Prac. Natl. Acad. Sci. 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/DNA-dependent ATPase/single-strand binding protein/phage ¢X174/poly(dT)]

KEN-ICHI ARAI AND ARTHUR KORNBERG

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

Contributed by Arthur Kornberg, June 4, 1979

ABSTRACT _ Priming of phage ¢X174 DNA synthesis is ef-
fected simply by dnaB protein and primase when the DNA is
not coated by single-strand binding protein (SSB). The five
prepriming proteins (n, n’, n”, i, and dnaC protein) required for
priming a SSB-coated ¢X174 DNA circle are dispensable. The
dnaB protein-primase priming system is also active on uncoated
phage G4 and M13 DNAs and on poly(dT). Multiple RNA

rimers, 10-60 nucleotides long, are transcribed with patterns

istinctive for each DNA template. Formation of a stable dnaB
protein-DNA complex in the presence of primase and ATP
supports the hypothesis that dnaB protein provides a mobile
replication promoter signal for primase.

 

Pathways for conversion of various single-stranded 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 (dnaG protein) (6-
8).
By contrast, primer synthesis on SSB-coated DNA of phage
$X174 (@X) 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 dnaC. The
stable replication intermediate catalyzes the synthesis of mul-
tiple primers on the @X 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 @X 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
IH! holoenzyme. Uncoupled from DNA replication, dnaB
protein and primase, in the presence of ATP, form a complex
with a single-stranded 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.

MATERIALS AND METHODS

Materials. Buffer A is 100 mM Tris-HCI (pH 7.5)/20%
(wt/vol) sucrose/40 mM dithiothreitol/200 yg of bovine serum
albumin per ml. Buffer B is 20 mM Tris-HCl (pH 7.5)/8 mM
MgCl./0.1 mM EDTA/20 mM KC1/5% (wt/vol) sucrose/100
eg of bovine serum albumin per ml/5 mM dithiothreitol. 3H-
and 52P-labeled rNTPs and dNTPs and sodium boro{*H]hy-
dride were purchased from New England Nuclear or from
Amersham Corp.

Extensively purified Escherichia coli replication proteins
were: SSB [fraction IV, 2 X 104 units/ml, 4 X 104 units/mg
(16)]; dnaB protein (fraction V, 1.3 X 10° units/ml, 6.2 < 10°
units/mg (15)]; dnaC protein (fraction V, 1 X 10° units/ml, 6
X 10‘ units/mg); protein n’ (fraction VII, 5 X 104 units/ml, 2.4
X 10° units/mg); proteins n + n” (fraction V,* 1.5 X 10°
units/ml, 5 X 105 units/mg); protein i (fraction V, 1.5 X 105
units/ml, 8 X 10° units/mg); primase [fraction VI, 5 X 10°
units/ml, 1.1 X 108 units/mg (17)]; DNA polymerase ITI ho-
loenzyme [fraction V, 8 X 104 units/ml, 3.8 X 10° 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 0°C in the following
order; 5 yl of buffer A; 0.2 zmol of MgCl; 1.2 nmol each of
dATP, dCTP, and dGTP; 0.45 nmol of [SH]dTTP (specific
activity, 1500 dpm/pmol); 2.5 nmol each of GTP, CTP, and
UTP; 20 nmol of ATP; 40 nmol of spermidine-HC); 0.5 yg of
SSB; 60 ng of dnaB protein; 50 units of dnaC protein; 35 ng of
protein i; 40 units of proteins n + n”; 30 ng of protein n’; 60 ng
of primase; 0.2 4g of DNA polymerase III holoenzyme; 250
pmol (as nucleotide) of ¢X DNA; and water to 25 yl. For assay
of DNA synthesis catalyzed by dnaB protein, primase, and
DNA polymerase IIE holoenzyme, spermidine-HCI was omitted
from the reaction-mixture. Incubation was at 30°C.

Assay of RNA Primer Synthesis, For RNA primer synthesis
catalyzed by dnaB protein and primase, the reaction mixture
contained in 25 yl: 5 yl of buffer A; 0.2 nmol of MgCl; 0.5-0.75.
nmol (as nucleotide) of DNA as indicated; 1.25 nmol each of
(SH|GTP, CTP, and UTP (each at 4000 dpm/pmol); 20 nmol
of ATP; 0.1 ug of rifampicin; 0.4 ug of dnaB protein; and 0.12
ug of primase. When poly(dT) (4 nmol, as nucleotide) was used
as template, 20 nmol of (]HJATP (at 1000 dpm/pmol) was
present, and GTP, CTP, and UTP were omitted. Incubation
was at 30°C. The amount of RNA primer synthesized was de-
termined by the DEAE-cellulose filter procedure (12).

 

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4308

Abbreviations: ¢X, phage @X174; SSB, single-strand binding pro-

tein.

* Protein n fraction used here was further resolved into two replication
enzymes (protein n and n”) by J. Shlomai.
Biochemistry: Arai and Kornberg

Formation and Isolation of 6X DNA-:dnaB Protein Com-
plex. The reaction mixture contained in 25 yl: 5 yl of buffer A,
0.2 pmol of MgCle, 1.5 nmol (as nucleotide) of ¢X DNA, 20
nmol of ATP, L.5 ug of H-labeled dnaB protein (8 104
cpm/g), and, when present, 0.4 ug of primase. After incu-
bation at 30°C for 20 min, the amount of dnaB protein bound
to 6X 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 24°C) 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 boro[*H]hydride (7.2 Ci/
mmol; 1 Ci = 3.7 X 10!° becquerels) and formaldehyde. The
labeled dnaB protein (8 X 10* cpm/yg) 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’77 M
urea and autoradiography were carried out as described (20,
21).

RESULTS

The Reconstituted System for Converting ¢X DNA to the
Duplex Form. ¢X viral DNA incubated with purified prepa-
rations of SSB, dnaB protein, dnaC protein, protein i, proteins
n +n’, protein n’, primase, DNA polymerase III 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’, n”, primase, or DNA
polymerase III holoenzyme) abolished the activity (Table 1).
The product of the reaction was the duplex replicative form II
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).

dnaB Protein, Primase, and DNA Polymerase III Ho-
loenzyme Suffice for DNA Replication in the Absence of
SSB. With uncoated ¢X DNA as template, there was no re-
quirement for proteins i, n, n’, or n” (Table 2); dependency on

Table 1. Requirements for conversion of ¢X DNA to replicative
form II with purified replication proteins

 

dNMP incorporated,
Condition pmol
Complete 210
Minus dnaB protein 1
Minus dnaC protein 2
Minus protein i 3
Minus protein n + n” 2
Minus protein n’ 7
Minus primase 4
Minus DNA polymerase HI
holoenzyme <i
Minus SSB 51
Minus spermidine 205
Minus SSB and spermidine 180

 

Incubation was 10 min.

Proc. Natl. Acad. Sci. USA 76 (1979) 4309

Table 2. Requirements for conversion of ¢X DNA to replicative
form II] in the absence of SSB and spermidine

 

dNMP incorporated,
Condition pmol

Complete 95
Plus rifampicin (0.5 pg) 82
Plus SSB (0.5 ug) 70
Minus dnaB protein 6
Minus dna protein 38
Minus protein i 90
Minus protein n + n” 94
Minus protein n’ 81
Minus primase 8
Minus DNA polymerase III

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 III 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 III holoenzyme. It would
seem reasonable to expect that this system also catalyzes the
conversion of other single-stranded 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 III 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

 

dNMPs INCORPORATED (pmo!)

 

 

 

 

0 0.5 1.0 15

SSB (ug)

Fic. 1. Inhibition by SSB of DNA synthesis catalyzed by dnaB
protein, primase, and DNA polymerase III holoenzyme. DNA syn-
thesis was measured as described in Materials and Methods except
that spermidine was omitted and 250 pmol (as nucleotide) of @X, G4,
or M13 DNA was used as template. The reaction mixtures (25 ul)
contained 0.1 ug of dnaB protein, 60 ng of primase, 0.2 ug of DNA
polymerase III holoenzyme, and amounts of SSB as indicated. Incu-
bation was 15 min.
4310 Biochemistry: Arai and Kornberg

Table 3. Template specificity of the purified replication systems
DNA synthesis, pmol

 

DNA template G4 system* oX systemt
oX 2 140
G4 220 180
M13 1 8

 

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 III ho-
loenzyme, and SSB were included.

t Prepriming proteins n, n’, n”, i, and dnaC were included, as well as
dnaB protein, primase, DNA polymerase III holoenzyme, and SSB.

protein on G4 DNA was completely inhibited by anti-dnaB
protein antibody but not by anti-SSB antibody. This result ex-
cludes the possibility of a SSB contaminant in the dnaB protein
preparation. The products of the reaction were duplex repli-
cative form II (data not shown). As anticipated, this replication
system failed to discriminate among phage templates that were
not covered by SSB. However, in the presence of SSB, DNA
synthesis on X and M183 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, SSB-coated ¢X DNA 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 M18
DNA, which remained inactive (Table 3). Thus, SSB determines
template specificity during the priming process.

dnaB Protein and Primase Catalyze the Synthesis of
Multiple Primers on @X, G4, and MI3 DNAs. BNA primer
formation as the basis 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 syntliesis 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

 

 

oX DNA
RNA synthesized,
Condition pmo}
Complete 189
Minus dnaB protein 2
Minus primase 1
Minus dneB protein and primase <i
Minus ¢X DNA <1
Plus SSB 9

 

RNA synthesis was measured as described in Materials and
Methods except that 625 pmol (as nucleotide) of 6X DNA was used
as template. Incubation was 30 min.

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

FR eee
1 80}

   

   

5 ; §
a vo Mn + poiyiaT) |

ie 8.8 G5 05 Bi HB 68 6G ab a4
S86 iugi Bb 828 0.76 LS HB ots Lh 9a 22

 

Fic. 2. Electrophoresis of RNA primer transcripts. Reaction
mixtures (25 1) contained: 5 yl of buffer A; 0.2 mol of MgClo; 1.25
nmol each of [a-3?P]GTP, CTP, and UTP (each at 7000 dpm/pmol);
20 nmol of ATP; 0.1 yg of rifampicin; 500 pmol (as nucleotide) of
phage DNA; 0.15 ug of primase; and additional components as indi-
cated. The poly(dT) reaction mixture contained [a-32P]ATP (1000
dpm/pmol) and 4 nmol of poly(dT). Incubation was at 30°C for 30
min. The reaction was terminated by cooling to 0°C. To each sample
were added 50 yl of 95% ethanol (— 20°C), 2.5 4] of 3 M sodium acetate
(pH 5.5), and 30 yg (1 wl) of E. coli tRNA. After 4 hr at —20°C, the
precipitates were collected by centrifugation and resuspended in 25
ul 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 6X 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
SSB-Coated #X DNA. In the presence of prepriming proteins,
SSB-coated ¢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 SSB did not affect the level of RNA
synthesis, consistent with results obtained in DNA synthesis
(Table 1).
Biochemistry: Arai and Kornberg

Table 5. Requirements for RNA primer synthesis on SSB-coated

 

 

#X DNA
RNA synthesized,
Condition pmol
Complete 33.1
Minus proteins n and n” 5.0
Minus protein n’ <0.2
Minus protein i 4,4
Minus dnaC protein <0.2
Minus SSB 30.3
Minus proteins n, n’, n’, i,
and dnaC 4.7
Minus proteins n, n’, n”, i,
dnaC, and SSB 68.8

 

For RNA synthesis, components were mixed at 0°C in the following
order: 5 4] of buffer A; 0.2 zmol of MgClo; 500 pmol (as nucleotide)
of @X DNA; 1.25 nmol each of (3H}CTP, GTP, and UTP (each at 4000
dpm/pmol); 20 nmol of (]HJATP (4000 dpm/pmol); 0.1 yg of rifam-
picin; 0.8 wg of dnaB protein; 0.2 zg 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 yg of SSB. Final volume, 25 yl; incubation, 30 min. The
amount of RNA synthesized was determined by the DEAE-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 III 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 ¢X DNA-dnaB Protein:Primase Complex.
Interaction of dnaB protein with 6X DNA was measured after
separation of DNA-dnaB protein complex from unassociated

dnaB PRIMASE
PROTEIN PRIMER

dnaB
PROTEIN

PRIMASE M13

 

Fic. 3.
and ¢X DNAs coated with SSB.

 

Proc. Natl. Acad. Sei. USA 76 (1979) 4311

dnaB protein by gel filtration. In the absence of primase, less
than 0.3 molecule of dnaB protein was bound per 6X DNA
circle (data not shown). Addition of primase markedly stimu-
lated formation of the dnaB 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 °H-labeled primase showed that
primase also remained bound to #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
(dnaG protein) acting on phage G4 DNA, and (iii) primase
acting on a form of ¢X DNA produced by the action of dnaB
protein and other replication proteins (dnaC, i, n, n’, n”); the
#X 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 #X DNAs. When the template is cov-
ered by binding protein, the dnaB protein-primase system is

RNA

POLYMERASE PRIMER

 

PRIMASE

PRIMER

PRIMASE

   

PROTEINS:

n,n’,n",i,dnaB,dnaC,
PRIMASE

Illustration of nonspecific priming by dnaB protein and primase on uncoated DNA and the specific priming systems for M13, G4,
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 (4) studies with a tempera-
ture-sensitive mutant (22) that fails to sustain phage or host
chromosome replication in vivo and (it) studies in vitro (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
excess 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 was supported in part by grants from the National Insti-
tutes of Health and the National Science Foundation. K.-i.A. is'a fellow
of the American Cancer Society.

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