© 2001 European Molecular Biology OrganizationEMBO reports vol. 2 | no. 11 | pp 1003–1006 | 2001 1003
A hybrid bacterial replication origin
Harald Seitz, Michaela Welzeck & Walter Messer+
Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany
Received July 26, 2001; revised and accepted September 18, 2001
We constructed a hybrid replication origin that consists of the
main part of oriC from Escherichia coli, the DnaA box region
and the AT-rich region from Bacillus subtilis oriC. The AT-rich
region could be unwound by E. coli DnaA protein, and the
DnaB helicase was loaded into the single-stranded bubble.
The results show that species specificity, i.e. which DnaA
protein can do the unwinding, resides within the DnaA box
region of oriC.
Bacterial initiator protein DnaA binds to iterated binding sites in
replication origins, forming specialized nucleoprotein structures
(Echols, 1990; Kornberg and Baker, 1992). The net result is the
local unwinding of an AT-rich region, which in the case of
Escherichia coli consists of an AT cluster and three 13mer
repeats (Bramhill and Kornberg, 1988). This is the crucial
reaction in the initiation of replication. The unwound region is
the entry site for the replicative helicase, which in E. coli is
loaded as a double-hexamer of the helicase DnaB and the loader
protein DnaC (Fang et al., 1999). DnaA and interaction between
DnaA and DnaB are required for the loading reaction (Marszalek
et al., 1996; Seitz et al., 2000). DnaB helicase extends the initial
bubble allowing primase to enter the complex (Fang et al.,
1999). The resulting primer is extended by the DNA polymerase
III holoenzyme (Kornberg and Baker, 1992).
We have shown recently that the mechanism of the unwinding
reaction relies on a cooperative binding of the ATP-complexed
form of DnaA protein to special binding sites in the AT-rich
region of E. coli oriC, 6mer ATP–DnaA boxes, using the adjacent
9mer DnaA box as an anchor. ATP–DnaA then binds to the
exposed single-stranded ATP–DnaA boxes, thereby stabilizing
the single-stranded region (Messer et al., 2001; Speck and
The region corresponding to the E. coli AT-rich 13mers in
Bacillus subtilis oriC is a 27mer containing exclusively A and
T residues, and AT-rich sequences close to it. The two origins
could only be unwound by the homologous DnaA protein
(Krause et al., 1997). The regions of oriC that are unwound by
DnaA protein in E. coli and B. subtilis are thus AT rich, but other-
wise quite different in sequence. Alignment of both regions
using the adjacent DnaA box reveals, however, that the spatial
pattern of unwinding is very similar. In both cases the first
unpaired nucleotide, measured by its sensitivity to oxidation by
KMnO4, is 14 bp from the border of the DnaA box, R1 in the case
of E. coli. The following 28 bp are unwound without SSB, and
16–18 additional base pairs in the presence of SSB, both in
E. coli and B. subtilis oriC (Figure 1) (Krause and Messer, 1999).
In addition, the ATP–DnaA boxes, which mediate cooperative
binding of ATP–DnaA to the AT-rich region, are present at
corresponding positions in both origins (Figure 1) (Speck and
This similarity prompted us to construct a hybrid replication
origin that contains the AT-rich region from B. subtilis and the
main part of oriC from E. coli, to the right of DnaA box R1
(Figure 1), and to analyze which initiation reactions can be
observed with such an origin.
Structure of pOCBS
For the construction of such an E. coli–B. subtilis hybrid origin
we used plasmid pOC180. It contains the oriC region from
E. coli and the replication origin of pBR322 (Skarstad et al.,
2000). The AT-rich region from this plasmid, encompassing the
AT cluster and the three 13mers (E. coli oriC coordinates 6–79)
(Buhk and Messer, 1983), was replaced by the PCR-amplified
AT-rich region from B. subtilis, positions 2216–2064 in
Moriya et al. (1985) (Figure 1). The fragment from B. subtilis
oriC is larger than the E. coli fragment in order to include the
complete region of helical instability (DNA unwinding
element) (Krause et al., 1997).
+Corresponding author. Tel: +49 30 8173788; Fax: +49 30 84131385; E-mail: email@example.com
1004 EMBO reports vol. 2 | no. 11 | 2001
H. Seitz, M. Welzeck & W. Messer
The hybrid origin is unwound by
E. coli DnaA protein
Unwinding of the AT-rich region was monitored using the
reactivity of unpaired T residues to KMnO4 as described previ-
ously (Krause et al., 1997; Krause and Messer, 1999). Super-
coiled pOCBS was incubated with E. coli protein HU and
different concentrations of purified E. coli DnaA protein, and
subjected to oxidation by KMnO4. The positions of modified
bases were determined by primer extension using 5′-32P-end-
labelled primer A (see Methods), electrophoresis in a sequencing
gel and detection in a PhosphorImager. A sequencing reaction
with the same primer and pOCBS as template was run in
parallel. The plasmid with the E. coli oriC, pOC180, served as a
With E. coli DnaA protein modified bases were observed with
the hybrid origin from 14 to 41 bases upstream of the DnaA box,
with signals of lower intensity up to 51 bases (Figure 2). This
region of unwinding is identical to the unwound region
observed with a plasmid containing a bona fide B. subtilis repli-
cation origin using B. subtilis DnaA protein (Krause et al., 1997).
The hybrid origin thus shows a normal unwinding pattern. With
pOCBS we did not observe any modified bases using purified
DnaA protein from B. subtilis (data not shown).
DnaA can load helicase to the
unwound hybrid origin
The next step after the unwinding reaction is the loading of heli-
case. Therefore we tested whether helicase can be loaded to the
hybrid origin. Loading of DnaB helicase, as a DnaB–DnaC
double-hexamer, and the subsequent action of helicase can be
monitored using the FI* test. DnaB helicase action on a nega-
tively supercoiled plasmid in the presence of DNA gyrase
changes the linking number of the plasmid. Such plasmids can
be detected as a faster moving band (FI*) in agarose gels (Baker
et al., 1987; Konieczny and Helinski, 1997).
Plasmids pOCBS and pOC180 were incubated with purified
proteins from E. coli: DnaA, DnaBC, SSB, HU and DNA gyrase.
The reaction was stopped with EDTA–SDS and subjected to
agarose electrophoresis. With the complete mixture, a FI* band
was observed with both pOC180 and pOCBS (Figure 3, lanes 5
and 11). This shows that helicase can be loaded to the hybrid
origin. Omission of DnaA, DnaBC, SSB or DNA gyrase abol-
ished the reaction (Figure 3, lanes 2–4). Without protein HU,
there was only a small amount of a FI* band with pOC180
(Figure 3, lane 6), which was further reduced by increasing the
DnaA concentration or adding IHF protein (data not shown).
With pOCBS, nearly as much FI* band was observed with and
without HU (Figure 3, lane 12). The results show that the hybrid
origin cannot only be unwound by DnaA protein, but DnaA-
mediated loading of helicase is also possible.
The hybrid origin does not replicate in vivo
The ability of the hybrid replication origin to replicate in vivo
was tested by transformation of a polA1 strain. No transformants
were found in multiple experiments, whereas the same plasmid
preparation resulted in >1000 transformants per plate with a
polA+ strain. pOC180 as a control gave comparable numbers of
transformants (>1000 per plate) with both strains. The results
show that DnaA-mediated unwinding and helicase loading are
not sufficient to allow replication in vivo.
We show here that a hybrid replication origin, consisting of the
DnaA box region from E. coli oriC and the AT-rich region from
B. subtilis oriC, can be unwound in vitro by E. coli DnaA
protein. The unwound bases, measured by KMnO4 footprinting,
cover an identical part in the AT-rich region of the hybrid as in
B. subtilis oriC. In addition, helicase, E. coli DnaBC protein can
be loaded onto such a bubble substrate, mediated by DnaA
protein (Seitz et al., 2000). DnaB helicase then extends the
Fig. 1. Alignment of AT-rich regions from E. coli, B. subtilis and pOCBS oriC. The regions are aligned using the adjacent DnaA box (shaded). AT cluster, 13mers
and 27mers are shaded. Potential ATP–DnaA boxes are underlined. KMnO4-reactive pyrimidines are indicated by arrows. Escherichia coli sequences from
positions 10 to 93 (DDBJ/EMBL/GenBank accession No. K00826) and B. subtilis sequences positions 2706 to 2623 (DDBJ/EMBL/GenBank accession
No. X02369) are shown. Colons are at every 10th position.
EMBO reports vol. 2 | no. 11 | 2001 1005
A hybrid bacterial replication origin
bubble (Fang et al., 1999). Since this is a pre-requisite for the
formation of the FI* structure, we can also conclude that this step
occurs in the hybrid origin.
It has been shown previously that the unwinding reaction is
species specific. Unwinding of E. coli oriC requires E. coli DnaA
protein, likewise B. subtilis oriC is only unwound by B. subtilis
DnaA (Krause et al., 1997). The observation that the hybrid
origin is unwound by E. coli DnaA protein, and only by E. coli
DnaA, suggests that the species specificity resides in the DnaA
box part of the origin, probably in the spatial arrangement of
Although it is possible to replace the AT-rich region of E. coli
oriC by the corresponding region of another species, it cannot be
completely random, and AT richness is not sufficient for its func-
tion (Speck and Messer, 2001). We have shown that several
6mer consensus sequences, ATP–DnaA boxes that are different
from the 9mer consensus sequences of regular DnaA boxes,
mediate the cooperative binding of the ATP-complexed form of
DnaA protein. Such ATP–DnaA boxes are present in the AT-rich
regions of both E. coli and B. subtilis origins in the same spatial
arrangement (Figure 1). Binding of six or more ATP–DnaA
protein molecules destabilizes the helix. ATP–DnaA protein
then binds with high affinity to the exposed single-stranded
ATP–DnaA boxes (Messer et al., 2001; Speck and Messer, 2001),
preparing them for the loading of helicase.
It is not clear why the hybrid origin is unable to replicate
in vivo. One specific requirement for replication initiation
in vivo is transcriptional activation (Lark, 1972; Messer, 1972).
Crucial for transcriptional activation is the gidA promoter at the
left boundary of oriC, and the spacing between gidA and oriC
(Asai et al., 1990). The different spacing between gidA and the
AT-rich region in the hybrid origin is likely to be one reason for
its inactivity in vivo.
Unwinding at the replication origin is the central reaction in
the initiation of most origins. Most cellular origins, prokaryotic
and eukaryotic, have AT-rich regions that are unwound with the
help of an initiator protein, DnaA in the case of bacteria. The
analysis of the hybrid B. subtilis/E. coli replication origin allows
us to define that the DnaA box region of oriC contains the
species-specific elements of this reaction.
Bacterial strains and plasmids. Escherichia coli WM1963
[XL1-blue] (endA1, gyrA46, hsdR17, lac, recA1, relA1, supE44,
thi; F′lac: lacIQ, lacZ∆M15, Tn10, proAB+) (Sambrook et al.,
1989) and SCS1 [F–, endA1, hsdR17(rK–, mK+), supE44, thi-1,
recA1, gyrA96, relA1; Stratagene, La Jolla, CA] were used for
cloning and in the polA tests as control. WM1933 (wild-type
except for polA1) was the strain used for the polA tests.
Plasmid pOC170 (Roth and Messer, 1995) contains the oriC
region (coordinates –176 to +1497; coordinates refer to Buhk
and Messer, 1983), the replication origin of pBR322 and the bla
gene from pT7-7. Plasmid pOC180 is pOC170 with 24 bp of the
multicloning region deleted. Plasmid DNA was purified using
Qiagen purification kits (Qiagen, Hilden, Germany).
Proteins. Restriction enzymes, DNA polymerases and T4 DNA
ligase were from New England Biolabs (Beverly, MA). Thermo-
stable polymerase for PCR was from Promega (Madison, WI),
DNA gyrase from Gibco BRL and E. coli SSB from Promega.
DnaA protein was overproduced from expression vector
pdnaA116 and purified as described previously (Schaper and
Messer, 1995; Krause et al., 1997). DnaB and DnaC were
simultaneously overproduced from plasmid pPS562 and the
Fig. 2. KMnO4 footprinting of pOCBS and pOC180 oriC. The footprinting
reaction was done with 1 µg ccc plasmid DNA and 0, 250, 500 and 750 ng
DnaA as described in the Methods. Sequencing reactions of pOCBS were used
as standards and obtained using the same labelled primer A as for the KMnO4
footprints. The bar indicates the 28 bp region that is unwound in E. coli and
B. subtilis (Krause and Messer, 1999) (also see Figure 1).
Fig. 3. FI* helicase loading assay. The FI* assay was done as described in
1006 EMBO reports vol. 2 | no. 11 | 2001
H. Seitz, M. Welzeck & W. Messer
DnaB–DnaC complex separated from DnaB and DnaC by chro-
matography on a Mono Q-column and gel filtration as described
previously (San Martin et al., 1995).
KMnO4 footprinting. In vitro KMnO4 footprinting was performed
as described previously (Krause et al., 1997) with minor modific-
ations. All analysis contained 1 µg oriC plasmid, pOC180 or
pOCBS, and as assisting protein 100 ng of E. coli HU protein in
75 µl. Purified DnaA protein was added on ice. Open complex
formation and subsequent KMnO4 modification was allowed for
2 min each as described previously (Krause et al., 1997). Primer
extension was performed using a 5′-32P-labelled oligonucleotide
complementary to the top strand of oriC (primer A, oriC
coordinates 142–119: 5′-CATTCACAGTTAATGATCCTTTCC) as
primer. The samples were run on sequencing gels and analyzed
by a PhosphorImager (Molecular Dynamics).
FI* assay. Reactions (25 µl) contained 40 mM HEPES–KOH pH
7.6, 11 mM Mg++-acetate, 2 mM ATP, 1 mM DTT, 0.1 pmol
plasmid DNA, 23 pmol SSB protein (440 ng), 2.1 pmol HU
protein (40 ng) and 10 pmol DnaA (540 ng). After incubation for
5 min at 4°C and 5 min at 32°C, 0.8 pmol DnaB*C complex
(410 ng) and 0.65 pmol DNA gyrase (125 ng) were added and
incubation continued for an additional 30 min at 32°C. The
reactions were stopped by adding 5 µl stop mix (10 mM EDTA,
2.5% SDS) followed by incubation at 65°C for 2 min. Twenty
microlitres of the reaction was loaded onto a 0.8% agarose gel
in 0.5× TBE buffer with 0.1 µg/ml ethidium bromide. The
samples were electrophoresed for 20–24 h at a constant 1 V/cm,
then stained with Vistra Green™ and scanned.
This work was in part supported by grant Me659/6-1 from the
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