OF BIOLOGICAL Vol. 263, July 15, pp. 9818 … › content › 263 › 20 › 9818.full.pdfleading and lagging strand DNA synthesis, the unwinding of the helix to enable DNA polymerase

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 20, Issue of July 15, pp. 9818-9630,1988 Printed in U. S A .

Leading and Lagging Strand Synthesis at the Replication Fork of Bacteriophage T7 DISTINCT PROPERTIES OF T7 GENE 4 PROTEIN AS A HELICASE AND PRIMASE*

(Received for publication, January 11, 1988)

Hiroshi NakaiS and Charles C. Richardson From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021 15

Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork. A minimum of three proteins is required to catalyze leading and lagging strand synthesis. The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities. A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity. Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis. Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases. Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis. The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms. Removal of the large form results in the loss of primase activity but not of helicase activity. Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms. These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms. The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.

Three basic reactions are necessary for the movement of a replication fork: the polymerization of nucleotides for both leading and lagging strand DNA synthesis, the unwinding of the helix to enable DNA polymerase to proceed through duplex DNA, and primer synthesis to initiate multiple rounds of lagging strand synthesis. These reactions are catalyzed by three proteins at the replication fork of bacteriophage T7: the T7 gene 4 protein, T7 gene 5 protein, and Escherichia coli thioredoxin.

The polymerization of nucleotides is catalyzed by the phage-encoded DNA polymerase, the gene 5 protein. By itself,

* This investigation was supported by United States Public Health Service Grant AI-06045 and Grant NP-1P from the American Cancer Society, Inc. This is Paper 44 in a series entitled “Replication of Bacteriophage T7 Deoxyribonucleic Acid.” The previous paper is White and Richardson (1988). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Damon Runyon-Walter Winchell Cancer Fund Fellowship DRG-814.

it has very low processivity, dissociating from a primed single- stranded DNA template after incorporating only 1-50 nucleotides (Tabor e t al., 1987). E. coli thioredoxin, bound tightly to the gene 5 protein in a 1:l stoichiometry (Modrich and Richardson, 1975a, 1975b), confers high processivity by keep- ing the gene 5 protein tightly bound to the primer-template (Tabor e t al., 1987; Huber et al., 1987). Hereon, we will simply refer to the gene 5 protein-thioredoxin complex as T7 DNA polymerase.

The gene 4 protein serves as both a helicase and a primase at the replication fork (Richardson, 1983). It exists in two forms of molecular weight 56,000 and 63,000, the smaller one arising from an internal ribosome binding site and initiation codon (Dunn and Studier, 1983). In leading strand synthesis, unwinding of the helix by the gene 4 protein (Matson et al., 1983) is coupled to the polymerization of nucleotides by T7 DNA polymerase (Kolodner e t al., 1978; Lechner and Rich- ardson, 1983). The rate of fork movement is 300 nucleotides/ s at 30 “C (Lechner and Richardson, 1983). As a primase it catalyzes the synthesis of tetraribonucleotides pppACCC/A and pppACAC at the major recognition sites T/GGGTC and GTGTC on single-stranded DNA, primers which are extended at the site of their synthesis by T7 DNA polymerase (Scher- zinger et al., 1977a; Romano and Richardson, 1979; Tabor and Richardson, 1981; Nakai and Richardson, 1986b).

The gene 4 protein is a single-stranded DNA-dependent NTPase,’ hydrolyzing NTPs to NDP and Pi (Kolodner and Richardson, 1977; Matson and Richardson, 1983), and this activity plays a central role in gene 4 protein’s function as a helicase and primase. In the presence of an NTP (preferably dTTP), the gene 4 protein binds to single-stranded DNA, a process not dependent on NTP hydrolysis (Matson and Rich- ardson, 1985). NTP hydrolysis is required for translocation of gene 4 protein unidirectionally 5’ to 3’ along the DNA (Tabor and Richardson, 1981; Matson and Richardson, 1983). To promote strand-displacement DNA synthesis ( i e . leading strand synthesis) at the replication form, the gene 4 protein, bound to the displaced DNA strand, translocates in a 5’ to 3‘ direction to unwind the DNA helix (Matson e t al., 1983). Leading strand synthesis may then be catalyzed by T7 DNA polymerase, which cannot by itself catalyze DNA synthesis

The abbreviations used are: NTPase, nucleoside 5”triphospha- tase; kb, kilobase(s); NTP, nucleoside 5”triphosphate; NDP, nucleoside 5”diphosphate; dNTP, deoxyribonucleoside 5’-triphosphate; rNTP, ribonucleoside 5”triphosphate; N, nucleoside residue; M13 RF DNA, M13 replicative form DNA (duplex DNA); RFII, circular duplex DNA with a nick or small gap; RFIII, linear duplex DNA; FCA,c, preformed replication forks consisting of M13mp6 RFII DNA with single-stranded poly(dA,dC) tails arising at random sites; FCS, preformed replication forks consisting of M13 RFII DNA with a 33- nucleotide SV40 sequence arising at the unique BamHI sites.

9818

Distinct Properties of T7 Gene 4 Helicase and Primase 9819

through duplex DNA (Lechner and Richardson, 1983). Trans- location of gene 4 protein along DNA is also involved in its search for primase recognition sites (Tabor and Richardson, 1981; Matson and Richardson, 1983).

T7 DNA polymerase and gene 4 protein interact specifically to catalyze strand-displacement and RNA-primed DNA synthesis. Other DNA polymerases such as the T7 DNA polymerase cannot catalyze DNA synthesis through duplex regions together with the T7 gene 4 protein (Kolodner et al., 1978; Lechner and Richardson, 1983) and cannot efficiently extend primers synthesized by gene 4 protein (Scherzinger et al., 1977a, 197713). In RNA-primed DNA synthesis the affinity of gene 4 protein for T7 DNA polymerase as well as the inde- pendent binding of these proteins to single-stranded DNA play an important role in the transition from primer synthesis to DNA synthesis (Nakai and Richardson, 1986a, 1986b).

The dual role of the T7 gene 4 protein as helicase and primase raises two important questions. The first major question concerns the distribution of the multiple activities of gene 4 protein between the two molecular weight forms. Are the helicase and primase activities contained in one of the molecular weight forms or in a complex composed of both forms? Can a gene 4 protein molecule (or complex) acting as helicase also catalyze primer synthesis to initiate lagging strand synthesis? The second major question concerns the processivity of gene 4 protein as it serves as a helicase or primase. Can a single gene 4 protein molecule catalyze multiple rounds of primer synthesis as it translocates long DNA?

The smaller molecular weight form of gene 4 protein has been purified from a clone that produces the small form but not the large form.' The purified protein has DNA-dependent NTPase and helicase activities but no primase activity (Bern- stein and Richardson, 1988). In this paper we describe the purification of the small form of gene 4 protein from an equimolar mixture of the two molecular weight forms. The small form preparation has greatly reduced levels of primase activity but normal levels of helicase activity. Nevertheless, submolar amounts of large form are sufficient to restore high levels of primase activity present in a mixture of both forms. The results suggest that the gene 4 primase is an oligomer composed of both forms.

In addition, we show that the helicase activity of gene 4 protein functions in a highly processive manner. In contrast, the primase activity is distributive. The gene 4 primase cannot catalyze multiple rounds of primer synthesis without dissociating from the template. The results suggest the possibility that the large form is a distributive component of a primase complex, dissociating from the template after each round of primer synthesis.

EXPERIMENTAL PROCEDURES

Many of the material and methods used in this paper were described previously (Nakai and Richardson, 1986a, 1986b).

Materials Enzymes-Purified T7 DNA polymerase (gene 5 protein-thiore-

doxin complex, each of the components present in equimolar amounts) was furnished by Stanley Tabor (Harvard Medical School). It was purified from cells overproducing both T7 gene 5 protein and E. coli thioredoxin (Tabor et al., 1987). Purification of the T7 gene 4 protein is described below. Calf intestine phosphatase and proteinase K were purchased from Boehringer Mannheim, and restriction endonuclease Hue11 was from New England Biolabs.

Other Materials-Norit (activated charcoal) was from Matheson, Coleman, and Bell, and yeast tRNA was from Boehringer Mannheim.

S. Tabor, J. Bernstein, and C. C. Richardson, unpublished results.

Methods Preparation of the Preformed Replication Fork-Construction of a

preformed replication fork was previously described (Lechner and Richardson, 1983). It is a circular duplex of 7.2 X lo3 base pairs (phage M13mp6 DNA) with a protruding 5"single-stranded tail of 237 nucleotides (SV40 DNA). In this procedure a linearized M13 duplex DNA (RF DNA) with an SV40 DNA insert is denatured and annealed to single-stranded M13mp6 DNA. We have constructed two alternate templates to serve as preformed replication forks that can be prepared by a simpler procedure in greater yield. In the preparation of both templates, a synthetic oligonucleotide primer is annealed to M13mp6 DNA and extended with T7 DNA polymerase to yield a circular duplex DNA molecule. The oligonucleotide primer is not fully complementary to M13 DNA such that the resulting duplex (a forked circle, FC) has a protruding 5"single-stranded tail. The use of a DNA polymerase in the preparation of forked molecules raises the possibility that some of these molecules have a small single-stranded gap between the 3'-end and the 5"single-stranded tail of the template. We have not extensively characterized these forked templates for the presence of small gaps; however, our previous studies of T7 DNA polymerase have indicated it would efficiently fill in these gaps (Engler and Richardson, 1983).

The first template (FCA,C) is prepared using a random heteropolymer poly(dA,dC) (average length, 170 nucleotides; purchased from Pharmacia LKB Biotechnology Inc.) as primer. A reaction mixture (300 pl) containing 130 mM Tris-HC1 (pH 7.5), 33 mM MgCl,, 170 mM NaCl, 100 pg of M13mp6 DNA, and 120 pmol of poly(dA,dC) (amount expressed in terms of the number of primer 3'-ends; primer:template ratio, 3:l) was incubated at 65 "C for 10 min and allowed to cool slowly at room temperature for 20 min. The reaction mixture was adjusted to a volume of 1 ml, adding 75 pg of T7 DNA polymerase, the four dNTPs to a final concentration of 1.5 mM each, bovine serum albumin to 50 pg/ml, and dithiothreitol to 10 mM. Incubation was at 30 "C for 20 min. The reaction was stopped by adding EDTA to a final concentration of 20 mM. The majority of the templates (>70%) was formed by the use of a single primer by T7 DNA polymerase on an M13 template, judged by assessing the amount of full-length complementary strands synthesized on M13 DNA by alkaline agarose gel electrophoresis.

The second template (FC13) is similar to the preformed fork previously described (Lechner and Richardson, 1983). The only differ- ence is that the 5"single-stranded tail is only 33 nucleotides in length rather than 237 nucleotides. A synthetic 50-mer (synthesized and purified by Alexander Nussbaum, Harvard Medical School) is annealed to M13mp6 DNA. 17 bases on the 3'-end on this oligonucleotide are complementary to M13mp6 DNA at the BamHI site. The other 33 bases on the 5'-end represent the SV40 DNA sequence, corresponding to the sequence of the preformed fork previously described (Lechner and Richardson, 1983). Annealing of the primer to the template was conducted in the 300-pl annealing mix as described above except that the mixture was heated in a heating block to 90 "C and slowly cooled over a 1-h period to 30 "C. Primer extension to yield the duplex molecule was carried out in 1-ml reaction mixtures as described above except that incubation was at 37 "C.

The product of DNA synthesis was deproteinized by incubation in 100 pg/ml proteinase K and 1% sodium dodecyl sulfate at 50 'C for 30 min followed by extraction with pheno1:chloroform (1:l) and chloroform. The DNA was precipitated with ethanol and dissolved in 10 mM Tris-HC1 (pH 7.5), 1 mM EDTA (TE). DNA was purified by hydroxylapatite chromatography and high-salt sucrose gradient centrifugation as previously described (Lechner and Richardson, 1983). 90-150 pg of preformed fork were prepared by this procedure. A notable modification of this procedure was in performing the sucrose gradient centrifugation. DNA pooled from the hydroxylapatite chromatography was dialyzed extensively against TE and precipitated with ethanol. DNA was resuspended in 400 pl of TE containing 1 M NaCl and 2 pg/ml ethidium bromide. 200-p1 samples were layered onto linear 11-ml sucrose gradients of 5-25% sucrose in 50 mM Tris- HCl (pH 7.5), 5 mM EDTA, 1 M NaCl, and 2 pg/ml ethidium bromide. These gradients, formed in Beckman SW 41 tubes, were centrifuged at 40,000 rpm for 7 h at 15 "C. The major band (visualized using an ultraviolet lamp), resolving halfway through the gradient, consisted of the preformed replication fork. It was collected by side puncture using a syringe and needle. Ethidium bromide was removed from the preparation by extraction with water-saturated butanol. The preformed fork was precipitated with ethanol, resuspended in TE, and stored at 4 "C.

9820 Distinct Properties of T7 Gene 4 Helicase and Primase

Enzyme Assays-Gene 4 protein was assayed by three methods: the RNA-primed DNA synthesis assay, primer synthesis assay, and strand-displacement synthesis assay.

The RNA-primed DNA synthesis assay measures gene 4 primase activity by measuring its ability to prime DNA synthesis on single- stranded M13 DNA (Matson and Richardson, 1983). The reaction mixtures (50 pl) contained reaction buffer (40 mM Tris-HC1, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, and 50 pg/ml bovine serum albumin), 300 p~ each of [3H]dTTP (70-90cpm/pmol), M T P , dCTP, dGTP, ATP, and CTP, 20 pg/ml M13 DNA, 125 ng of (27 nM) T7 DNA polymerase, and 0-100 ng of gene 4 protein. Incubation was at 37 "C for 20 min, and the reaction was stopped by adding 5 pl of 200 mM EDTA (pH 8). Incorporation was measured by determining the amount of radioactivity retained on DE81 paper as previously described (Nakai and Richardson, 1986a). Background levels of incorporation, measured in reaction mixtures containing no gene 4 protein or ATP and CTP, were subtracted in the calculation of results. One unit of activity catalyzes the incorporation of 1 nmol of total deoxyribonucleotides under these assay conditions.

The primer synthesis assay measures the incorporation of CMP into an oligonucleotide by gene 4 protein in the absence of T7 DNA polymerase. The assay measures conversion of radioactivity in [CI-~~PICTP into a form that is adsorbable to Norit after treatment with calf intestine phosphatase. Reaction mixtures (25 pl) contained reaction buffer, 20 pg/ml M13 DNA, 300 p~ ATP, 50 p~ [CI-~~PICTP (1.8 Ci/mmol), 1 mM dTTP, and 0-100 ng of gene 4 protein. After incubation at 37 "C for 20 min, the reaction was stopped by addition of 1.4 pl of 200 mM EDTA (pH 8) followed by heating at 92 "C for 5 min. The mixture was then adjusted to a volume of 100 pl, which contained a final concentration of 50 mM Tris-HC1 (pH 8), 10 mM MgClz, 0.1 mM ZnS04, and 1 mM spermidine. 80-100 units of calf intestine phosphatase were added, and the mixture was incubated at 37 "C for 30 min and then chilled to 0 "C. A 90-pl portion of the reaction mixture was added to 2.0 ml of a solution containing 0.5 M HC1,50 mM sodium pyrophosphate, 100 mM phosphoric acid, and 2% (packed volume) Norit. The suspension was mixed thoroughly and placed at 0 "C for 5-15 min. The Norit was collected by filtration through Whatman GF/C glass filter paper (2.4-cm diameter) and washed with six 2-ml aliquots of cold 1 M HC1, 100 mM sodium pyrophosphate. Filters were dried and counted in a nonaqueous liquid scintillation mixture. The background, measured using reaction mixtures containing no gene 4 protein, was subtracted in the calculation of results. One unit of activity catalyzes incorporation of 10 pmol of CMP under these assay conditions.

The strand-displacement DNA synthesis assay measures gene 4 helicase activity by measuring its ability to stimulate DNA synthesis catalyzed by T7 DNA polymerase on the preformed replication fork. Reaction mixtures (20 pl) contained reaction buffer, 5 pg/ml of the preformed fork (FCA,~) , 300 p~ each of [3H]dTTP (70-90 cpm/pmol), dATP, dCTP, and dGTP, 125 ng of (68 nM) T7 DNA polymerase, and 0-100 ng of gene 4 protein. Incubation was at 37 "C for 20 min, and the reaction was stopped by the addition of 2 pl of 200 mM EDTA (pH 8). Incorporation was determined by measuring the retention of radioactivity on DE81 paper. Background levels of incorporation, measured in reaction mixtures containing no gene 4 protein, were subtracted in the calculation of the results. One unit of activity catalyzes the incorporation of 100 pmol of total deoxyribonucleotides under these assay conditions.

Unless otherwise indicated, all enzymatic reactions were initiated by combining a mixture of reaction buffer, DNA template, and NTPs with a mixture of enzymes in the standard enzyme diluent (50 mM Tris-HC1, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, and 500 pg/ ml bovine serum albumin).

Purification of Gene 4 Protein-The gene 4 protein was purified from extracts of cells harboring a plasmid expressing gene 4 (constructed and provided by Stanley Tabor, Harvard Medical School). Expression of the cloned gene 4 is accomplished using a T7 RNA polymerase/promoter system (Tabor and Richardson, 1985). By a conservative estimate, the induced cells produced at least as much gene 4 protein as T7-infected cells, and the two molecular weight forms of gene 4 protein are produced in equimolar amount^.^

The procedure for growth and induction of the cells was similar to that already described for the overproduction of the gene 5 protein (Tabor et al., 1987). 10 liters of cells were grown with aeration in a New Brunswick fermentor at 30 "C in 2% Tryptone, 1% yeast extract, 0.5% NaC1, 0.2% glucose, 0.2% casamino acids, and 50 mM potassium

S. Tabor and C. C. Richardson, unpublished results.

phosphate (pH 7.4). The temperature was rapidly shifted to 42 "C during exponential growth (A690 = 4), and this temperature was maintained for 20 min. The temperature was then lowered to 37 "C, and aeration was continued for 90 min. Cells were harvested and washed as previously described (Tabor et al., 1987). The cell paste (126 g) was resuspended in 50 mM Tris-HC1 (pH 7.5), 10% sucrose, 2 mM EDTA, frozen in liquid nitrogen, and stored at -70 "C.

Purification of the gene 4 protein was performed according to the procedure of Fischer and Hinkle (1980) up to the preparation of Fraction IV (DEAE-Sephadex A-50 chromatography). All steps were performed at 4 "C unless otherwise indicated. Gene 4 protein was monitored during purification using the RNA-primed DNA synthesis assay. The procedure with minor modifications is summarized below.

The frozen cell suspension was thawed overnight on ice and incubated at 0 "C for 45 min with 250 pg/ml lysozyme in the presence of 100 mM NaCl and protease inhibitors (10 mM benzamidine hydro- chloride and 0.5 mM phenylmethylsulfonyl fluoride). The mixture (divided into six aliquots) was incubated in a 37 "C water bath until the temperature reached 20 'C and was then quickly cooled on ice to 4 "C. The lysate was clarified by centrifugation in a Beckman 45Ti rotor at 40,000 rpm for 45 min. The supernatant (175 ml) was Fraction 1.0.1 volume of 40% (w/v) streptomycin sulfate was added to Fraction I, and the nucleic acid precipitate was removed by low speed centrifugation. To the supernatant 0.35 g of ammonium sulfate/ml was added, and the protein precipitate, collected by centrifugation, was dissolved in 500 ml of buffer A (20 mM potassium phosphate, pH 7.0, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol) to yield Fraction 11. This volume was necessary to lower the conductivity of the solution to a conductivity equal to that of buffer A containing 0.1 M KCl.

Fraction I1 was applied to a phosphocellulose column (12.0 cm2 X 15.5 cm) equilibrated with buffer A containing 50 mM KC1 (flow rate, 160 ml/h). The column was washed with 200 ml of buffer A containing 0.1 M KCl, and gene 4 protein was eluted with a 2-liter linear gradient of 100-500 mM KC1 in buffer A. Column fractions were assayed using the RNA-primed DNA synthesis reaction. Fractions with a specific activity of greater than 200 units/mg were pooled and dialyzed for 12 h at 0 "C against 4 liters of 20 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA, 0.1 mM dithiothreitol, 20% (w/v) glycerol (Fraction 111, 315 ml).

Fraction I11 was applied to a DEAE-Sephadex A-50 column (5.3 cm2 X 7.5 cm) equilibrated with buffer B (20 mM Tris-HC1, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol) containing 0.1 M NaCl. The column was washed with 20 ml of buffer B containing 0.1 M NaCl, and gene 4 protein was eluted with a 400-ml linear gradient of 100-600 mM NaCl in buffer B. Column fractions with a specific activity of greater than 1000 units/mg were pooled and dialyzed for 12 h at 4 "C against 2 liters of 20 mM potassium phosphate (pH 7.4), 1 mM sodium citrate, 1 mM dithiothreitol, and 20% glycerol (Fraction IV, 40 ml).

Further purification of gene 4 protein by hydroxylapatite chromatography is described under "Results." A summary of the purification of gene 4 protein is presented in Table I. Purification of gene 4 protein from E. coli Dl10 (thy, end, polAI) infected with T73,6LG12 (see Kolodner et al., 1978) was also carried out by this procedure. The experiments presented in this paper were performed with gene 4 protein from phage-infected cells and from the clone purified by this procedure. These protein preparations were interchangeable in all of these experiments.

Analysis Involving Gel Electrophoresis-Electrophoresis on alkaline agarose gels was performed as previously described (Maniatis et al., 1982). DNA products from DNA synthesis reactions were denatured in preparation for electrophoresis as follows. Reaction mixtures of relatively small volume (up to 20 pl) were adjusted to 150 mM NaOH. The mixture was adjusted to contain 5-10% glycerol and bromocresol green and loaded onto the gel. When reaction mixtures were of a greater volume (20 p1 and greater), products were treated with 100 pg/ml proteinase K in the presence of 1% sodium dodecyl sulfate for 30 min at 50 "C. The solution was then adjusted to a final concentration of 0.3 M sodium acetate (pH 8), and DNA products were precipitated with 10 pg of yeast tRNA carrier by addition of 2.5 volumes of ethanol. The precipitate was gently resuspended in 50 mM NaOH, 1 mM EDTA, 5% glycerol, and bromocresol green.

Initiation sites for RNA-primed DNA synthesis were examined as previously described (Nakai and Richardson, 1986b). Reactions were carried out in the presence of radiolabeled ATP and CTP (label in [Y-~'P]ATP or [CI-~~PICTP) and unlabeled dNTPs to specifically label RNA primers. RNA/DNA products were digested with HaeII, denatured, glyoxalated, and resolved on 1.4% agarose gels. Gels were


soaked for 30 min with gentle agitation in 10% (v/v) acetic acid before being dried down for autoradiography.

Proteins were resolved on polyacrylamide slab gels by electrophoresis in the presence of sodium dodecyl sulfate as previously described (Laemmli, 1970). Bands were visualized by silver staining according to the procedure of Morissey (1981).

RESULTS

Purification of Gene 4 Protein

The gene 4 protein exists in two forms of molecular weight 56,000 and 63,000 (Dunn and Studier, 1983). It has been purified as an equimolar mixture of the two forms from phage- infected cells (Scherzinger et al., 1977a; Fischer and Hinkle, 1980; Matson and Richardson, 1983). It has also been purified from infected cells as a preparation enriched for the lower molecular weight form of gene 4 protein (Kolodner et al., 1978). Partial separation of the two forms by phosphocellulose chromatography has been reported (Fischer and Hinkle, 1980). Resolution of the two molecular weight forms has nevertheless been an elusive task.

In our purification of gene 4 protein, we have observed an enrichment for the smaller molecular weight form of gene 4 protein. We have taken advantage of this finding to obtain gene 4 protein preparations that consist predominantly of the small form as well as preparations that consist of equimolar amounts of the two molecular weight forms. Gene 4 protein was purified according to the procedure of Fischer and Hinkle (1980) up to the preparation of Fraction IV as described under “Experimental Procedures.” Fractions for purification were pooled based on specific activity measurements from the RNA-primed DNA synthesis assay, an assay that measures gene 4 protein’s ability to prime DNA synthesis by T7 DNA polymerase on single-stranded DNA templates. The final purification step, chromatography on hydroxylapatite, re- solves the small form from an equimolar mixture of both molecular weight forms,

We have purified gene 4 protein from both T7-infected cells and from a clone expressing gene 4 by this procedure. Purifi- cation of gene 4 protein from the clone is presented below (summarized in Table I).

Hydroxylapatite Chromatography-Fraction IV of gene 4 protein (1.92 mg; see “Experimental Procedures”) was loaded onto a hydroxylapaptite column (2.7 cm2 x 4 cm) equilibrated with buffer C (20 mM potassium phosphate, pH 7.0, 0.1 mM

TABLE I Purification of gene 4 protein

The activity of the gene 4 protein was measured by the RNA- primed DNA synthesis assay except for measurements marked “SD” (strand-displacement DNA synthesis assay using the preformed fork) and “ P R (primer synthesis assay measuring CMP incorporation). The assays and unit definitions are described under “Experimental Procedures.” Fraction VA, both molecular weight forms of gene 4 protein; Fraction VB, the lower molecular weight form of gene 4 protein (see Fig. 1).

Fraction Step Protein Total Specific activity

mg units unitslmg I Extract (from 126 g 5,100

of cell paste) 11. Ammonium sulfate 2,900 111. Phosphocellulose 50 27,000 540 IV. DEAE-Sephadex 1.9 7,600 4,000 VA. Hydroxyapatite 0.089 1,500 17,000

74,000 (SD) 44,000 (PR)

91,000 (SD) 6,000 (PR)

VB. 0.13 360 2,800

dithiothreitol, 1 mM sodium citrate, 10% (w/v) glycerol) a t a flow rate of 10.5 ml/h. The column was washed with 15 ml of buffer C, and gene 4 protein was eluted with a 120-ml linear gradient of 20-500 mM potassium phosphate in buffer C. The profile of gene 4 primase activity is shown in Fig. 1B. The first half of the primase peak corresponds to fractions containing approximately equimolar amounts of the large and small forms of gene 4 protein (Fig. LA). A shoulder of primase activity elutes at higher phosphate concentrations (280-400 mM potassium phosphate). These fractions contain predominantly the small form of gene 4 protein (Fig. lA, Fractions 40-48). The large form is not detectable in these fractions on the sodium dodecyl sulfate-polyacrylamide gel shown in Fig. L4.

The gene 4 protein eluting from the hydroxylapatite column was pooled into two fractions. The pooled fractions were dialyzed for 12 h against 2 liters of buffer A containing 20% (w/v) glycerol and 12 h against 2 liters of buffer A containing 50% (v/v) glycerol (final volumes: Fraction VA, 8.5 ml; Frac- tion VB, 10.2 ml; stored at -20 “C). Fraction VA (column fractions 24-32) consisted of approximately equimolar amounts of the two molecular weight forms. Fraction VB (column fractions 41-52) consisted predominantly of the small form. Gene 4 protein in both these fractions was greater than 90% pure, judged by resolving polypeptides on a sodium dodecyl sulfate-polyacrylamide gel.

The Small Form of Gene 4 Protein Is Deficient in Primer Synthesis-The specific activities of purified gene 4 protein preparations were measured by three assays, designed to compare the helicase and primase activities (Table I). The small form (Fraction VB) is as active, if not more active, as the mixture of the two forms (Fraction VA) in promoting strand-displacement DNA synthesis by T7 DNA polymerase on the preformed replication fork, a measure of its helicase activity. However, it has 6-7-fold less primase activity as measured by the RNA-primed DNA synthesis assay and the

A Frocf;on L 16 18 20 22 24 26 28 30 32 34 35 38 40 42 44 46 4R .. .

4A - 48- *

0 1 0 20 30 40 50 60 Fraction

FIG. 1. Resolution of gene 4 protein by hydroxylapatite chromatography. Fraction IV (1.92 mg) of gene 4 protein, prepared as described under “Experimental Procedures,” was loaded onto a hydroxylapatite column (2.7 cm2 X 4 cm). Gene 4 protein was eluted with a linear gradient (120 ml) from 20 to 400 mM potassium phosphate (+) in buffer C as described in the text, and 2-ml fractions were collected. 5-111 portions of each fraction were resolved on a sodium dodecyl sulfate-polyacrylamide (10% acrylamide) gel ( A ) , and primase activity (El) in each fraction was measured by assaying 4 p1 of each fraction in the RNA-primed DNA synthesis reaction (B; see “Experimental Procedures”). 4A, large form of gene 4 protein; 4B, small form of gene 4 protein; L, Fraction IV, which was loaded onto the hydroxylapatite column.


+ 0 ng large form

0 20 40 60 8 0 100 Small Form, Gene 4 Protein (ng)

FIG. 2. Submolar amounts of the large form of gene 4 protein complement the small form to restore high specific activity of primase. Primase activity was measured by the RNA-primed DNA synthesis assay as described under “Experimental Procedures.” 0 (m), 5 (+), an 10 ng (E) of the large form of gene 4 protein was added to reactions by addition of Fraction VA (equimolar mixtures of both forms; see Fig. 1 and Table I), 50% of the protein in this fraction being the large form. At each concentration of the large form, the amount of short form was varied by the addition of Fraction VB (small form). Reaction mixtures were incubated at 37 “C for 20 min. The results were plotted, taking into account the contribution of small form from both Fraction VA and Fraction VB. We were unable to achieve a sufficiently high concentration of the small form that was saturating with respect to the large form in this primase assay.

primer synthesis assay. The latter assay measures CMP incorporation by the gene 4 protein in the absence of T7 DNA polymerase.

Submolar Amounts of the Large Form of Gene 4 Protein Greatly Stimulate Primase Activity of the Small Form-We were prompted to examine the effect of the two preparations of gene 4 protein on each other for two reasons. First, the small form, isolated from cells that do not produce the large form: has been shown to have DNA-dependent NTPase and helicase activities but no primase activity (Bernstein and Richardson, 1988). The preparation of small form (Fraction VB) described in this paper has a low but detectable primase activity even though contamination of this preparation with large form can be no more than 0.5% (estimated from the gel analysis shown in Fig. lA). We wished to determine whether trace amounts of large form can account for the low levels of primase activity. Second, the number of total units (RNA- primed DNA synthesis assay) in the pool of the two molecular weight forms (Fraction VA, Table I) is 4-5 times greater than expected from assays of the individual column fractions (Fig. 1B). Pooling of the fractions increases the specific activity of the gene 4 primase. On the other hand, the pool of fractions containing predominantly the small form (Fraction VB, Table I) has approximately 2 times more activity than that present in the individual column fractions.

In the experiment shown in Fig. 2, RNA-primed DNA synthesis was catalyzed in the presence of varying amounts of small form (Fraction VB) in reaction mixtures containing a constant amount of Fraction VA (hence a constant amount of the large form of gene 4 protein). The amount of DNA synthesis catalyzed is plotted as a function of the concentration of small form, taking into account the contribution of small form from Fraction VA. A linear relationship is maintained as the concentration of small form is varied at a constant concentration of large form, the ratio of small form

S. Tabor, J. Bernstein, and C. C. Richardson, unpublished results. ~~

to large form ranging from 1:l to 16:l. The slope of this linear relationship is increased as the amount of large form added to each reaction is increased, reflecting an increase in specific activity. A mixture of 10 ng of large form and 87 ng of small form has a specific activity of 14,000 units/mg, greater than 80% the specific activity of 17,000 units/mg of Fraction VA (equimolar mixture). We obtained identical results mixing Fractions VA and VB and measuring activity with the primer synthesis assay, in which we measure CMP incorporation by gene 4 protein (data not shown). Thus, submolar amounts of the large form of gene 4 protein are sufficient to restore high specific activity of primase present in a mixture of the two molecular weight forms.

The results suggest that trace quantities of large form may account for primase activity found in Fraction VB. We have verified that the small form, purified from cells that do not produce the large form (Bernstein and Richardson, 1988), can greatly stimulate primase activity present in Fraction VA (equimolar mixture) although it has no primase activity by itself (data not shown). These results indicate that the small form, although deficient in primase activity, can be a component in the assembly of an active primase. We will examine the significance of these results under “Discussion.”

In most of the experiments that follow, Fraction VA (both molecular weight forms) of gene 4 protein is used. Thus, we simply refer to this fraction as gene 4 protein, specifically referring to the small form of gene 4 protein when Fraction VB is used.

Leading Strand Synthesis on the Preformed Replication Fork The Preformed Replication Fork-The preformed fork is a

circular duplex DNA with a protruding 5”single-stranded tail. T7 DNA polymerase and gene 4 protein catalyze strand displacement synthesis at a rate of 300 nucleotides/s on this template (Lechner and Richardson, 1983). The original procedure for preparing the preformed fork involves annealing single-stranded M13 DNA with denatured and linearized M13 RF DNA, to which has been inserted a 237-base pair fragment of SV40. This is a fairly laborious procedure in which 2 mg of M13 DNA and 400 pg of M13 RF DNA are required to yield 32 pg of the preformed fork (Lechner and Richardson, 1983). We have modified the procedure such that at least 100 pg of purified preformed fork can be prepared from 200 pg of M13 DNA. By this procedure synthetic oligonucleotides are annealed to M13 DNA. These primed M13 DNA molecules are then converted to circular duplexes by DNA synthesis catalyzed by T7 DNA polymerase. The oligonucleotides used as primers are not fully complementary to the template so that the resulting duplex has a protruding 5”single-stranded tail.

Two types of oligonucleotides are used as primers. One is the commercially available heteropolymer poly(dA,dC) (average length, 170 bases). Preformed forks prepared using these oligonucleotides have a protruding single-stranded poly(dA,dC) sequence at the 5’-end. These templates have single-stranded tails of varying lengths arising at random sites on the M13 duplex DNA with the majority of the templates having only a single tail. This template is ideal for use in assaying gene 4 protein’s ability to promote DNA synthesis by T7 DNA polymerase through a duplex region.

However, in some experiments it is preferable to have a more defined template. For this purpose we utilize a synthetic 50-mer as a primer. 17 bases on the 3’-end of this molecule are complementary to a unique site (BamHI site) on M13mp6 DNA. Conversion of M13 DNA primed with this oligonucleotide results in a preformed fork analogous to the preformed fork previously described (Lechner and Richardson, 1983) but


having a 33-nucleotide instead of a 237-nucleotide SV40 sequence as a tail. In the experiments we will describe, the two types of preformed forks are interchangeable except that DNA synthesis may be initiated slightly more efficiently with the preformed fork bearing the poly(dA,dC) tail, most likely due to the longer single-stranded tail the average template has for the gene 4 protein to bind.

Leading Strand Synthesis Is a Highly Processive Reaction- As shown in Fig. 3, T7 DNA polymerase and gene 4 protein catalyze a rolling circle mode of strand-displacement DNA synthesis on a preformed fork, resulting in the accumulation of DNA products greater than 7.2 kb in length. The product of 7.2 kb corresponds to the linear strand of the preformed fork, most likely labeled at the 3'-ends by the alternating action of the 3'- to 5'-exonuclease and polymerase of T7 DNA polymerase. In the presence of limiting amounts of DNA polymerase (Fig. 3, reactions 1-9), high molecular weight products accumulate with similar kinetics. These results indicate that the DNA polymerase is catalyzing DNA synthesis by a highly processive mechanism, in which i t polymerizes greater than 40 kb of nucleotides without dissociating from the template. A similar experiment was performed by limiting the amount of gene 4 protein (Fig. 3, reactions 10-18). I t appears that high molecular weight products accumulate faster when the gene 4 protein concentration is lower, indicating that the rate of initiation of DNA synthesis or the rate of fork movement is slower at the higher gene 4 protein concentration.

Leading Strand Synthesis Is Resistant to Challenge with Single-stranded DNA Competitor-One difficulty in inter- preting the experiments shown in Fig. 3 is that the structure

A ) T7 DNA Polymerase B) Gene 4 Protein

nM 0.65 6.5 65 8 33 67 nnn nnn

Minutes 2 4 10 2 4 10 2 4 10 2 4 10 2 4 10 2 4 10 kb 40 -

12 - 9.4 -

7.2(M13) - 6.2 - 5.2 -

2.2 -

1 2 3 4 5 6 7 8 9 10 I t 12 13 14 15 1617 18 FIG. 3. Time course of leading strand synthesis at varying

concentrations of T7 DNA polymerase and gene 4 protein. Reaction mixtures (20 pl) contained reaction buffer, 50 mM NaCI, 600 p~ each of [ c ~ - ~ ' P ] ~ A T P (400 mCi/mmol), dCTP, dGTP, and dTTP, 0.1 pg of the preformed fork (FC33; 1.0 nM, in equivalents of 3'-ends), and the following amounts of T7 gene 4 protein and DNA polymerase. A , 52 ng (43 nM) of gene 4 protein plus 1.2 ng (0.65 nM), 12 ng (6.5 nM), or 120 ng (65 nM) of T7 DNA polymerase; B, 120 ng (65 nM) of T7 DNA polymerase plus 10 ng (8 nM), 40 ng (33 nM), or 80 ng (67 nM) of gene 4 protein (Fraction VA). Reactions were initiated by addition of reaction buffer, NTPs, and template to a mixture (8 pl) of gene 4 protein and DNA polymerase in the standard enzyme diluent. Incubation was at 30 "C. 6-pl portions were withdrawn at 2,4, and 8 min and mixed with 2 pl of 200 mM EDTA (pH 8). DNA was denatured and resolved on an alkaline agarose gel (0.796, see "Experimental Procedures"). For reference, in the reaction shown in lane 6, approximately 10% of the forked molecules have been extended greater than 40 kb.

of the replication fork is altered during the course of DNA synthesis. With the movement of the replication fork, the protruding single-stranded tail increases in length, thus increasing the target to which gene 4 protein may bind and gain access to the fork. I t is, therefore, possible that the gene 4 protein dissociates after unwinding a limited stretch of DNA but more readily reassociates with a replicated template, which bears a much longer single-stranded tail than unrepli- cated templates. In order to eliminate this possibility, we have performed competition experiments with single-stranded DNA.

When gene 4 protein and DNA polymerase are added to a mixture of the preformed fork and a 10-fold excess of single- stranded circular DNA of bacteriophage fl, DNA synthesis on the preformed fork is inhibited (Fig. 4) . That is, the excess single-stranded template competitively inhibits strand-displacement DNA synthesis. High molecular weight products greater than 40 kb in length do not accumulate in the presence of excess single-stranded competitor DNA (reactions 1-3) as they do in its absence (reactions 4-6). The major product of DNA synthesis in reaction mixtures which contain fl DNA is fl linear duplex DNA (RFIII). Small amounts of single- stranded linear fl DNA which contaminates the fl DNA preparation prime its own replication by the DNA polymerase.

f l DNA Competitor

kb 40 -

12.8 ( f l RFIII) - 7.2 (MI31 +

unm

6.4(f l ) - ,

1 2 3 4 5 6 FIG. 4. Single-stranded competitor DNA blocks initiation

of leading strand synthesis. Reaction mixtures (75 pl) contained reaction buffer, 50 mM NaCI, 300 p~ each of [ C ~ - ~ ' P ] ~ A T P (400 mCi/ mmol), dCTP, dGTP, and dTTP, 800 ng of gene 4 protein, 345 ng of T7 DNA polymerase, 1 pg of preformed replication fork, (FCA.~), and 0 pg (reactions 4-6) or 10 pg (reactions 1-3) of single-stranded fl DNA. The reaction was initiated by addition of a mixture (15 pl) of gene 4 protein and T7 DNA polymerase in standard enzyme diluent to a mixture (60 pl) of DNA, nucleotides, reaction buffer, and NaCI. Incubation was a t 30 "C. 2 0 4 portions were withdrawn at the indicated times, mixed with 2 p1 of 200 mM EDTA (pH 8), and chilled on ice. DNA products were denatured and resolved on an alkaline agarose gel (0.7%).


The product is a hairpin, which, after denaturation, is twice the length of the fl template.

Preincubation of gene 4 protein, DNA polymerase, and the preformed fork in the absence of dNTPs permits a very small amount of DNA synthesis to initiate upon addition of dNTPs and competitor DNA (Fig. 5, reaction 3; a discrete band of higher than 40 kb is clearly visible upon long exposure). In the absence of dNTPs, the gene 4 protein cannot bind to single-stranded DNA, and hence it is likely bound via its affinity for T7 DNA polymerase, which is bound to the template in the absence of dNTPs (Nakai and Richardson, 1986a). Clearly, the amount of DNA synthesis is very small compared to reactions which contain no competitor DNA (reactions 4-6) or reactions in which the addition of competitor DNA is delayed until 0.5 min (reactions 7-9) or 1 min (reactions 10-12) after DNA synthesis has been initiated by the addition of [cu-32P]dNTPs.

The presence of dTTP in the preincubation mix promotes the formation of more initiating replication complexes of gene 4 protein and T 7 DNA polymerase to be established at the fork, enabling a higher amount of DNA synthesis to proceed in the presence of excess competitor DNA (Fig. 6, reactions 1-3; cf. Fig. 5, reactions 1-3, when preincubation is in the absence of NTPs). Still, the addition of competitor DNA reduces the number of replication complexes (Fig. 6, reactions 1-3; cf. reactions 4-6 with no competitor DNA). As in the experiment shown in Fig. 5, the number of replication complexes is increased if DNA synthesis is allowed to initiate before addition of competitor DNA (Fig. 6, lanes 7-12). Once initiated, leading strand synthesis proceeds with almost identical kinetics in the presence or absence of competitor DNA (reactions 1-12). We conclude from these results that leading strand synthesis is a highly processive reaction in which greater than 40 kb of nucleotides are polymerized without dissociation of gene 4 protein or DNA polymerase from the template. If it were not processive, single-stranded competitor

A B C D

f i DNA No f l DNA f l DNA ot 0 min f l DNA ot 0.5 min at I min

Mnufes 2 4 10 2 4 10 2 4 10 2 4 10

kb 40 -

i2.8 ( f l RFIU) -.

7.2 (M13) -. 6.4(fl)-.

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

FIG. 5. A stable complex of gene 4 protein, DNA polymerase, and the preformed fork is not formed in the absence of NTPs. Stage I, the preformed fork (1.0 pg of FCA,c), gene 4 protein (400 ng), and T 7 DNA polymerase (345 ng) were preincubated in 25- p1 reaction mixtures containing reaction buffer and 50 mM NaCl for 5 min. Stage 11, DNA synthesis was initiated by addition of a mixture (15 p l ) containing reaction buffer and [ C X - ~ ~ P I ~ A T P (400 mCi/mmol), dCTP, dGTP, and dTTP to a final concentration of 300 p~ each. In reaction A single-stranded competitor DNA (10 pg) was introduced at this stage with the dNTPs. Stage 111, a mixture (20 pl) containing reaction buffer and 300 p~ each of [a-"PIdATP (400 mCi/mmol), dCTP, dGTP, and dTTP, and 0 pg (A, B ) or 10 pg (C, D) of f l DNA was added. The mixture was introduced 1 min (A, B, D) or 0.5 min (C) after initiating DNA synthesis. Reaction mixtures were incubated during Stages 1-111 a t 30 "C. 15-p1 portions of reactions A-D were withdrawn at the indicated times after initiating DNA synthesis, mixed with 1.5 pl of 200 mM EDTA, and chilled on ice. DNA products were denatured and resolved on an alkaline agarose gel (0.7%).

-. w a ..

" W U .I.-- . . . . . . . -. .. I .* .. H

A B C D

f l DNA No f l DNA f l DNA of 0 min f l DNA ot 0.5 min ot 1 min

Mnufl5 2 4 10 2 4 10 2 4 10 2 4 10

H ' sa

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

FIG. 6. Formation of a replication fork complex resistant to single-stranded competitor DNA in the presence of dTTP. Reactions were carried out as indicated in the legend to Fig. 5 except that 1 mM d T T P was present in the preincubation mixture during Stage I.

DNA should greatly reduce the rate of fork movement. I t is likely that 40 kb is a conservative estimate for the processivity of this reaction; we are constrained by the limited resolution of DNA greater than 40 kb on agarose gels.

The presence of dTTP enables gene 4 protein to bind directly to the single-stranded tail of the preformed fork. I t is likely that this binding promotes the formation of a stable gene 4 protein-DNA polymerase-template complex for the catalysis of processive strand-displacement synthesis. The complex most stable to challenge by single-stranded competitor DNA is formed when DNA synthesis is allowed to initiate (Fig. 5, reactions 7-12; Fig. 6, reactions 7-12). The presence of three dNTPs (including dTTP) during preincubation al- lows limited DNA synthesis to proceed but does not result in the formation of a complex more resistant to competitor DNA than that formed in the presence of only dTTP (data not shown). Thus, the gene 4 protein-DNA polymerase complex that is actively engaged in catalyzing leading strand synthesis is the most resistant to disruption by competitor DNA.

Leading and Lagging Strand Synthesis on the Preformed Fork

In the previous section we specifically examined the helicase activity of gene 4 protein at the replication fork by omitting rNTPs from the reaction. When ATP and CTP are included in the reaction, gene 4 protein may catalyze RNA primer synthesis to initiate lagging strand synthesis (Fig. 7, reaction 1). Most of the Okazaki fragments are 1-6 kb in length, consistent with the size range of 1-6 kb for Okazaki fragments characterized in uiuo (Masamune et al., 1971; Sternglanz et al., 1976; Shinozaki and Okazaki, 1977; Seki and Okazaki, 1979). I t is of interest to note that the small form of gene 4 protein (Fraction VB) also efficiently catalyzes leading strand synthesis (Fig. 7, reaction 2); however, the reduced primase activity present in Fraction VB is reflected in the relatively small number of Okazaki fragments, which on average are higher in molecular weight than those geneated by both forms. The frequency with which lagging strand synthesis is initiated is increased if the concentration of Fraction VA or VB is raised (data not shown). That is, raising the gene 4 protein concentration increases the number of Okazaki fragments synthesized and tends to decrease their size.

We can confirm that the observed Okazaki fragments arise from lagging strand synthesis on the displaced strand of the preformed fork. Fig. 8A shows a map of the preformed fork indicating the location of the major gene 4 recognition sites


kb 40 - 1 2 - 9.4 -

7.2(M13) * - 6.2 - 5.2

2.2 - 2.0 - 1.4 - 1.0 -

1 2 FIG. 7. Leading and lagging strand synthesis catalyzed by

the gene 4 protein on the preformed replication fork. Reaction mixtures (10 pl) contained reaction buffer, 600 FM each of [a-'*P] dATP (400 mCi/mmol), dCTP, dGTP, and dTTP, 300 FM each of ATP and CTP, 50 ng of preformed fork (FC-33; 1.0 nM in equivalents of 3'-ends), 62 ng of T 7 DNA polymerase (68 nM), and 10 ng of gene 4 protein (17 nM). Either Fraction VA (reaction 1, equimolar mixture of the two molecular weight forms) or Fraction VB (reaction 2, the smaller molecular weight form of gene 4 protein) was used. Incubation was a t 37 "C for 10 min. The reaction was stopped by adding 1 pl of 200 mM EDTA and chilling on ice. DNA products were denatured and resolved on an alkaline agarose gel (1%).

on this strand. The gene 4 primase initiates DNA synthesis by synthesizing tetraribonucleotide primers (pppACNN) a t the major recognition sequences T/GGGTC and GTGTC; it initiates DNA synthesis less frequently a t minor recognition sites, which consist of other sites with the pentanucleotide sequence NNGTC (Tabor and Richardson, 1981; Nakai and Richardson, 1986b). To map the sites at which RNA-primed DNA synthesis is being initiated, we carried out reactions in the presence of [y3*P]ATP to specifically label Okazaki fragments at the 5'-ends. RNA/DNA products of the reaction were then digested with restriction endonuclease HaeII, which cleaves at the six sites designated in Fig. 8A. Such treatment yields labeled fragments of a prescribed length, the 5'-end determined by the primase recognition site and the 3'-end by the restriction site. In the presence of ATP and CTP, the gene 4 protein initiates RNA-primed DNA synthesis predominantly a t the major primase recognition sites on the lagging strand template (Fig. 8B, reaction 1). The presence of GTP and UTP as well as ATP and CTP does not dramatically alter the frequency of usage of gene 4 recognition sites (Fig. 8B, reaction 2).

Lagging Strand Synthesis Is Inhibited by Challenge with Single-stranded DNA Competitor-We established in a previous section that T7 gene 4 protein, DNA polymerase, and the preformed fork form a stable complex so that leading strand synthesis is resistant to challenge by competitor DNA. An important question is whether such a stable complex is

HI64091

bl167l

B bases

- I2039 - 9360 -6165 - 5i69

a 'b- * -2215 - 2009

- 1353 c"

8 -975 9 - '-111 0-53*

- 114

t \H13039) HI27101 I 2

FIG. 8. Initiation sites for RNA-primed DNA synthesis on the preformed fork. A , map of the preformed replication fork: Sites of the major recognition sequences GGGTC (d, f ) , TGGTC (a, e, g), and GTGTC (b , c ) on the lagging strand template are shown. The solid arrows indicate cleavage sites of restriction endonuclease HaeII. B, extension of labeled primers, synthesized by gene 4 protein, with T7 DNA polymerase. The reaction mixture (25 pl) contained reaction buffer, 0.5 pg of the preformed fork (FCA,~) , 50 mM NaCI, 500 ng of T 7 DNA polymerase, 200 ng of gene 4 protein, 300 p M each of M T P , dCTP, dGTP, and dTTP, 50 p~ [y-"P]ATP (80 Ci/mmol), and either 50 p~ CTP (reaction 1) or 50 p M each of CTP, GTP, and UTP

stopped by addition of 2.5 p1 of 200 mM EDTA (pH 8). RNA/DNA (reaction 2). Incubation was at 30 "C for 10 min. Reactions were

products were digested with restriction endonuclease HaeII, denatured, glyoxalated, and resolved on a 1.4% agarose gel (buffer system: 10 mM sodium phosphate, pH 7.0; see "Experimental Procedures").

formed for the catalysis of lagging strand synthesis. In the experiment shown in Fig. 9, leading and lagging

strand synthesis catalyzed by T 7 DNA polymerase and gene 4 protein was allowed to proceed on the preformed fork for 5 min in the presence of unlabeled ATP, CTP, and dNTPs. Single-stranded fl DNA competitor and either [a-"PIdATP or [cY-~'P]CTP are simultaneously added to the actively rep- licating fork, and the mixture was incubated for an additional 10 min. When [a-"PIdATP is added with competitor DNA (Fig. 9, reaction 2), leading strand synthesis, represented by the high molecular weight product (>40 kb), is inhibited to approximately 10-20% the level observed in the absence of competitor (reaction 1). When [a-"PICTP is introduced to specifically label RNA primers, RNA/DNA products of the reaction are digested with HaeII and resolved on a denaturing gel. When lagging strand synthesis is examined in this way, it is evident that the number of primers synthesized and extended on the preformed fork in the presence of excess competitor is reduced to less than 1% the level in its absence. RNA-primed DNA synthesis proceeds almost exclusively on the more abundant fl template.

The 10-20-fold greater inhibition of lagging strand synthesis indicates that although leading strand synthesis proceeds by a highly processive mechanism, lagging strand synthesis does not. Either gene 4 protein or T 7 DNA polymerase, or both, dissociate from the template after each round of lagging strand synthesis. The experiment shown in Fig. 9 has been performed at a wide range of T 7 DNA polymerase concentrations (data not shown). Even when the actively propagating fork is challenged with competitor DNA in the presence of a large excess of DNA polymerase, lagging strand synthesis is severely inhibited. These results suggest that the gene 4 protein molecules acting as helicase and promoting strand- displacement DNA synthesis are not efficiently catalyzing primer synthesis for lagging strand synthesis. RNA-primed

9826

A

a-32Pl dATP m

Preformed fork t + - f1DNA - + +

Distinct Properties of T7 Gene 4 Helicase and Pr imase

B

[ a-32P 1 CTP

+ + - - t t

m

kb 40 - a-1

bases

12.8(fl RFII), ~

1 2 3

i

m - 86- 2600 a'b

00 - i400

- 750 e -600 d

- 320f - 1909

rrL 4 5 6

FIG. 9. Effect of single-stranded competitor DNA on leading and lagging strand synthesis at the replication fork. Stage I, reaction mixtures (20 pl) contained reaction buffer, 50 mM NaC1, 600 p~ each of dATP, dCTP, dGTP, and dTTP, 300 p~ each of ATP and CTP, 0 pg (reactions 3 and 6) or 0.1 pg (reactions 1, 2,4, and 5) of the preformed replication fork (FC33), 31 ng of T7 DNA polymerase, and 190 ng of gene 4 protein. Incubation was a t 30 "C for 5 min, allowing leading and lagging strand synthesis to proceed in the presence of unlabeled dNTPs and rNTPs. Stage 11, 20-pl mixtures containing reaction buffer, [cx-~*P]~ATP or [CX-~~PICTP, and 0 pg (reactions 1 and 4) or 1.0 pg (reactions 2,3,5, and 6) of fl DNA were added to each reaction mixture. The final concentrations of NTPs are as follows: reactions 1-3,600 p~ each of [ cx -~*P]~ATP (400 mCi/ mmol), dCTP, cGTP, and dTTP and 300 p~ each of ATP and CTP; reactions 4-6: 600 p~ each of dATP, dCTP, dGTP, and dTTP, 300 p~ ATP, and 50 p~ [w3'P]CTP (20 Ci/mmol). Incubation was a t 30 "C for 10 min. Reactions 1-3: RNA/DNA products were denatured with NaOH and resolved on an alkaline agarose gel (0.5%). Reactions 4-6: RNA/DNA products were digested with restriction endonuclease HaeII, denatured, glyoxalated, and resolved on a 1.4% agarose gel (buffer system: 10 mM sodium phosphate, pH 7.0; see "Experimental Procedures"). Labeled fragments correspond to initiation of RNA- primed DNA synthesis a t the primase recognition sites (a-g) shown in Fig. 8A.

DNA synthesis is catalyzed on the more abundant fl competitor DNA.

Leading Strand Synthesis Is Resistant to Dilution-We wished to confirm that the gene 4 protein acting as helicase does not efficiently act as primase. In the previous sections we demonstrated that when an actively propagating fork is challenged with single-stranded competitor DNA, lagging strand synthesis is more severely inhibited than leading strand synthesis. Both the gene 4 protein (in the presence of NTPs) and T7 DNA polymerase binds to single-stranded DNA (Nakai and Richardson, 1986a); therefore, single- stranded competitor DNA may restrict the availability of both T7 DNA polymerase and gene 4 protein to the replication fork. To examine the processivity of gene 4 protein as a helicase and primase independently of the availability of T7 DNA polymerase to the fork, we performed dilution experiments, which avoids the use of competitor DNA.

As shown in Fig. 10, leading strand synthesis is resistant to

Preincubotion No NTPs dCTP dTTP 4 dNTPs

findvo/umef'/l 20 200 20 200 20 200 20 200

kb a - m- a- ""

40 -

7.2 (M13)-,

1 2 3 4 5 6 7 8 FIG. 10. The complex of T7 replication proteins that cata-

lyze leading strand synthesis is resistant to disruption by dilution. Stage I: reaction mixtures (10 pl) contained reaction buffer and 50 mM NaCl plus no NTPs (reactions 1 and 2), 1 mM dCTP (reactions 3 and 4), 1 mM dTTP (reactions 5 and 6), or 600 p~ each of M T P , dCTP, dGTP, and dTTP (reactions 7 and 81,O.l pg of the preformed fork (FC& 125 ng of T 7 DNA polymerase (140 nM), and 21 ng of gene 4 protein. Incubation was a t 30" C for 5 min. Stage 11, reaction mixtures were diluted to 20 pl (reactions 1, 3, 5, and 7) or 200 pl (reactions 2,4,6, and 8). The diluent contained reaction buffer, 50 m" NaCI, 140 nM DNA polymerase, and enough dATP, dCTP, dGTP, and dTTP to adjust the reaction mixture to a final concentration of 600 p~ each and enough [a-"P]dATP (600 Ci/mmol) to adjust the specific activity of [cx-~'P]~ATP to 400 mCi/mmol. Incubation was a t 30 "C for 10 min. Reactions were stopped by addition of EDTA (pH 8) to a final concentration of 10 mM. DNA products were resolved on an alkaline agarose gel (1%).

dilution provided that a gene 4 protein-DNA polymerase- preformed fork complex is allowed to form in the presence of dTTP (reactions 5 and 6). Consistent with the results obtained with the use of competitor DNA (Fig. 6), complexes formed in the presence of dTTP (reactions 5 and 6) are slightly less resistant to dilution than the actively propagating fork (reactions 7 and 8). When the preformed fork is preincubated with gene 4 protein and DNA polymerase in the absence of NTPs or in the presence of dCTP, which is unable to promote binding of gene 4 protein to single-stranded DNA (Matson and Richardson, 1985), DNA synthesis proceeds efficiently only in the concentrated reactions (20 pl; reactions 1 and 3) and not in the dilute reactions (200 pl; reactions 2 and 4). This result indicates that the gene 4 protein and DNA polymerase have not had a chance to form an active complex at the replication fork before dilution. Since the diluent contains DNA polymerase to maintain the polymerase concentration, it is the gene 4 protein that is not efficiently associating with the single-stranded tail of the preformed fork in the dilute reactions. However, the results of the experiment shown in Fig. 10 are identical if no DNA polymerase is present in the diluent (data not shown).

Lagging Strand Synthesis Is Inhibited by Dilution-In the experiment shown in Fig. 11, leading and lagging strand synthesis was allowed to proceed on the preformed replication fork in the presence of unlabeled ATP, CTP, and dNTPs. The reaction was then diluted, introducing [ c ~ - ~ ~ P ] ~ A T P label and maintaining concentrations of all components except that of the template and gene 4 protein. As expected from the previous experiment, the radioactivity in the high molecular weight product (>40 kb), representing leading strand synthesis, does not diminish with greater dilution. In contrast, the Okazaki fragments that are 2-6 kb in length diminish in number with greater dilution. The Okazaki fragments synthesized become longer in length with greater dilution. We have confirmed that RNA-primed DNA synthesis on the lagging strand template is inhibited by dilution by repeating this analysis using [a-"PICTP label, digesting RNA/DNA prod-

Distinct Properties of T7 Gene 4 Helicase and Primase

::- 1 2 - 9.4-

7.2(M13)+ 6.2- 1 5.2 -

a c

c

cc 2.2 - 2.0 - 1.4-

1.0 -

1 2 3 4 FIG. 11. The number of Okazaki fragments generated at the

replication fork is diminished by dilution. Stage I, reaction mixtures (10 pl) contained reaction buffer, 50 mM NaC1.600 p M each of dATP, dCTP, dGTP, and dTTP, 300 p~ ATP, 50 p~ CTP, 0.1 pg of the preformed fork (FC33), 125 ng of T7 DNA polymerase (140 nM), and 21 ng of gene 4 protein. Incubation was at 30 "C for 5 min to allow leading and lagging strand synthesis to proceed in the presence of unlabeled NTPs. Stage 11, reaction mixtures were diluted to a final volume of 20 pl (reaction l ) , 50 pl (reaction 2), 100 pl (reaction 3), and 200 pl (reaction 4), and [a-"PIdATP was introduced. The diluent contained reaction buffer, 50 mM NaCI, 140 nM T7 DNA polymerase, 600 p~ each of dATP, dCTP, dGTP, and dTTP, 300 phi ATP, 50 p~ CTP, and enough [a-32P]dATP (600 Ci/mmol) to adjust the specific activity of [a-"PIdATP in each reaction to 400 mCi/ mmol. Incubation was at 30 "C for 10 min. The reaction was stopped by addition of EDTA (pH 8) to a final concentration of 20 mM and chilling the reaction mixture on ice. DNA products were denatured and resolved on an alkaline agarose gel (1%).

ucts with HaeII, and resolving labeled fragments on an agarose gel (Fig. 12).

The results indicate that gene 4 protein acting as helicase for processive leading strand synthesis does not catalyze multiple rounds of primer synthesis for lagging strand synthesis. Moreover, the results (along with results presented in Fig. 9) show the distributive nature of gene 4 primase. In the experiments shown in Figs. 10 and 11, leading and lagging strand synthesis is initiated in a concentrated reaction mixture (10 pl). This can be confirmed by including [ L Y - ~ ~ P I ~ A T P or [LY-~~PICTP in the reaction mixture (data not shown). Gene 4 protein can continue to catalyze primer synthesis to initiate lagging strand synthesis so long as the reaction mixture is maintained in concentrated form (20 pl; Figs. 11 and 12, reaction 1). RNA-primed DNA synthesis on the lagging strand template is inhibited upon dilution (200 pl; Fig. 11, reaction 4, and Fig. 12, reaction 2). Thus, the gene 4 protein cannot catalyze multiple rounds of primer synthesis as it translocates 5' to 3' along single-stranded DNA without dissociating from the template.

DISCUSSION

An intriguing property of the T7 gene 4 protein is that it is both a helicase and a primase. The consolidation of primase and helicase activities has striking parallels in other replica-

a/b- - c - =

e- d- ' -

f - 9-

1

9827

200

2 FIG. 12. Initiation of lagging strand synthesis at the repli-

cation fork is inhibited by dilution. Stage I, reaction mixtures (10 pl) contained reaction buffer, 50 mM NaC1,600 p~ each of dATP, dCTP, dGTP, and dTTP, 300 p~ ATP, 50 p~ CTP, 0.1 pg of the preformed fork (FC33), 125 ng of T7 DNA polymerase (140 nM), and 21 ng of gene 4 protein. Incubation was at 30 "C for 5 min. Stage 11, reaction mixtures were diluted to a final volume of 20 pl (reaction 1) and 200 pl (reaction 2), and [a-"PICTP was introduced. The diluent contained reaction buffer, 50 mM NaCl, 600 p~ each of dATP, dCTP, dGTP, and dTTP, 300 p~ ATP, 50 p~ CTP, and enough [a-32P]CTP (600 Ci/mmol) to adjust the specific activity to 20 Ci/mmol. Incuba- tion was at 30 "C for 10 min. The reaction was stopped by addition of EDTA (pH 8) to a final concentration of 20 mM and chilling the reaction mixture on ice. RNA/DNA products were digested with restriction endonuclease HaeII, denatured, glyoxalated, and resolved on a 1.4% agarose gel (see "Experimental Procecures"). Fragments marked a-g correspond to initiation of RNA-primed DNA synthesis at the major primase recognition sites depicted in Fig. 8A.

tion systems. The dnaB protein, the mobile constituent of the E. coli priming apparatus, is a helicase as well as a target site for the dnaG primase (McMacken et al., 1977; Arai et al., 1981; LeBowitz and McMacken, 1986). The gene 41 protein of bacteriophage T4 is a helicase which, as a complex with the gene 61 protein (an inefficient primase by itself), forms an efficient primase (Venkatesan et al., 1982; Hinton et al., 1985; Cha and Alberts, 1986; Hinton and Nossal, 1987; Nossal and Hinton, 1987). Both the E. coli dnaB protein and the T4 gene 41 protein are helicases which, like the T7 gene 4 protein, translocate 5' to 3' along single-stranded DNA (LeBowitz and McMacken, 1986; Venkatesan et dl., 1982).

The consolidation of helicase and primase activities is an important property to consider in understanding the design of the DNA replication apparatus. This is especially signifi- cant in light of models which envision the existence of a replisome, a multiprotein complex consisting of two DNA polymerase molecules, a helicase, and a primase and catalyz-

9828 Distinct Properties of T7 Gene 4 Helicase and P r i m a e

ing not only leading strand synthesis but also lagging strand synthesis without dissociating from the template (Sinha et al., 1980; Kornberg, 1982; Alberts, 1984). In these models the proteins catalyzing continuous leading strand synthesis provide the anchor to which proteins catalyzing lagging strand synthesis are bound. Hence, these proteins would be able to catalyze multiple rounds of RNA-primed DNA synthesis without dissociating from the replication fork.

Most consistent with these models is a mechanism in which the same gene 4 protein molecules serve as both helicase and primase at the replication fork. Preincubation of the preformed replication fork, gene 4 protein, and T7 DNA polymerase together with dTTP forms a replication fork complex which catalyzes leading strand synthesis by a highly processive mechanism (Figs. 6 and 10). Such a stable complex would be an ideal core of a replisome. However, our results indicate that the gene 4 protein acting as helicase in this complex does not efficiently serve as primase for lagging strand synthesis (Figs. 9, 11, and 12). Furthermore, the gene 4 protein cannot catalyze multiple rounds of primer synthesis as it translocates 5' to 3' along single-stranded DNA without dissociating from the template. Our dilution experiments indicated that lagging strand synthesis can proceed only so long as the template and gene 4 protein are maintained in a concentrated form in the reaction mixture (Figs. 11 and 12). Lagging strand synthesis is severely inhibited upon dilution whereas leading strand synthesis is not. Thus, multiple rounds of primer synthesis are dependent on multiple association/dissociation steps by gene 4 protein.

These results indicate that helicase action and primer synthesis are distinct reactions that are catalyzed by distinct molecules of gene 4 protein. Does each of the two molecular weight forms of gene 4 protein simply catalyze each of these two reactions? The small form has been purified from a clone which produces the small form but not the large form; pro- duction of the large form was eliminated by deleting the portion of gene 4 encoding its amino terminu~.~ The small form is a helicase and NTPase but is defective in primer synthesis, demonstrating that the large form is required for primer synthesis (Bernstein and Richardson, 1988). The question raised by these results is whether the small form partic- ipates in the catalysis of primer synthesis.

In this paper we have described the purification of the small form of gene 4 protein from an equimolar mixture of both forms. As it is separated from the large form, the small form retains helicase activity but loses primase activity (Table I and Fig. 7). Nevertheless, addition of submolar amounts of the large form to the small form is sufficient to restore the high levels of primase activity present in an equimolar mixture of both forms (Fig. 2). In reaction mixtures containing a constant amount of the large form, primase activity is pro- portional to the concentration of the small form over a wide range of ratios of small form to large (from 1:1 to 16:l). These results indicate that the small form can be a component of an active primase.

We have not yet purified the large form of gene 4 protein free of the small form. Thus, we do not know if the large form by itself may serve as helicase and primase. Three lines of evidence suggest that the small form as well as the large form of gene 4 protein is involved in the catalysis of primer synthesis. 1) The small form of gene 4 protein has no primase activity (Table I; Bernstein and Richardson, 1988); 2) submolar amounts of the large form can complement the small form in the formation of an active primase (Fig. 2); and 3) there is a tendency to enrich for the small form in gene 4

' S. Tabor, J. Bernstein, and C . C. Richardson, unpublished results.

protein purifications using the RNA-primed DNA synthesis assay (Fig. lA; see lane L, Fraction IV). However, conclusions as to whether the small form is an obligate component of primase or whether the large form may serve as helicase or primase by itself must await purification and characterization of the large form.

In proposing a mechanism for the dual role of gene 4 protein as helicase and primase at the replication fork, it is important to keep in mind a number of questions. What is the significance of consolidating helicase and primase activities? Why do two molecular weight forms of gene 4 protein exist? How does such a design facilitate the catalysis of reactions at the replication fork?

A central issue is whether helicase action for leading strand synthesis and primer synthesis for lagging strand synthesis are catalyzed in a coordinated manner. The consolidation of helicase and primase activities in the gene 4 protein may reflect a means for coordinating leading and lagging strand synthesis. It may also reflect economical usage of T7-encoded proteins. Gene 4 protein's ability to translocate 5' to 3' along single-stranded DNA is exploited not only for helicase action but also for primer synthesis, in which translocation facili- tates gene 4 protein's search for primase recognition sites along single-stranded DNA (Tabor and Richardson, 1981; Matson and Richardson, 1983).

Our results indicate that distinct molecules of gene 4 protein are required to provide helicase and primase activities at the fork. However, it is possible to propose a mechanism by which primer synthesis and helicase action are performed in a CO- ordinated fashion (Fig. 13). As we noted, the small form of gene 4 protein is fully active in promoting leading strand synthesis (Table I and Fig. 7; Bernstein and Richardson, 1988). Although deficient in primase activity, it contributes to the formation of an active primase (Fig. 2). We may postulate that the small form serves as helicase for processive leading strand synthesis. The large form of gene 4 protein oligomerizes with the small form acting as helicase to form an active primase (Fig. 13A). The small form may be envi- sioned as a counterpart of the bacteriophage T4 gene 41 protein and the E. coli dnaB protein, and its function as a helicase includes providing a target for the large form to bind. As the small form-large form complex encounters a primase recognition site while translocating 5' to 3' along single- stranded DNA, the large form binds at that site to catalyze primer synthesis (Fig. 13B). Upon initiating primer synthesis the large form dissociates from the small form, which continues to translocate 5' to 3' along single-stranded DNA. Upon synthesis of the primer and its extension by T7 DNA polymerase, the large form dissociates from the template and is available to catalyze primer synthesis at another replication fork, (Fig. 13C).

By this model the continuous leading strand synthesis need not stall momentarily for gene 4 protein to catalyze primer synthesis for lagging strand synthesis. Rather, leading strand synthesis may be a continuous process with the large form of gene 4 protein simply dissociating from the small form to catalyze primer synthesis. Lagging strand synthesis is inhibited by dilution, but leading strand synthesis is not (Figs. 11 and 12). Dilution of reaction mixtures may reduce the rate at which the large form of gene 4 protein oligomerizes with the small form. The distributive nature of primase activity may be an attribute of the large form of gene 4 protein, which moves from one template to another. By this model we may postulate the necessity of two molecular weight forms of gene 4 protein. The large form is required for primer synthesis. This protein stalls at the primase recognition site to synthe-


A

Gene 4 Protein

6

51 / /

3' 5'

\ / \ \ I

I I

I

I Y Thioredoxin

I

I

I Large Form I Smnll Form I

I I

I

Primer 3' 5' I

/ I

Ly /

FIG. 13. Model for the propagation of the replication fork. A , the small form of the gene 4 protein serves as helicase for leading strand synthesis by T7 DNA polymerase (gene 5 protein-thioredoxin complex). The large form of gene 4 protein binds to the small form to form an active primase. B , as the small form-large form complex encounters a primase recognition site, the large form binds at that site, dissociating from the small form, and catalyzes the synthesis of a tetraribonucleotide primer. The small form continues to serve as helicase for leading strand synthesis. Its movement 5' to 3' along single-stranded DNA is uninterrupted by primer synthesis. C, as DNA synthesis catalyzed by T7 DNA polymerase extends the primer, the large form dissociates from the template and is available to catalyze primer synthesis at another replication fork.

size a primer. It would thus be analogous to the gene 61 protein of bacteriophage T4 and the dnaG primase of E. coli. The small form, deficient in primase activity, is necessary for processive leading strand synthesis, for it would not stall at primase recognition sites.

This model does not exclude the possibility that the large form, in the absence of the small form, has both helicase and primase activities. For example, if the active primase complex consists of an oligomer of gene 4 protein components that include the large form, such an active primase complex may be composed entirely of the large form components. We have previously proposed that the amino terminus of the large form, not present in the small form, contains a domain that recognizes the primase recognition site (Bernstein and Rich- ardson, 1988). This domain contains a potential zinc finger that may bind specifically to the primase site. The small form, which lacks this domain, cannot catalyze template-directed primer synthesis; however, the small form as well as a mixture of both forms catalyze at low levels the random polymerization of rNTPs to dinucleotides in the absence of DNA template (Bernstein and Richardson, 1988). A domain that binds specifically to primase sites may impede the processive move-

ment of gene 4 protein along single-stranded DNA. The small form may be required not only to provide processive helicase action but also to serve as a processive component of the primase complex, providing a target to which the large form readily associates.

If both molecular weight forms of gene 4 protein are required for the assembly of an active primase, the formation of such a complex may be the rate-limiting step in primer synthesis. This is especially true if an active primase complex were disassembled after each round of primer synthesis. At a constant submolar concentration of the large form, a linear relationship between the concentration of small form and the level of primase activity is established (Fig. 2). Raising the concentration of small form may increase the rate of binding of small form to large, thus increasing the rate at which an active primase is formed.

The structure of gene 4 protein as a helicase or primase is not yet known. Is the helicase or primase an oligomer? Does the helicase or primase consist of one or both molecular weight forms? Sedimentation analysis on glycerol gradients has sug- gested the existence of oligomeric forms of gene 4 protein that are more active as primase than the monomeric forms (Hil- lenbrand et al., 1979). An important question is, can we form an oligomeric structure that can serve both as helicase and primase? The distributive nature of primase activity implies that such a structure would fall apart upon initiating a single round of RNA-primed DNA synthesis. If there is a replisome at the T7 replication fork which efficiently recycles proteins necessary for lagging strand synthesis, a major problem is how it efficiently reassembles an active primase for multiple rounds of primer synthesis. In proposing a model for the movement of the replication fork (Fig. 13), we have focused on the role of the two molecular weight forms of gene 4 protein in the propagation of the replication fork. Absent is a proposal for how DNA polymerase for lagging strand synthesis is recycled. Models for the coordinated catalysis of leading and lagging strand synthesis envision a replisome in which two DNA polymerase molecules are bound together at the replication fork (Sinha et al., 1980; Kornberg, 1982; Alberts, 1984). It is not yet clear whether two T7 DNA polymerase molecules may be bound together at the T7 replication fork.

It is possible that additional proteins are involved in promoting efficient recycling of proteins for lagging strand synthesis. We are currently examining whether additional T7- encoded proteins may promote efficient recycling of DNA polymerase and primase. In the accompanying paper (Nakai and Richardson, 1988) we describe how the T7 gene 2.5 protein, a single-stranded DNA-binding protein, greatly increases the efficiency with which lagging strand synthesis is initiated. The gene 2.5 protein may play an important role in recycling T7 DNA polymerase and gene 4 protein by inter- acting specifically with these proteins.

Acknowledgments-We thank Stan Tabor for helpful discussions throughout this work. We thank Julie Bernstein and Hans Huber for their critical reading of this paper.

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OF BIOLOGICAL Vol. 263, July 15, pp. 9818 … › content › 263 › 20 › 9818.full.pdfleading and lagging strand DNA synthesis, the unwinding of the helix to enable DNA polymerase