1111.

Dialyzed Fraction III (15 ml, representing one-half the total) wasclarified (15,000 rpm, 10 min in a Sorvall SS34 rotor, 0) and dilutedto conductivity

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dnaG Gene Product of E. coli 761

46,300 -

DEAE CELLULOSE FRAC TION, 11 , 12 , 13 , 14 , 15 , 16 , 17

35,800 - ;, .,.Y

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dnaG Gene Product of E. colistrength. Inhibition of 50% occurred when 50 mM NaCl wasadded to the buffers in the assay compon ents, estimated to benear 20 mM in NaCl in conductivity value. RNA sy nthesis byprimase is sensitive to phosphate (40% inhibition at 40 PM).

Stoichiometry of Primase and Template- The rate andextent of RNA synthesis on G4 DNA was influenced by theamount of primase present (Fig. 6). It appears that at leastone molecu le of primase is required per circle to attain thesynthesis of a complete primer. That no more than one copyof primer is synthesized per circle is suggested by the limit ofincorporation of rNTPs at 30 residues per circle. Additionalprimase increase s the rate at which RNA priming of all thecircles is completed. When either primas e or DNA was inexcess (1.5, or 0.2 primase molec ules/circle, respectively) onlya full length RNA product was observed (by polyacrylamidegel electrophoresis (17)) throughout the course of the reaction;products of intermediate size were not found. Thus , the rate-limiting step appears to be initiation of RNA synthesis ratherthan elongation of the RNA ch ain.Inhibitors of RNA Synthesis Activity-Primase was notinhibited by several of the well known inhibitors which bind

Y I I I IP IA)g 24Pt 20 -

AT P C T P , G T P , U T P

FIG. 4. Influence of ribonucleos ide triphosphate concentrationson primase activity. RNA synthesis assays were performed asdescribed under Materials and Methods, except that in A, AT Pwas added to the indicated concentrations in the presence of 20 pMeach of CTP, GTP, and UTP (a ll 3H-labeled, 3000 cpm/pm ol nucleo-tide), and in B, the concentration of the 3H-labeled CTP, G TP, UTPmixture was varied, and ATP was 20 PM. Primase was DEAE-Fraction 13 (Fig. l), 1 ~1, 108 ng. Incubations were for 3.5 min at30.

I I I I I24 -

16 -

4-A0 I I I I ,

0 2 4 6 8 10 12Mg2+, mM

FIG. 5. Effect of MgZ+ on RNA polymerase action of primase.Source of primase was as in Fig. 4. Incubations were for 10 min at30.

E. coli RNA polymer-ax (Table IV) further indicating thatcontamination by the latter enzyme is not responsible for theobserved RNA synthesis. Actinomycin D, which binds G-Cpairs in DNA (40, 411, inhibits both enzymes. E. coli DNA-binding protein, in an amount sufficient to cover the G4DNA, was essential for primase action, but almost completelyinhibited transcription by RNA polymerase.Influence of Template on RNA Polymerase Action-l%?-mase in the presence of DBP, was highly specifi c for G4 DNA(Table V). DNA of the closely related phage ST-1 was theonly other template which showed significant act ivi ty; poly-acrylamide gel electrophoretic analysis indicated that theRNA product was the same size as that for G4 DNA (data notshown). Lack of transcriptional act ivi ty by primase in thecase of Ml3 DNA and 4X DNA is probably due to the

I I I I I I40 d-G PROTEIN/G4 CIRCLE:

32

24

16

0 -.----.-m--L0 10 20 30 40 50 60

T I ME (MI NIFIG. 6. RNA s ynthesis at different levels of primase. RNA syn-thesis (see Materials and Methods) in 25 ~1 containing 2300 pmolof 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. Incubationwas at 30. At indicated times, 2 ~1 (containing 184 pmol of DNA)were removed. The number of primase molec ules per circle is basedon a purity of 25%. The preparation, originally 80% pure, haddecayed to 32% of its original activity (110,000 to 35,000 units/ml).One microliter of Fraction 15 (containing an estimated 25 ng or 2.5x 10 molec ules of active primase) is sufficient to prime 2300 pmolof DNA (2.6 x 10 single-stranded circles) at a level of one monomerper circle.

TABLE IVEffect of RNA polymerase inhibitors on primase action

RNA synthesis by primase (see Materials and Methods) was in25 ~1 containing 460 pmol of G4 DNA, 0.85 pg of DBP, and 1 ~1 ofprimase (DEAE-Fraction 15, see Fig. 1). RNA synthe sis by RNApolymerase was in 25 ~1 containing 230 pmol of G4 DNA and 1 ~1 ofRNA polymerase holoenzyme (Fraction PC 10, 1 mg/ml, 22,200units/mg). Inhibitors were added before enzyme as indicated. Incu-bation was for 20 min at 30. Control values (NMP/circle in absen ceof inhibitor) were: 26.3 for nrimass and 555 for RNA aolvmerase.

InhibitorInhibition of RNA synthesis

Concentration RNA poIymer-as ! Primase

pglml %Rifampicin 40 99 0Heparin 40 99 29Streptolydigin 40 72 0Streptovaricin C 40 96 10Actinomycin D 40 94 99DBP 16 99 0

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dnaG Gene Product of E. coli 763TABLE V

Influence of templates on primuse and RNA polymerase actions inpresence of DNA-binding protein

RNA synthesis (see Materials and Methods) was in 50 ~1containing DNA and DBP as indicated, and 4 ~1 of primase (DEAE-Fraction 17 (Fig. 1)) or 1 ~1 of RNA polymerase; (see Table IV).Incubation was for 30 min at 30. Reactions with G4 SS DNA, Ml3SS DNA, $X SS DNA, pf-1 SS DNA, poly(dA), and poly(dC),contained 920 pmol of nucleo tide residues and 3.2 pg of DBP.Reactions with G4 RF I DNA, I$X RF I DNA, T4 DNA, ColEl DNA,A DNA, and Escherichia col i DNA, contained 1840 pmol of nucleotideresidues and 3.2 pg of DBP. The reaction with the trp attenuatorDNA contained 185 pmol of nucleotide and 3.2 pg of DBP. V alues ofNMP/molecule template were based on template size of 5.5 kb (kb= 1000 residues for SS DNA or 1000 base pairs for duplex DNA) forC4, M13, $X, and pf-1 DNAs (421, 6.5 kb for ColEl DNA (271, 46 kbfor A DNA (43), 144 kb for T4 DNA (44), and 0.56 kb for trpattenuator DNA.4 For E. coli, poly(dA) and poly(dC) DNA tem-plates, the number of residues synthesized per 5.5 kb of inputtemplate is given. The se numbers represent 47%, 0.178, and 1.3%transcription of the template, respectively. A value for NMP/mole-cule template of zero indicate s incorporation of radioactivity lessthan 30% above the background value obtained in absen ce ofenzyme.

DNA template RNA synthesisFrimase RNA polymeraseNMPlmokcule oftemplate

G4 ss 21.8 4.0ST-l SS 4.0 417Ml3 SS 0 353c#ax ss 0 40Pf-1 ss 0 100G4RFI 0 11,000$XRFI 0 11,200ColEl 1.4 4,020A 0 10,700T4 0 33,400E. coli 0 2,600trp attenuator 0.8 44Poly(dA) 0 11Poly(dC) 0 79

inability of the protein to bind to these DNAs3 RNA tran-scribed by RNA polymerase on trp attenuator DNA (29)contains a hairpin region with seven G-C base pairs4 resem-bling the RNA primer synthesized from G4 DNA (17); howeverprimase did not transcribe trp attenuator DNA to a significantextent under the conditions tested. The duplex DNA templatestested were all inert for primase action (~0.03% of thetemplate transcribed) with the possible exception of ColElDNA, the small amount of RNA synthesized in this case hasnot been further characterized.

DISCUSSIONThe product of the dnaG gene is needed continuously duringreplication of the E. col i chromosome (45). Studies withlysates of temperature-sensitive dnaG mutants suggested afunction in the initiation of nascent (Okazaki) fragments (46).Previous enzymatic studies o f the dnaG protein showed it tobe an RNA polymerase whose product served as a primer forDNA replication (8). The enzyme has been renamed primasebecause we recognize its role in forming primers for DNAreplication at very special loci. The importance of this proteinand its remarkable properties made its isolation in quantities

4 F. Lee and C. Yanofsky, personal comm unication.

adequate for physical and functional analysis an importantobjective.Using the dependence of G4 DNA replication upon primerformation as an assay for primase, we have purified theenzyme 26,000-fold to near-homogeneity. From the specif icactivity of the purified enzyme and the number of units in themost active cell extract, there appear to be only 50 to 100molecules of primase per cel1.j In confirmation of an earlierreport (9) the native protein is a polypeptide of 60,000 daltons.It is unlike the large, multisubunit E. coli RNA polymerasenot only in size and organization but also in its insensitivityto rifampicin and other inhibitors that bind and inactivatethe p subunit of the large polymerase.The RNA-synthesizing activity of primase coinc idesthroughout the purification procedure with its complemen ta-tion activity for an extract of a dnaG mutant, as well as withits priming capacity for DNA replication. This is a strongindication that all these activi ties reside in the primasemolecule and, along with other distinctions, eliminates anypossibili ty that the potent, rifampicin-sensi tive RNA polym-erase contaminates our preparations.Perhaps the most remarkable feature of primase is thegreat transcriptional spec ific ity it displays for certain tem-plates. Among single-stranded DNA circles coated with DNA-binding protein, only phage G4 (or ST-l) DNA can serve andit can sustain the synthes is of a unique 29-residue, transcript(17). The DNA-binding protein may promote formation of aparticular secondary structure in the DNA or it may preventnonspecific, unproductive binding of primase to the DNA. Forthe 4X DNA circle to be utilized by primase, masking byDBP does not suf fice. Instead, a replication intermediatecontaining one dnaB protein molecule in a complex with theDNA (11) (produced through the action o f dnaC protein,proteins i and n, and ATP) is essential (12). Both the G4 and4X systems afford attractive opportunities for understandingthe actions of primase, the dnaB protein and other replicationproteins responsible for initiation events in replication of theE. coli chromosome.The succeeding paper (17) describes the structure of the 29-residue RNA transcript synthesized by primase on G4 DNAwhen deoxynucleoside triphosphates are excluded. When theribo- and deoxynucleoside triphosphates are all present, theenzyme synthesizes a shorter, hybrid transcript, which iseven more abbreviated when priming DNA synthes is (18).

Acknowuledgments - We would like to acknowledge Dr. JoelWeiner and John Scott for developing the early stages of theprimase purification procedure, and Janey Beuchel fo r prepa-ration of G4 phage.

Note Added in Proof-J. Sims, D. Hourcade, and D. Dres-sler (personal communication) have found a DNA sequencecomplementary to the primer RNA at a region identified,through in viuo studies, as the origin of G4 negative strandsynthesis.REFERENCES

1. Schekman, R., Weiner, A., and Kornberg, A. (1974) Scienc e

5 The calculated number of molecules/cel l is based upon assump-tions of 5 X 10 cells/g; a spe cific activity of pure primase of 1.3 X106 units/me: a molecular weight of nrimase of 60,000 almol; andthat 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.

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764 dnaG Gene Product of E. coli2.3.4.5.6.7.8.9.

10.

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