Proc. Natl. Acad. Sei. USA
Vol. 82, pp. 3562-3566, June 1985
Biochevstry

Initiation of enzymatic replication at the origin of the Escherichia
coli chromosome: Contributions of RNA polymerase and primase

@NA/priminp/oriC/pIPW

TOHRU OGAWA*, TANIA A. BAKER, ARIE VAN DER ENDEt, AND ARTHUR KORNBERG
Dcparlrnent of Biochemistry. Stanford University School of Medicine, Stanford, CA 94305

Contributed by Arthur Kornberg, January 28, 1985

ABSTRACT  Replication of plasmids that depend on the
245-be-pair origin of the Escherichia coli chromosome (ana
requires many purified proteins that (i) direct initiation to oriC
(e.g., dnaA protein), (ii) influence initiations elsewhere (e.g.,
auxiUary proteins), and (iii! prime and extend DNA chains
(e.g., priming and synthesis proteins). For the RNA priming
and initiation of new DNA chains, the requirements for both
primase and RNA polymerase (RNA pol) [Kaguni, J. M. &
Kornberg, A. (1984) Cell 38, 183-1901 have been further
analyzed. Depending on the levels of auxiliary proteins
(topoisomerase I and protein HU), three priming systems can
operate primase done, RNA pol done, or both combined. At
low levels of auxiliary proteins, prime alone sustains an
effective priming system. At higher levels, primase don is
blocked, but RNA pol done   initiate replication, dbeit
feebly; at these high levels of auxiliary proteins, prima and
RNA pol act synergistically. When RNA pol is stalled by an
inhibitor or lack of a ribonucleoside triphosphate, prSmase
action is also inhibited. Based on these and other data [van der
Ende, A., Baker, T. A., Ogawa, T. & Kornberg, A. (1985)
Proc. Natl. Acad. Sci. USA 82, in press], RNA pol can
counteract inhibition by auxiliary proteins and thus activate the
origin for the priming by primase of the leadiig strand of the
replication fork.

~

Replication of plasmids in Escherichia coli that depend on the
chromosomal origin of replication (oriC) has been partially
reconstituted with purified proteins (1-6). These proteins fall
into three functional groups: initiation proteins (e.g., dnaA
protein) for recognition of the oriC sequence and assembly of
a replication complex, specificity (auxiliary) proteins for
suppression of initiation of replication at sites other than
oriC, and replication proteins for priming and elongation of
DNA chains. A key event in the initiation process is specific
binding of dnaA protein to the oriC sequence (2, 7). RNA
polymerase (RNkpol) involvement in initiation of replication
of the E. coli chromosome in vivo is based on inhibition of a
new cycle of replication by rifampicin (S), suppression of
dnaA mutations by rpoB mutations (9, lo), and increased
chromosomal copy number in rpoB or rpoC mutants (11,12).
In oriC replication in vitro, the RNA pol-dependent event
precedes DNA synthesis (1, 6), but its precise role has not
been clarified. In the absence of primase, RNA pol was
presumed to prime the synthesis of the leading strand at oriC
(6).
Under certain conditions examined in this and a companion
study (13), DNA synthesis takes place in the absence of RNA
pol. whereas primase is essential, indicating a role of RNA
pol in activating the origin for initiation of replication as
proposed for replication of bacteriophage A DNA (14). The
present study examines various parameters, particularly the

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auxiliary proteins, to evaluate the contributions of RNA pol
and primase to initiation of oriC replication. Three priming
systems have been revealed: primase alone, RNA pol alone,
and both combined. Further characterization of the solo
primase system is presented elsewhere (13). These studies
establish the capacity of primase to prime the leading strand
of DNA replication and point to a role for RNA pol in
activating the oriC sequence as the unique initiation site.

MATERIALS AND METHODS

Strains, Phages, and DNAs. E. coli CM987 (F-, rhi, asn,
recA) and oriC plasmid pCM959 (15) were generously pro-
vided by M. Meijer (University of Amsterdam, The Nether-
lands). pCM959 DNA (see below) was prepared by alkalime
lysis of CM987 cells that harbor the plasmids (16). Phages
M13oriC26 (17) and M13AE101(18) were from J. M. Kaguni
and D. S. Ray, respectively. Covalently closed supercoiled
replicative form (RFI) DNA of these phages was prepared
from infected K37 (Hfr) cells as described (19). The DNAs
were further purified by phenol/chloroform treatment, two
successive bandings in CsCl/ethidium bromide density gra-
dients, ethanol precipitation, and gel filtration through Bio-
Gel A-5m.
Reagents and Enzymes. Sources were ribonucleoside
triphosphates (rNTPs) and phosphocreatine from Sigma;
deoxyribonucleoside triphosphates (dNTPs) from P-L
Biochemicals; [a-32P]dTTP (>SO0 Ci/mmol; 1 Ci = 37 GBQ)
from Amersham; rifampicin from Calbiochem; strepto-
lydigin, a gift from Upjohn; highly purified DNA replication
proteins as described (6).
Reconstitution Assay for DNA Replication. The novel re-
quirement of oriC replication in a crude enzyme fraction for
a high concentration of a hydrophilic polymer, such as
polyvinyl alcohol or polyethylene glycol (20), has been
replaced in a reconstituted system by an increased level of the
gyrase A subunit (6) and the removal of inhibitors (unpub-
lished). In such a reconstituted system, stimulation of replica-
tion by crude enzyme fractions, such as flowthrough and
eluate fractions from an Amicon red-A agarose column (1,6),
is no longer significant (data not shown). Thus, maximal
DNA synthesis is achieved by a combination of highly
purified enzymes (see below). The minichromosome pCM959
(15), which consists solely of E. coli DNA encompassing onC
(bp -677 to bp +3335, where bp = base pair) (21), was used
as the oriC DNA template, except where indicated, to avoid
possible complications by sequences of the vector portion of

Abbreviations: RFI, covalently closed supercoiled replicative form;
rNTP and dNTP, rib and deoxyribonucleoside triphosphate; SSB,
single-stranded DNA binding protein; bp, base pair; RNA pol, RNA
polymerase; Top0 I, topoisomerase I; Pol 111, DNA polymerase 111.
*Present address: Institute of Molecular Biology, School of Science,

+Present address: Department of Cell Biology, Medical Faculty,

Nagoya University, Chikusa, Nagoya 464, Japan.

University of Utrecht, Utrecht, The Netherlands.

3562

Biochemistry: Ogawa et a/.

I I
I

C

Proc. Natl. Acad. Sci. USA 82 (1985)

3563

I I I
           I

D

previously used M13-oriC chimeric phage DNA (1-6) (un-
published).
The standard reaction (25 pl) contained HepesKOH (pH
7.6), 40 mM; magnesium acetate, 8 mM; phosphocreatine, 2
mM; dithiothreitol, 4 mM; ATP, 2 mM; GTP, CTP, and UTP,
500 pM each; dATP, dGTP, and dCTP, 100 pM each;
[a-3ZP]dTTP, 100 pM, 2o(Moo cpm/pmol; supercoiled tem-
plate DNA, 200 ng (600 pmol of nucleotide); bovine serum
albumin, 0.4 mg/ml; creatine kinase, 300 ng; protein HU, 20
ng; topoisomerase I (Topo I), 10 ng; RNase H, 1.4 ng; gyrase
A subunit, 360 ng; gyrase B subunit, 500 ng; RNA pol, 400 ng;
dnaA protein, 110 ng; single-stranded DNA binding protein
(SSB), 320 ng; dnaB protein, 100 ng; dnaC protein, 50 ng;
primase, 16 ng; and DNA polymerase 111 (Pol 111) holoen-
zyme, 100 ng. Primase was omitted in the solo RNA pol
reactions and RNA pol was omitted in the solo primase
reactions. The mixtures were assembled at 0C and incubated
for 30 min at 30C. Total nucleotide incorporation was
measured in a liquid scintillation counter after trichloroacetic
acid precipitation onto Whatman GF/C glass-fiber filters.

RESULTS

Replication Can Occur in the Absence of Either RNA pol or
Primase. Upon varying the levels of the reaction components,
significant replication was consistently observed when either
RNA pol or primase was omitted (Table 1). The solo primase
system appeared far more efficient than the solo RNA pol
system; yet they have the same requirements for the other
replication proteins.
Top I and a High Level of Protein HU Inhibit the Solo
Priiase System. As auxiliary factors, the effects of Top0 I
and protein HU on the solo primase system were most
striking (Figs. 1 and 2). Although high levels of Top0 I were
generally inhibitory, its action at lower levels [effective for
specificity function at oriC (5)] was most profound on the solo

80

60

1 40

-

0

!i

- 20
Y

IO



800

v)
Lu

I-
z

a

600

x

400

200

0

Table 1.  Requirements for replication in reactions with RNA
pol, primase, or both

DNA synthesis, pmol

                        RNA pol
omitted RNAwl Primase + Drimase

Component

None             54 489 853
Rotein HU          10 184 287
dnaA protein          3 3 5
Gyrase A subunit       14 6 49
Gyrase B subunit        1 1 3

dnaB protein          1 1 3
dnaC protein          1 2 3
Pol 111 holoenzyme       0 0 1

SSB 7 28 41

Complete reaction contains Top I and RNase H to maintain
specificity for oriC DNA and dependence on dnaA protein. In-
complete dependence of incorporation on protein HU and gyrase A
subunit is partly due to the contamination of small amounts of these
proteins in the other protein fractions. Less than 5 pmol of incor-
poration was observed when both RNA pol and prirnase were
omitted from the complete reaction mixture.

primase system. This inhibitory effect was due in large part
to prolonging the lag time of the reaction (Fig. 2) (13).
Protein HU, which stimulated all reactions when present at
a low level, became inhibitory at elevated levels, especially
on the solo primase system (Fig. 1). At twice the fully
inhibitory level, protein HU (at a 1:l weight ratio to DNA)
converts form I of simian virus 40 into a nucleosome-like,
beaded structure in the presence of a type I topoisomerase
(22) and enhances transcription of phage A DNA (23). At an
optimal level of protein HU (1:20 weight ratio to DNA), the
solo primase system appeared to be as efficient as the one
containing both primase and RNA pol. In contrast, the
system lacking primase was inefficient at all concentrations

I I I I I

A B

t

\ \ RNA pol + PRIMASE

+PRIMASt \ -0

0 10 20 0 50 100 150

TOPOISOMERASE 1 (ng)

PROTEIN HU (ng)

FIG. 1.

Effect of Top0 I and protein HU on the reactions with RNA pol, primase, or both. Titration of Topo I was performed in the presence

of 20 ng of protein HU. Titration of protein HU was performed in the absence of Top0 I.

3564 Biochemistry: Ogawa et al.

5100

1

U
2
0

Proc. Natl. Acad. Sci. USA 82 (1985)

A 1 I 1

-

RNA pol

1OOC

I I 1 I 1 1

B
- 9

20 40 60 0 20 40 60

TIME (min)

FIQ. 2.  Time course of replication and the influence of Topo I. Reaction mixtures (225 jd) of the three priming systems were assembled in
the presence (0) or absence (0) of Top0 I. DNA synthesis was measured on 25+l samples taken at the indicated times of incubation at 3VC.

of Top0 I and protein HU. The strong dependence on RNA
pol observed previously in reconstituted systems (6) was due
in large part to the high levels of Top0 I and protein HU that
were used.
The Initiation Systems Depend on dnaA Proteii and oriC
DNA. RNase H (4) and Topo I (5) have been identified as
specificity proteins that suppress DNA synthesis that is not
dependent on dnaA protein and the oriC sequence. The solo
primase system did not require these auxiliary proteins to
maintain dnaA protein dependence of replication (Table 2)
nor was replication observed when DNA templates lacked
the oriC sequence (Table 3). On the other hand, a low level
of DNA synthesis in the absence of dnaA protein was
detected in reactions that contained RNA pol but lacked
auxiliary proteins, particularly RNase H (Table 2). DNA
synthesis was also observed with DNA lacking the oriC
sequence (to an extent depending on the template) in re-
actions that contained RNA pol but did not contain auxiliary
proteins (Table 3). The contribution to the total DNA
synthesis of this nonspecific DNA synthesis was most sig-
nificant in the solo RNA pol system, where only a small
amount of dnaA protein- and oriC-dependent replication was
observed. RNase H had no effect on the dnaA protein-
dependent DNA synthesis in the solo primase system but
stimulated the dnaA protein-dependent reaction by 50-100%
when both RNA pol and primase were present (data not
shown). The stimulation by auxiliary proteins of the com-
bined RNA pol and primase reaction with the oriC DNA
template (Table 3) was due to this activity of RNase H.
Inhibition of the solo primase reaction with M13oriC26 by
auxiliary proteins was especially great (Table 3), presumably
due to a prolonged lag time caused by Top0 I on this very
large template (13).
Solo RNA pol System Is Inefficient. In contrast to the solo
primase system, the solo RNA pol system was rather feeble
and could not be enhanced by systematic adjustments of the
reaction components. To insure that this limited level of

Table 2.  Effect of auxiliary proteins on replication of pCM959
DNA in the absence of dnaA protein

DNA synthesis, pmol

Auxiliary dnaA RNA pol
proteins protein RNA pol Primae + primase

None + 54 489 853
                 47 5 43
None -
                  31 4 27
Topo I -
                  3 3 5
RNase H -
Top0 I and             3 3 5
RNase H -

replication was not due to traces of primase in the other
reagents, antibody against primase was shown to selectively
inhibit primase-dependent replication but not to affect the
RNA pol-dependent reaction (Table 4). Thus, RNA pol can
prime DNA synthesis under certain conditions that depend
on the oriC sequence, dnaA protein, and other key replica-
tion proteins. However, the inefficiency of this reaction
suggests that primase is an essential component of the
replication machinery at a growing fork and not replaceable
by RNA pol.
rNTPs, Other Than ATP, Are Not Required in the Sob
primnse System. The amount of replication observed upon
omission of one or more of the rNTPs was strikingly Werent
in the three initiation systems (Table 5). The solo RNA pol
reaction was totally dependent on all four rNTPs, while the
solo primase reaction required only ATP and was inhibited by
adding ffiTP, rCTP, and rUTP; ATP is essential for the
functions of gyrase, dnaB protein and Pol I11 holoenzyme, as
well as primase. This result is consistent with the capacity of
primase to prime DNA synthesis on the SSB-coated single-
stranded circular DNA of bacteriophage M13 in the presence
of only ATP and the dNTPs (24). The enhanced replication,
in the absence of ffiTP, CTP, and rUTP, suggests that
extensive primer synthesis competes with use of the template
for efficient replication.
In contrast to the solo primase system, the reaction that
contained both RNA pol and primase was inhibited by

Table 3.  Comparison of temulate DNAs

Template
(size, kb)

Auxiliary
proteins

pCM959

M13oriC26

(12.3)
4x174
(5.4)
pBR322
(4.4)

M13AE101
(6.3)

(4.0)

DNA synthesis, pmol

RNA pol

163
41
173
68
115
7
65
10
168

8

Primase

1095
409
665

63
6
4
10
7
29
16

RNA pol
+ primase

553
811
279
351
174

8
84
15

269
20

Reactions were performed with or without auxiliary proteins
(Top0 1 and RNase H). Only pCM959 and M13oriC26 contain the
minimal oriC sequence, flanked by only E. coli DNA in pCM959 and
by E. coli, G4, and M13 sequences in M13oriC26; 4x174 is the phage
replicative form (RFI); pBR322 is a ColEl-like plasmid; M13AEl01
(lacking the origin for complementary strand synthesis) and
M13oriC26 are RFI DNAs. kb, Kilobase pairs.

Biochemistry: Ogawa et al.

Table 4.  Effect of primase antibody

Proc. Natl. Acad. Sci. USA 82 (1985)

3565

RNA pol, Primase, Antiserum, DNA synthesis,

Reaction ng ng 4 pmol

RNA pol 400 0 0 35
         400 0 0.025 40
          400 0 0.05 42

Primase 0 16 0 502
           0 16 0.025 23
           0 16 0.05 12
           0 40 0.025 441

RNA pol 400      16 0 470
+ primase 400      16 0.025 45
          400      16 0.05 38
          400     40 0.025 460

Reactions were performed with the indicated amounts ofRNA pol,
primase, and antiserum (prepared in mice with DEAE-purified
primase and heated 15 min at 56'C).

omitting rCTP, rGTP, and rUTP, especially at higher levels
of RNA pol (Fig. 3). Similarly, replication was blocked when
RNA pol was inhibited by rifampicin or streptolydigin (Fig.
3). Thus, the primase reaction is blocked when RNA pol is
inhibited. When core RNA pol (lacking the ofactor) was used
in place of RNA pol holoenzyme, no significant inhibition
was observed either upon omission of rGTP, rCTP, and rUTP
or by addition of rifampicin or streptolydigin (data not
shown). These observations may be explained by RNA pol
being fixed at a promoter(s) in the presence of an inhibitor or
stalled for lack of rNTPs and thus blocking a stage in
replication. Inhibition of transcription by an RNA pol-rif-
ampicin complex bound to a promoter has been reported
(25). When RNA synthesis by RNA pol was allowed to
proceed (for 10-20 min), then stopped by rifampicin and
streptolydigin, while at the same time DNA synthesis was
started by addition of dNTPs, replication was not inhibited
(ref. 6; data not shown). These findings suggest that the role
of RNA pol is limited to the initiation stage of the reaction.

DISCUSSION

More than 10 purified proteins are needed to reconstitute the
specific replication of plasmids that depend on the unique
origin (oriC) of the E. coli chromosome (6). Among these
proteins are some that direct initiation of DNA strands to the
oriC sequence and others (auxiliary, specifcity proteins) that
suppress initiation elsewhere. In this study, evaluating the
contributions of RNA pol and primase to the priming of new
DNA chains, three systems have been revealed: (12 solo
primase, (it] solo RNA pol, and (iii) primase and RNA pol
combined.
The solo primase system can support efficient replication
provided that the auxiliary protein, Top0 I, is absent or at a
low level and that protein HU is at a weight ratio to DNA of
about 1:20; at a ratio of 1:2, protein HU inhibits primase

Table 5. Requirement for rNTPs

DNA synthesis, pmol

                          RNA pol
rNTPs RNA pol Primase + primase

A, G, C, U        84 241 673
- G, C, U         3 3 5
A, - C, U         7 685 173
A, G, - -         9 448 128
A- __         6 1045 191

except for omission of rNTPs as indicated.

Reactions were performed as described in Materials and Merhods

"

0 0.4 0.8 1.2 1.6

RNA POLYMERASE (ug)

FIG. 3.  Effect of omission of rGTP, rCTP, and rUTP or addition
of rifampicin and streptolydigin on the reaction containing primase
and various amounts of RNA pol. Complete reactions were per-
formed as described in Materials and Methods except that the
amounts of RNA pol were changed. In the "ATP ONLY" reactions,
rGTP, rCTP, and rUTP were omitted. In the "RIF + STREP"
reactions, 20 pg of rifampicin per ml and 200 pg of streptolydigin per
ml were added. Similar results were obtained with either rifampicin
alone or streptolydigin alone.

action completely (Fig. 1). Neither these nor other auxiliary
proteins (e.g., RNase H) are needed to insure oriC specifcity
and the absolute dependence of replication on dnaA protein
bound to the oriC sequence (7) (Tables 2 and 3). Further
characterization of the solo primase system and divisibility of
initiation into prepriming and priming stages are presented
elsewhere (13).
In contrast to the solo primase system, the solo RNA pol
system is only l/lOth as active and is relatively unaffected by
the auxiliary proteins, except when they are needed to
suppress initiation at non-oriC regions of an oriC template or
on supercoiled DNAs that do not contain oriC (Fig. 1, Table
3).
In the system that includes both RNA pol and primase, the
RNA pol relieves much of the inhibition of the primase
system imposed by high levels of Top0 I and protein HU but
has little effect on replication at low levels of these auxiliary
proteins. However, the auxiliary proteins are needed for
specificity to block the use of non-oriC DNA whenever RNA
pol is present. When RNA pol transcription is inhibited by
rifampicin or streptolydigin or stalled for lack of a rNTP,
initiation of replication by either primase or RNA pol is
prevented (Fig. 3). These inhibitory effects are not observed
once transcription has taken place.
Reactions containing primase show a lag of 10-15 min,
whereas the lag time of the solo RNA pol reaction is only 2-5
min (Fig. 2). All reactions proceed for >60 min. Prior
incubation of the primase-dependent reaction mixtures in the
absence of dNTPs removes the lag and a burst of replication
is observed upon addition of dNTPs (6, 13). Prior incubation
of the solo RNA pol reaction mixture, on the other hand,
results in only a small reduction in the lag time (data not
shown). When an assay system is used that allows only a
limited period for initiation and a single round of elongation,
only 10-20% of the input DNA molecules serve as templates
in reactions that contain primase, while even fewer (4%) are
used in the solo RNA pol reaction (ref. 13; data not shown).

3566 Biochemistry: Ogawa et al.

Proc. Natl. Acad. Sci. USA 82 (1985)

Thus, several features distinguish the primase-independent
reaction from those that contain primase.
These and other findings (13) suggest that primase has a
major role in priming the leading and lagging strands of
replication forks. Although MA pol transcription can also
prime DNA synthesis, it is far less efficient than primase and
may make its chief contribution by activating the oriC region
for priming by primase. Possibly, a factor involved in
determining the frequency of initiation of replication of the E.
coli chromosome could control the synthesis of RNA that
activates the origin.

How the properties of primase and RNA pol in these
reconstituted reactions apply to physiological conditions is
difficult to judge. RNA pol has been reported to participate in
initiation of replication of the E. coli chromosome (8-12,20).
However, inferences of involvement of RNA pol based on
actions of inhibitors (e.g., rifampicin) in cells (8) and crude
enzyme systems (20) may be unsound because of the capacity
of an inhiiitor-RNA pol complex to block initiations by pri-
mase as well (Fig. 3). Independence from RNA pol in replica-
tion of R1 plasmids has been demonsmed (26) and implies a
reliance an primase for priming this double-stranded template.
Despite the multiplicity of proteins in the replication
system, it seems likely that the true oriC system demands still
more. For example, several partially purified fractions are
known to stimulate the rate or extent of replication (1, 6).
These fractions were not replaced by available replication
proteins, such as proteins n, n', n", and i, dnd protein, dnaK
protein, rep protein, helicase 11, DNA ligase, and DNA
polymerase I.

We thank LeRoy L. Bertsch for expert assistance in preparing this
manuscript. This work was supported by grants from the National
Institutes of Health and the National Science Foundation. A.v.d.E.
was Supported by the Netherlands Organization for the Advance-
ment of Pure Research (Zuiver Wetenschappelijk Ondcrzoek) with a
North Atlantic Treaty Organization Science Fellowship.

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