Proc. Natl. Ad. Sd. USA
Vol. 76, No. 9, pp. 4308-4312, September 1979
Biochemistry

A general priming system employing only dnaB protein and primase
for DNA replication

[mobile replication promoter/DNAdependent ATPase/single-strand binding protein/phage &X174/poly(dT)]

KEN-ICHI ARAI AND ARTHUR KORNBERC
Department of Biochemistry. Stanford University School of Medicine, Stanford, California 94305
Contributed by Arthur Kmberg, June 4,1979

ABSTRACT  Priming of phage 4x174 DNA synthesis is ef-
fected sim ly by dn8B protein and primase when the DNA is
not coatelby single-strand bindin protein (SSS). The five
prepriming proteins(n, n', n", i, and &aCprotein)required for
priming a SSBcoated 9x174 DNA circle are dispensable. The
dnaB protein-  ase priming system is also active on uncoated
phage G4 anE13 DNAs and on poly(dT). Multiple RNA
$rimers, 10-80 nucleotides long, are transcribed with patterns

istinctive for each DNA template. Formation of a stable dnnB
proteinaDNA com lex in the resence of primase and ATP
sup orts the hyposesis that 28B protein provides a mobile
repication promoter signal for primase.

Pathways for conversion of various singlestranded phage DNAs
to the duplex form are distinguished by the three different
mechanisms employed in priming initiation of DNA synthesis
(1-3). Priming replication of M13 DNA coated with single
strand binding protein (SSB) depends on synthesis of a unique
transcript by RNA polymerase (4,5). With coated G4 DNA,
primer synthesis is effected by primase (dnoC protein) (6-
8).
By contrast, primer synthesis on SSB-coated DNA of phage
4x174 (dX) demands the numerous host replication proteins
presumably required for initiations in discontinuous chromo-
some replication (nascent, Okazaki fragments). Resolution and
reconstitution of this multi-enzyme system provides insights
into how these proteins participate in the priming process
(9-11). DNA synthesis as well as RNA primer synthesis (12)
depends on SSB coating of @X DNA. In the process that pre-
cedes primase action, $X DNA is converted to a nucleoprotein
intermediate that includes dnaB protein and SSB (13-15).
Formation of the intermediate requires participation of at least
five additional proteins: proteins n, n`, n", i, and dnnc. The
stable replication intermediate catalyzes the synthesis of mul-
tiple primers on the 4X circle when primase is added (12,
14).
Upon extensive purification of each of the replication pro-
teins and their use in the reconstitution of the 4X system, a novel
feature of the priming reaction becomes apparent. In the ab-
sence of SSB, conversion of any single-stranded DNA to the
duplex form can be achieved simply by the joint action of dnaB
protein and primase to initiate synthesis by DNA polymerase
I11 holoenzyme. Uncoupled from DNA replication, dnaB
protein and primase, in the presence of ATP, form a complex
with a singlestranded DNA template and catalyze the synthesis
of short primers on the DNA. This report describes these find-
ings and discusses their significance for the physiological role
of dnaB protein as a mobile replication promoter.

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this fact.

MATERIALS AND METHODS

Materials. Buffer A is 100 mM Tris-HC1 (pH 7.5)/20%
(wt/vol) sucrme/40 mM dithiothreitol/ux) pg of bovine serum
albumin per ml. Buffer B is 20 mM Tris-HCI (pH 7.5)/8 mM
MgC12/0.1 mM EDTA/20 mM KC1/5% (wt/vol) sucrose/lOO
pg of bovine serum albumin per m1/5 mM dithiothreitol. 3H-
and 32P-labeld rNTPs and dNTPs and sodium bor~[~H]hy-
dride were purchased from New England Nuclear or from
Amersham Corp.
Extensively purified Escherichia colt replication proteins
were: SSB (fraction IV, 2 X 104 units/ml, 4 X 104 units/mg
(16)]; dnaB protein [fraction V, 1.3 X 106 units/ml, 6.2 x 105
units/mg (15)]; dnaC protein (fraction V, 1 X 105 units/ml, 6
X 104 units/mg); protein n' (fraction VII, 5 X 104 units/ml, 2.4
X 105 units/mg); proteins n + n" (fraction V,* 1.5 X 105
units/ml, 5 X l@ units/mg); protein i (fraction V, 1.5 X ld
units/ml, 8 X 105 units/mg); primase [fraction VI, 5 X 105
units/ml, 1.1 X 1@ units/mg (17)); DNA polymerase 111 ho-
loenzyme [fraction V, 8 X 104 units/ml, 3.8 X 105 units/mg
(18)]. Procedures for purification of proteins n, n'. n", and i and
dnaC protein will be described elsewhere.
Assay of DNA Replication. DNA synthesis was assayed as
described (16). Components were added at 0C in the following
order; 5 pl of buffer A; 0.2 pcmol of MgC12; 1.2 nmol each of
dATP, dCTP, and dGTP; 0.45 nmol of [3H]dTTP (specific
activity, 1500 dpm/pmol); 2.5 nmol each of GTP, CTP, and
UTP; 20 nmol of ATP; 40 nmol of spermidineHC1; 0.5 pg of
SSB; 60 ng of dnaB protein; 50 units of dnoC protein; 35 ng of
protein i; 40 units of proteins n + n"; 30 ng of protein n'; 60 ng
of primase; 0.2 pg of DNA polymerase 111 holoenzyme; 250
pmol (as nucleotide) of @X DNA; and water to 25 pl. For assay
of DNA synthesis catalyzed by dnaB protein, primase, and
DNA polymerase I11 holoenzyme, spermidineHCI was omitted
from the reaction mixture. Incubation was at 30C.
Assay of RNA Rimer Synthesis. For RNA primer synthesis
catalyzed by dnaB protein and primase, the reaction mixture
contained in 25 p1: 5 pl of buffer A; 0.2 pmol of MgC12; 0.5-0.75
nmol (as nucleotide) of DNA as indicated; 1.25 nmol each of
[3H]GTP, CTP, and UTP (each at 4000 dpm/pmol); 20 nmol
of ATP; 0.1 pg of rifampicin; 0.4 pg of dnaB protein; and 0.12
pg of primase. When poly(dT) (4 nmol, as nucleotide) was used
as template, 20 nmol of 13H]ATP (at 1000 dpm/pmol) was
present, and GTP, CTP, and UTP were omitted. Incubation
was at 30C. The amount of RNA primer synthesized was de-
termined by the DEAEcellulose filter procedure (12).

Abbreviations: 4X. phage 6x174; SSB, single-strand binding pro-
tein.
* Prdein n fraction used here was further resolved into two replication

enzymes (protein n and n") by J. Shlomai.

4308

Biochemistry: Arai and Kornberg

Formation and Isolation of 4X DNA*dnnB Protein Com-
plex. The reaction mixture contained in 25 pl: 5 pl of buffer A,
0.2 pmol of MgCle, 1.5 nmol (as nucleotide) of $X DNA, 20
nmol of ATP, 1.5 pg of 3H-labe1ed dnaB protein (8 X lo4
cpm/pg). and, when present, 0.4 pg of primase. After incu-
bation at 30C for 20 min, the amount of dnaB protein bound
to t$X DNA was determined by filtering the reaction mixture
through Bio-Gel A-5m agarose (0.2 X 15 cm) (equilibrated with
buffer B containing 0.5 mM ATP at 24C) and collecting the
excluded volume. The radioactivity in each fraction was de-
termined in a liquid scintillation spectrometer.
Labeling of dnaB protein was performed according to Rice
and Means (19) by using sodium b~ro[~H]hydride (7.2 Ci/
mmol; 1 Ci = 3.7 X 1Olo becquerels) and formaldehyde. The
labeled dnaB protein (8 X lo' cpmlpg) retained more than
90% of its activity in DNA replication. Details of the labeling
procedure will be described elsewhere.
Other Methods. Polyacrylamide gel electrophoresis in 7 M
urea and autoradiography were carried out as described (20,
21).

RESULTS

The Reconstituted System for Converting 4X DNA to the
Duplex Form. t$X viral DNA incubated with purified prepa-
rations of SSB. dnaB protein, dmC protein, protein i, proteins
n + n", protein n', primase, DNA polymerase 111 holoenzyme,
and all the rNTPs and dNTPs was almost completely converted
within 10 min to the duplex form. Omission of any one of sev-
eral proteins (e.g., dnaB, dnaC, i, n, n', nu, primase, or DNA
polymerase I11 holoenzyme) abolished the activity (Table 1).
The product of the reaction was the duplex replicative form I1
as judged by autoradiography and ethidium bromide fluores-
cence after separation by agarose gel electrophoresis (data not
shown).
Unlike the results obtained with less purified preparations
(9), the requirement for SSB was only partial and omission of
spermidine had no effect on overall synthesis. Moreover, the
requirement for SSB almost completely disappeared in the
absence of spermidine. The possibility that SSB was supplied
as a contaminant in some of the replication protein preparations
was eliminated by the lack of inhibition by anti-SSB antibody,
which inhibits DNA synthesis completely when SSB is present
(data not shown).
dnnB Protein, Primase, and DNA Polymerase 111 Ho-
loenzyme Suffice for DNA Replication in the Absence of
SSB. With uncoated t$X DNA as template, there was no re-
quirement for proteins i, n, n'. or n" (Table 2); dependency on

Proc. Natl. Acod. Sd. USA 76 (1979)

4309

Table 1.

Requirements for conversion of &X DNA to replicative

form 11 with purified replication proteins

Condition pmol

Complete 210
Minus dnaB protein 1
Minus dnaC protein 2
Minus protein i 3
Minus protein n + n*
Minus protein n' I
Minus primase 4
Minus DNA polymerase 111

holoenzyme <1
Minus SSB 51
Minus spermidine 205
Minus SSB and spermidine 180

dNMP incorporated,

2

Incubation was 10 min.

Table 2.  Requirements for conversion of &X DNA to replicative
    form I1 in the absence of SSB and spermidine

dNMP incorporated,

Condition pmol

Complete 95
Plus rifampicin (0.5 pg) 82
Plus SSB (0.5 pg) IO
Minus dnaR protein 6
Minus dnoC protein 38
Minus protein i 90
Minus protein n + n"              94
Minus protein n' 81
Minus primase 8
Minus DNA polymerase Ill

holoenzyme 1

DNA synthesis was measured as described in Materials and
Methods except that SSB and spermidine were not included in the
complete mixture. Incubation was 10 min.

dnaC protein was only partial. By contrast, dnaB protein,
primase. and DNA polymerase 111 holoenzyme were absolutely
required. Rifampicin had no demonstrable effect. Thus we
conclude that DNA synthesis is initiated by dnaB protein and
primase when DNA is not covered with SSB. These results
suggest that dnaB protein and primase provide primers that
are elongated by DNA polymerase 111 holoenzyme. It would
seem reasonable to expect that this system also catalyzes the
conversion of other singlestranded phage DNAs to their duplex
forms.
Template Specificity of a Phage DNA Replication System
Requires Coating of the DNA by SSB. The combination of
dnaB protein, primase, and DNA polymerase 111 holoenzyme
was effective for DNA synthesis on G4 and M13 phage DNAs
(Fig. 1). Again, initiation of DNA synthesis required dnaB
protein and primase. The stimulation of DNA synthesis by dnaB

I I I

a I

SSB CS)

FIG. 1.

     Inhibition by SSB of DNA synthesis catalyzed by dnaB
protein, primase, and DNA polymerase 111 holoenzyme. DNA syn-
thesis was measured as described in Materials and Methods except
that spermidine was omitted and 250 pmol (as nucleotide) of gX, C4,
or M13 DNA was used as template. The reaction mixtures (25 pl)
contained 0.1 pg of dnaB protein, 60 ng of primase, 0.2 pg of DNA
polymerase 111 holoenzyme, and amounts of SSB as indicated. Incu-
bation was 15 min.

4310 Biochemistry: Arai and Kornberg

Prac. Natl. Acud. Sd. USA 76 (1979)

Table 3.

Template specificity of the purified replication systems

DNA synthesis, pmol

DNA temolate G4 svstem* dX svstem'

4X 2 140
G4 220 180

DNA synthesis was measured as described in Materials and
Methods except that @X, G4, or M13 DNA (250 pmol, as nucleotide)
was used as template. Incubation was 10 min.
* No prepriming proteins; only primase, DNA polymerase 111 ho-

loenzyme, and SSB were included.
+ Prepriming proteins n, n', n', i, and dnoC were included, as well as
dnoB protein, primase, DNA polymerase 111 holoenzyme, and SSB.

M13 1 a

protein on G4 DNA was completely inhibited by anti-dnal
protein antibody but not by anti-SSB antibody. This result ex-
cludes the pibility of a SSB contaminant in the dnaB protein
preparation. The products of the reaction were duplex repli-
cative form 11 (data not shown). As anticipated, this replication
system failed to dimiminate among phage templates that were
not covered by SSB. However, in the presence of SB, DNA
synthesis on $X and M13 circles was profoundly inhibited (Fig.
1). Almost 90% of the activity was inhibited by an amount of
SSB that covered these single-stranded templates completely
(16). On the other hand, DNA synthesis on G4 circles was, as
expected, not significantly inhibited by any level of SSB.
These results suggest that, except for G4 DNA, SSB-coated
single-stranded DNA is inert for the priming system of dnaB
protein and primase. SSB-coated G4 DNA is unique in being
primed by primase itself (refs. 8 and 17 and Table 3). As ex-
pected, SSBcoated 4X WA showed the template specificity
observed in the complex replication system. In the presence of
prepriming proteins (n. n', n", i, and dnaC), SSB-coated DNA
was converted to the duplex form, except for SSB-coated M13
DNA, which remained inactive (Table 3). Thus, SSB determines
template specificity during the priming process.
dn8B Protein and Primase Catalyze the Synthesis of
Multiple Primers on +X, G4, and M13 DNAs. BNA primer
formation as thebasii for initiation of DNA synthesis by dnaB
protein and primase was demonstrated directly with $X DNA.
The amount of primer synthesized was surprisingly large; as
much as 30% of $X DNA could be converted to the DNA-RNA
hybrid form (Table 4). RNA synthesis was completely depen-
dent on dnaB protein, primase. and DNA template; it was
strongly inhibited by SSB. Similar results were also obtained
when G4 and M13 DNA were used as templates (data not
shown).
RNA primers were denatured and sized by electrophoresis
in a 12% polyacrylamide gel in 7 M urea (Fig. 2). On each of
the templates, multiple primers ranging in size from 10 to 60

Table 4.

Requirements for RNA primer synthesis on uncoated

$X DNA

RNA synthesized,

Condition pmol

Complete 189

Minus dnaB protein 2
Minus primase 1
Minus dnoB protein and primase       <I
Minus gX DNA <1
Plus SSB 9

RNA synthesis was measured as described in Moterials and
Methods except that 625 pmol (as nucleotide) of @X DNA was used
as template. Incubation was 30 min.

FIG. 2.  Electrophoresis of RNA primer transcripts. Reaction
mixtures (25 pl) contained 5 pi of buffer A; 0.2 pmol of MgClz; 1.25
nmol each of (a-32P]CTP, CTP, and UTP (each at 7000 dpm/pmol);
20 nmol of ATP 0.1 pg of rifampicin; 500 pmol (as nucleotide) of
phage DNA; 0.15 pg of primase; and additional components as indi-
cated. The poly(dT) reaction mixture contained [a-32P]ATP (loo0
dpm/pmol) and 4 nmol of poly(dT). Incubation was at 30C for 30
rnin. The reaction was terminated by cooling to ODC. To each sample
were added 50 pl of 95% ethanol (-2OoC), 2.5 pl of 3 M sodium acetate
(pH 5.51, and 30 pg (1 rl) of E. coli tRNA. After 4 hr at -2O"C, the
precipitates were collected by centrifugation and resuspended in 25
pl of deionized formamide. RNA transcripts were fractionated by
electrophoresis in a 15% polyacrylamide gel containing 7 M urea and
located by autoradiography (20,21). Approximate lengths are given
on the left in nucleotides. XC, xylene cyanol; BPB, bromphenol
blue.

nucleotides long were synthesized (Fig. 2). However, the size
distribution did not appear to be random. Unique patterns were
observed with $X, G4, and M13 DNAs, suggesting that certain
regions of each circle were preferentially transcribed.
SSB strongly inhibited the synthesis of primers on $X DNA
(Fig. 2). On the G4 circle, synthesis of multiple primers was
suppressed by SSB and a unique primer was synthesized (Fig.
2). which was indistinguishable from that synthesized by pri-
mase alone in the presence of SSB.
Prepriming Proteins Restore the Synthesis of Primers on
SSBCoated +X DNA. In the presence of prepriming proteins,
Shted $X DNA is active in directing the synthesis of RNA
primers. However, priming activity was less than half that
obtained with the simple dnaB protein and primase system on
uncoated DNA without prepriming proteins (Table 5). In the
presence of SSB, primer synthesis was dependent on proteins
n, n', n", i, and dnaC, in agreement with previous observations
(12). However, omission of SB did not affect the level of RNA
synthesis, consistent with results obtained in DNA synthesis
(Table 1).

Biochemistry: Arai and Kornberg

Proc. Natl. Acad. Sd. USA 76 (1979)

4311

Table 5.

Requirements for RNA primer synthesis on SSB-coated

$X DNA

Condition

Complete
Minus proteins n and n"
Minus protein n'
Minus protein i
Minus dnaC protein
Minus SSB
Minus proteins n, n'. n', i.

and dnaC
Minus proteins n, n', n". i.
dnaC. and SSB

RNA synthesized,
   pmol

33.1
5.0
<0.2
4.4
<0.2
30.3

4.7

68.8

For RNA synthesis, components were mixed at 0C in the following
order: 5 pl of buffer A; 0.2 pmol of MgC12; 500 pmol (as nucleotide)
of gX DNA; 1.25 nmol each of (3H]CTP. GTP, and UTP (each at 4000
dpm/pmol); 20 nmol of I3H]ATP (4000 dpm/pmol); 0.1 pg of rifam-
picin; 0.8 pg of dnaB protein; 0.2 pg of primase; 80 units of protein n
+ n"; 80 ng of protein n'; 70 ng of protein i; 90 units of dnaC protein;
and 1.5 pg of SSB. Final volume, 25 pl; incubation, 30 min. The
amount of RNA synthesized was determined by the DUE-cellulose
filter procedure (12).

Primer Synthesis on Poly(dT). Among synthetic polynu-
cleotides tested, poly(dT) is an active template in directing
poly(dA) synthesis by dnaB protein, primase, and DNA poly-
merase 111 holoenzyme. dnaB protein and primase catalyzed
a large amount of oligo(rA) synthesis on poly(dT) (data not
shown); as much as 30% of the template was transcribed. RNA
synthesis was completely dependent on dnaB protein, primase,
and poly(dT); it was inhibited by SSB. Electrophoresis of RNA
products indicated that primers synthesized on poly(dT) were
significantly shorter than those synthesized on phage DNA (Fig.
2). Poly(rA) longer than 50 nucleotides was not detected, and
oligo(rA). 10-15 nucleotides long, was the most abundant
product.
Formation of 4X DNA-dnaB Protein-Primase Complex.
Interaction of dnaB protein with c$X DNA was measured after
separation of DNA.dnaB protein complex from unassociated

dnaB   PRIMASE
PROTEIN  I PRIMER

dnaB
PROZEIN
PRIMASE

4x174 0

0 4x174

dnaB protein by gel filtration. In the absence of primase, less
than 0.3 molecule of dnaB protein was bound per c$X DNA
circle (data not shown). Addition of primase markedly stimu-
lated formation of thednaB protein-DNA complex (1.3 mole-
cule of dnaB protein per circle). ATP was essential for the
binding reaction. When ATP was omitted during gel filtration,
a stable dnaB protein-DNA complex could not be observed.
Upon addition of primase and the four rNTPs. a condition
that sustains the synthesis of multiple primers, the bound dnaB
protein was not released; instead, binding of dnaB protein to
DNA was enhanced (1.9 molecule of dnaB protein per circle).
A similar experiment using 3H-labeled primase showed that
primase also remained bound to c$X DNA in the presence of
dnaB protein and rNTPs (data not shown). The binding of
dnaB protein to DNA was abolished by SSB.

            DISCUSSION
Initiation of DNA synthesis depends generally on RNA priming
(3). Three mechanisms are already known in uninfected E. coli
extracts for priming DNA synthesis on single-stranded DNA:
(i) RNA polymerase acting on phage M13 DNA, (ii) primase
(dnaC protein) acting on phage G4 DNA, and (iii) primase
acting on a form of c$X DNA produced by the action of dnaB
protein and other replication proteins (dnaC, i, n, n', n"); the
C#JX system is thought to be the one used by the cell for initiating
nascent fragments in the lagging strand at the replication fork
of its chromosome. Specific and effective priming in each in-
stance requires that the single-stranded DNA be covered with
single-strand binding protein (SSB) as discussed further
below.

We have now recognized another priming system, which,
in its simplicity and generality, may prove to be. a valuable
guide to the operation of the more complex systems. dnaB
protein forms a complex with virtually any uncoated single-
stranded DNA in the presence of ATP and primase to produce
primers for DNA synthesis (Fig. 3). Multiple short primers are
produced throughout the length of the DNA (Fig. 2), appar-
ently at preferred initiation points, as judged from distinctive
sizes with M13, G4, and c$X DNAs. When the template is cov-
ered by binding protein, the dnaB protein-primase system is

   RNA
POLY ME RASE

PR IM E R

   RNA
POLYMERASE

-

PRIMASE

PRIMER

dna8PRC.

PROTEINS:

ITEIN

3IMASE

MER

n,n',n",i,dnaB,dnaC,

PRIMASE

FIG. 3.

Illustration of nonspecific priming by dnoR protein and primase on uncoated DNA and the specific priming systems for M13. G4.

and gX DNAs coated with SSB.

4312 Biochemistry: Arai and Kornberg

blocked and the initiation systems specific for each template
are required (Fig. 3). A unique primer is produced on M13 and
on G4 DNA but, as will be described elsewhere, the size dis-
tribution of primers formed on @X DNA by the complex
prepriming system is remarkably similar to that formed by the
dnaB protein-primase system (data not shown).
In earlier studies, primer formation on a single-stranded
DNA template absolutely required SSB; priming by only dnaB
protein and primase was not observed in the absence of SSB.
Impurities in the partially purified protein preparations might
have degraded the template, the primers, or the ATP, and
spermidine may have protected against this. However, the es-
sential role of SSB is indicated by (1) studies with a tempera-
turesensitive mutant (22) that fails to sustain phage or host
chromosome replication fn o(w and (if) studies in oitro (Fig.
3) which show that SSB is needed for specific recognition of
origins of replication in M13 and G4 DNAs and for formation
of an active dnaB protein complex with @X DNA (see
below).
Formation of a complex of dnaB protein bound to @X DNA
in the absence of SSB requires the presence of primase and ATP.
Current studies show further that dnaB protein in this complex,
while stable enough to be isolated by gel filtration, can be dis-
placed by free dnaB protein in solution. However, the dnaB
protein in the complex formed with @X DNA by the multi-
protein prepriming system resists exchange even with a large
exes of free dnaB protein. These findings are consistent with
a proposal (14) that the unusual stability of dnaB protein
binding to DNA, combined with a mobility on DNA engen-
dered by its ATPase activity, enable the protein to function as
a mobile promoter in the discontinuous phase of chromosome
replication.

This work wassupported in part by grants from the National Insti-
tutes of HeaIth and the National Science Foundation. K.4.A. is a fellow
of the American Cancer Society.

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