From the Department of Biology, Center for Molecular
Genetics, University of California, San Diego,
La Jolla, California 92093-0322 and the
Department of
Molecular and Cellular Biology, Faculty of Biotechnology, University of
Gdansk, 24 Kladki, PL-80822 Gdansk, Poland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The requirement of DnaA protein binding for
plasmid RK2 replication initiation the Escherichia coli was
investigated by constructing mutations in the plasmid replication
origin that scrambled or deleted each of the four upstream DnaA boxes.
Altered origins were analyzed for replication activity in
vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I
footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein
binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative
with box 4 acting as the "organizer" for the formation of the
DnaA-oriV nucleoprotein complex. Interestingly, the
inversion of box 4 also abolished replication activity, but did not
result in a loss of binding to the other boxes. However, DnaA binding
to this mutant origin was no longer cooperative. These results
demonstrate that the sequence, position, and orientation of box 4 are
crucial for cooperative DnaA binding and the formation of a
nucleoprotein structure that is functional for the initiation of replication.
The DnaA protein is a key factor in the initiation of DNA
replication in prokaryotes and is highly conserved among a diverse group of eubacterial species (for reviews, see Refs. 1-3). DnaA binds
to conserved 9-base pair consensus sequences, DnaA boxes (5'-TT(A/T)TNCACA), which have been identified both in prokaryotic chromosomal origins, including those of Escherichia coli
(4), Pseudomonas putida (5), Pseudomonas
aeruginosa (6), Bacillus subtilis (7) and
Streptomyces lividans (8), and in plasmid origins of
replication, including R6K (9), RK2 (10, 11), F (12), pSC101 (13), P1
(14), R1 (15), ColE1 (16), and pSP10 (17).
Whereas the interactions of DnaA at oriC have been studied
in considerable detail (for a review, see Ref. 2), much less is known
about the formation of DnaA-nucleoprotein structures at the replication
origins of plasmid replicons. Unlike the more disperse arrangement of
DnaA boxes at oriC and replication origins of other
bacteria, the DnaA boxes at plasmid replication origins in general are
closely associated forming one or more clusters. In addition, the DnaA
protein must work in concert with a plasmid-encoded initiation protein
(for reviews, see Refs. 18 and 19)). Finally, unlike the case with
oriC, the ADP form of DnaA protein is sufficient for
replication in vitro of plasmids R1 (15) and P1 (20) and for
origin opening of F (21), RK2 (11), and R6K (22). These observations
likely reflect fundamental differences in the structure of the
nucleoprotein complexes formed by the DnaA protein at the replication
origin of plasmids and the bacterial chromosome.
The broad host range plasmid RK2 exhibits the remarkable ability to
replicate and maintain itself in a wide variety of Gram-negative bacteria (23) and, thus, is a particularly interesting system for
studying fundamental questions about the interactions between plasmid-
and host-encoded factors at a replication origin. The DnaA protein has
been shown to be required for RK2 replication (24, 25), but its exact
role has been less well studied. Only recently, it was demonstrated
that there are two DnaA boxes in addition to two previously described
boxes located in the leftward part of the RK2 origin, oriV,
and that all four boxes bind the E. coli DnaA protein (11).
The four DnaA boxes arranged as two pairs in an indirect orientation
can potentially form a cruciform structure (see Fig. 1). Boxes 2, 3, and 4 contain one mismatch from the stringent DnaA box consensus
sequence, whereas box 1 contains two mismatches.
Replication initiation of RK2 requires both DnaA and the
plasmid-encoded initiation protein, TrfA. The TrfA protein binds to the
iteron sequences in oriV (10, 26-28) and this binding results in the melting of all four 13-mers in the A+T-rich region in
the presence of the DnaA and HU proteins (11). The DnaA protein also
has the indispensable role of recruiting the DnaB-DnaC complex to the
RK2 origin through a protein-protein (DnaA-DnaB) interaction (29). The
loading and activation of the DnaB helicase at the open region requires
both the DnaA and TrfA proteins (29) and the correct positioning of the
DnaA boxes on the helical axis with respect to the rest of the origin
(30). How the DnaA protein and the TrfA protein cooperate to achieve
DnaB loading in not known.
Do the requirements for DnaA protein binding and function in plasmid
systems differ from that of bacterial chromosomes, specifically oriC? In this paper, we present the molecular and
biochemical characterization of the structural requirements for
functional binding of DnaA protein during plasmid replication
initiation, specifically plasmid RK2. Our results demonstrate that one
of the four DnaA boxes, box 4, directs the cooperative binding of DnaA
to the other DnaA boxes, essentially acting as the "organizer" in
the formation of a functional DnaA-oriV nucleoprotein
structure. These features of DnaA protein binding to a plasmid
replication origin may be important in understanding the differences
between plasmid and chromosomal replication initiation and, in the case of RK2, its broad host range properties.
Strains, Plasmids, and Proteins--
Plasmid pKD19L1, a dual
RK2-R6K mini-replicon, was used for constructing all mutations using
the QuickChange polymerase chain reaction-based site-directed
mutagenesis kit (Stratagene, Inc.) as described previously (30). All
oligonucleotide primers used for polymerase chain reaction were
synthesized by OPERON, Inc. The E. coli strains used in this
study were XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1
lac (F' proAB lacIqZDM15 Tn10))
from Stratagene, Inc., C600 (thr leu thi lacY supE44 tonA),
CC118 (araD139 D(ara, leu)7697
DlacX74 phoAD20 galE galK thi rpsE rpoB
argEam recA1), and CC118 DNase I Footprinting--
DNA probes were prepared as described
(11) with [
For footprinting of specific nucleoprotein complexes, reactions were
assembled as for gel mobility shift assays described below except that
the amount of probe used was increased. After incubating the reaction
containing labeled DNA template and DnaA protein for 15 min at
37 °C, DNase I was added (6.7 units/reaction) for 2 min at the same
temperature. Reactions were then run on a 5% nondenaturing
polyacrylamide gel. After electrophoresis, specific complexes were
identified and isolated from the gel and eluted overnight at 30 °C
in 1 M LiCl2, 20 mM Tris-HCl, pH
7.6, 0.2% SDS, 0.2 mM EDTA. Eluant was collected over a
spin column (Bio-Rad), extracted with phenol-chloroform,
ethanol-precipitated, and resuspended in loading buffer.
Gel Mobility Shift Assay--
Complexes between oriV
DNA and DnaA protein were formed and analyzed essentially as described
previously (11) except that the reaction mixture, based on standard
conditions used for RK2 in vitro replication (29), contained
40 mM Hepes/KOH, pH 8.0, 25 mM Tris-HCl, pH
7.4, 80 µg/ml bovine serum albumin, 4% sucrose, 4 mM
dithiothreitol, 11 mM magnesium acetate. Reactions also
contained 25 µg/ml poly(dI-dC) (Boehringer Mannheim) and 0.1%
Nonidet P-40. Incubation was at 34-37 °C for 20 min.
Requirement of DnaA Boxes for oriV Function--
To address the
importance of each DnaA box present in the RK2 origin (Fig.
1), individual box sequences were
replaced with the scramble sequence 5'-GATATCCTG, which is similar to a
sequence shown previously not to bind DnaA protein (35). Thus, mutants oriV A1, A2, A3, and A4 contain the scramble sequence
(5'-GATATCCTG) in place of the wild-type DnaA box sequence for boxes 1, 2, 3 and 4, respectively (see Fig. 1). Mutated origins were also
constructed that contained deletions of two, three, or all four DnaA
boxes. Mutagenesis was carried out using polymerase chain reaction (see under "Experimental Procedures") and the resulting origins were subcloned in plasmid pKD19L1 (30). All mutations were confirmed by
sequence analysis (data not shown). Plasmid pKD19L1 is a dual origin
(RK2-R6K
The mutated origins were also analyzed for replication activity
in vitro using an in vitro replication assay for
RK2 that has been described previously (33). This assay utilizes a 40% (NH4)2SO4 fraction from a soluble
E. coli extract to replicate supercoiled DNA containing an
RK2 origin in the presence of the initiator protein, TrfA. Fig.
2 shows DNA synthesis for wild-type and
altered origin templates. In vitro replication activity with the scrambled DnaA box origin templates was relatively similar to the
activity that was observed in vivo (Fig. 2A).
Scramble mutations in DnaA boxes 1 and 2 had no inhibitory effect on
DNA synthesis, whereas mutations in DnaA boxes 3 and 4 resulted in a
lower level of replication activity, with mutant oriV A4
displaying only 50% of wild-type levels. Replication activity of
origin templates with deletion mutations (Fig. 2B) again
showed that DnaA boxes 1and 2 can be deleted with out affecting
replication levels, whereas deleting all four DnaA boxes abolished DNA
synthesis. Replication of an origin template with only DnaA box 4 (oriV A Box 4 Is Essential for DnaA Binding to Other Boxes at the
Replication Origin--
We analyzed the binding of DnaA protein to
oriV fragments containing the DnaA box scramble mutations
using DNase I footprinting and a gel mobility shift assay. The DNase I
pattern of DnaA protection for wild-type oriV (Fig.
3, lanes 1-3) was as has been
previously shown for both linear and supercoiled oriV
templates (11). When a DnaA box contained the scrambled sequence, full
DNase I protection was not observed in that box. Thus, the replacement
of the wild-type sequence with the scramble sequence had altered DnaA
binding to the respective box. Interestingly, the scramble mutation in
DnaA box 4 (oriV A4) greatly reduced the amount of
protection observed for box 3 (Fig. 3, lanes 13-15). We
further analyzed DnaA binding to mutants oriV A3 and
oriV A4 using a gel mobility shift assay (Fig.
4). As expected for wild-type
oriV, increasing DnaA concentration led to four specific
nucleoprotein complexes (Fig. 4, lanes 1-5); however, on
the basis of the relative intensity of the various retarded bands,
their formation did not proceed sequentially. Even with smaller amounts
of DnaA protein, the appearance of complex I never occurred in the
absence of complex II, and usually there was much less of complex I
(data not shown and Ref. 11). Thus, binding of DnaA protein to linear
oriV DNA appears to be more stable when in the form of
complex II, suggesting the possibility of cooperative binding by DnaA.
DnaA binding to scramble mutants oriV A3 and A4 support this
idea. When DnaA box 3 was scrambled, only complex II and, to a lesser
extent, complex I was seen (Fig. 4, lanes 6-10). When DnaA
box 4 was scrambled, no nucleoprotein complexes were observed (Fig. 4,
lanes 11-15). This is consistent with the lack of
replication activity observed for mutant oriV A4 in
vivo (Table I) and the substantially reduced activity in vitro (Fig. 2A), suggesting that the function of DnaA
box 4 is to initiate DnaA binding to oriV.
DnaA Binds Initially to Boxes 4 and 3--
We further analyzed
DnaA binding by carrying out footprint analysis on specific
DnaA-oriV nucleoprotein complexes. DnaA was incubated with
wild-type oriV probe, treated with DNaseI, and run on a
native gel in order to isolate specific complexes. DnaA protein
concentrations were adjusted in order to obtain only complexes I and II
(see Fig. 4). DnaA-oriV complexes I and II were isolated, gel purified, and run on a denaturing gel in order to visualize DNaseI
protection patterns (Fig. 5). The
analysis of nucleoprotein complexes I and II showed that both DnaA
boxes 4 and 3 were protected from the DNaseI treatment. These results
demonstrate that upon the binding of DnaA to oriV, the first
boxes to be filled are boxes 4 and 3.
The fact that the protection pattern for complex I (which may be
expected to represent DnaA binding to a single DnaA box) shows that
both boxes 4 and 3 are bound by DnaA (Fig. 5, middle lane) suggests that the binding by DnaA to one box is
immediately followed by the binding to a second box. We should note
that similar results were obtained at even lower DnaA protein
concentrations (data not shown). This supports the idea that DnaA
binding to either box 4 or 3 alone is unstable compared with the
binding of both boxes. Because the treatment with DNase I is done prior to separation by gel electrophoresis, any subsequent disassociation of
DnaA protein from either box 4 or 3 would result in the isolation of
complex I, which contains a footprint of both boxes 4 and 3. We
investigated whether the appearance of complex I was simply a result of
the disassociation of DnaA protein from complex II in solution or
during gel electrophoresis by looking at the rate of complex I
formation; however, neither the incubation time nor the amount of gel
running time had any effect on the formation of complex I (data not
shown). It is possible that complex I contains DnaA bound to both boxes
4 and 3 but is structurally different than complex II and thus exhibits
an altered mobility during gel electrophoresis.
Cooperative DnaA Binding Is Dependent on the Position and
Orientation of Box 4--
Because DnaA box 4 appears to be critical in
initiating binding to the other DnaA boxes, we investigated whether it
is the actual DnaA box sequence and/or the position of box 4 that is crucial. Mutations were constructed as described above and analyzed for
in vivo activity (Table II)
and ability to bind DnaA protein using the gel mobility shift assay
(Fig. 6). When the sequence of box 4 was
replaced with the sequence of box 1 (oriV 4-1), in vivo activity was abolished and DnaA binding was greatly reduced (Fig. 6A). The results were the same if box 4 sequence was
reintroduced into this mutant construct in the box 3 position
(oriV 3-4, see Table II and Fig. 6B). If box 4 was flipped (oriV 4-Flip) so that it faced away from the
other boxes, the effect was even more dramatic in that complexes were
not formed at all with DnaA protein (Fig. 6C), and the
origin was completely inactive in vivo (Table II). Because
the sequences surrounding DnaA boxes in oriC have been shown
to effect DnaA binding (36), it was possible that DnaA could not bind
to box 4 in the oriV 4-Flip mutant because the adjoining
sequences were not also placed in the flipped position. Therefore, a
mutant was constructed that flipped box 4 and the adjoining sequences
(3 base pairs on either side of the box, oriV 4-FlipX). This
construct was also inactive in vivo (Table II), but
nucleoprotein complexes were observed with DnaA protein (Fig. 6D), suggesting that now DnaA could bind to box 4 as well as
the other boxes. However, the binding of DnaA to the boxes in this mutant template proceeded in a stepwise, noncooperative fashion, which
is not what is observed for a wild-type oriV template (see Fig. 4, lanes 1-5). Thus, the orientation of DnaA box 4 and
its adjoining sequences, relative to box 3, is critical for cooperative binding and it is this binding that is required for replication activity of oriV.
We have investigated the binding of the DnaA protein to the RK2
replication origin and the DnaA box requirement for plasmid RK2
replication. Our results clearly showed that in E. coli,
boxes 3 and 4 are essential for optimal replication activity, whereas boxes 1 and 2 are dispensable. The DnaA-oriV interaction is
dependent upon one critical box, box 4, as mutations in box 4 abolished stable DnaA binding to all four boxes. DNaseI footprinting of specific
DnaA-oriV complexes demonstrated that the first boxes to be
filled are boxes 4 and 3. These results, along with the nonlinear
progression of DnaA-oriV complex formation observed in the
gel mobility shift assay, indicate that DnaA binds cooperatively to the
boxes at the RK2 origin. This is in contrast to what has been observed
at oriC, where several approaches failed to detect cooperative DnaA binding (37, 38). DnaA was shown to bind preferentially to box R4, but the binding preference of DnaA to the
other sites was not altered when box R4 was mutated (38).
The DNase I footprinting experiments performed here do not reveal which
box in oriV, 4 or 3, actually binds DnaA first. We cannot
exclude the possibility that DnaA binds initially to either box 4 or
box 3 without a preference. This could account for the observed
footprint by DnaA in complex I (Fig. 5, middle lane), which
indicates a mixture of both boxes. However, we propose that because a
mutation in box 4 abolishes replication activity and the stable binding
of DnaA to the other boxes, DnaA probably binds first to box 4 and then
cooperatively, possibly via a conformational change in the DNA, to box
3. What is clear is that a fully functional origin requires the
formation of a complex II that contains DnaA protein bound to both
boxes 4 and 3.
Further analysis of DnaA box 4 argues that there are specific
requirements for both its sequence and position for oriV
functionality. If box 4 is replaced with the sequence of box 1 (mutant
oriV 4-1) the origin is inactive (Table II) and DnaA binding
is greatly reduced as judged by gel retardation analysis (Fig.
6A). The sequence of box 1 has less homology to the DnaA box
consensus than box 4, and in footprinting experiments more DnaA protein
is required to see protection in box 1 than in box 4 (Fig. 3 and data
not shown). Thus, the data suggest that box 4 has a higher affinity for
DnaA or forms a more stable complex with DnaA than the other DnaA boxes
and, in this capacity, acts to recruit DnaA protein to oriV.
However, the critical role of box 4 is also dependent upon its relative
position within the origin. In mutant oriV 3-4, the box 4 sequence replaces DnaA box 3 of the oriV 4-1 mutant (see
Table II); this mutant was also inactive and displayed reduced DnaA
binding, arguing for the importance of the location of box 4. It should
be emphasized that the gel mobility shift assay used in these
experiments is a measure of both protein affinity to the individual
boxes and the stability of the nucleoprotein complexes. It is,
therefore, possible that DnaA protein is still able to bind to the
boxes in these mutant templates but requires contact with box 4 in its
"correct" position to form stable complexes observable during gel electrophoresis.
It has been shown for oriC that the preference for DnaA box
R4 depended on it being in the correct orientation (38). We found for
oriV that if box 4 was inverted (mutant oriV
4-Flip), DnaA protein did not form stable complexes with any of the
DnaA boxes unless adjoining sequences were also inverted (mutant
oriV 4-FlipX) (Fig. 6). Thus, with the oriV
4-FlipX mutant, DnaA did bind to all four boxes, but this binding no
longer exhibited cooperativity. Possibly, in this case the binding of
DnaA to the high affinity but inverted box 4 increased the local
concentration of DnaA protein and, thus, enhanced the probability of
other DnaA-DnaA box contacts. Interestingly, the oriV
4-FlipX mutant was not active in vivo. Presumably when DnaA
is bound to the inverted box 4, it is now on the opposite face of the
DNA helix with respect to box 3, thus disrupting the structure and/or
critical protein contacts required for cooperative binding. These
results demonstrate that the correct orientation of box 4 is crucial
for directing not only the cooperative binding of DnaA but the
formation of a nucleoprotein structure that is required for a
functional initiation complex. More than likely, this specific
structure is critical for the loading of DnaB helicase. We have
previously demonstrated that when all four DnaA boxes are on the
opposite side of the DNA helix with respect to the rest of the origin,
DnaB helicase activity at oriV is reduced even though the
origin is fully open (30). Unlike replication initiation at
oriC, where origin opening and helicase loading occur as the
result of a single initiator protein (DnaA), which is responsible for
both steps, initiation at a plasmid origin usually requires two
initiator proteins (DnaA and a plasmid-encoded replication initiation
protein) and, therefore, may involve a fundamentally different
nucleoprotein structure for facilitating each step of replication initiation.
Most plasmid replication origins contain more than one DnaA box or
cluster of DnaA boxes. In the case of the narrow host range plasmids P1
(39) and R6K (40), one box is sufficient for a functional origin.
Evidence for cooperative DnaA binding to the DnaA boxes at the origin
of pSC101 has been demonstrated to occur at a distance (DNA looping)
with the combined action of the RepA initiator protein and IHF (41).
However, for RK2, the cooperative binding exhibited by the DnaA protein
occurs at boxes that are separated by only three to five base pairs and
is not dependent on the binding of additional proteins, including the
initiator protein, TrfA. Recently it was shown for plasmid R6K that the DnaA protein physically interacts with the R6K plasmid specific initiator protein and that this interaction is required for origin opening (22). There is no evidence for an interaction between DnaA and
the RK2 initiator protein, TrfA. Preliminary results using ELISA to
detect protein-protein interactions suggest that if an interaction does
occur, it is a relatively weak
interaction.2 An interaction
between the DnaA and TrfA proteins clearly is not required for binding
of the two proteins to their respective sites, DnaA boxes or iteron
sequences (11), or for the cooperative DnaA-oriV interaction
presented here. It is possible that these differences in molecular
interactions at the replication origin observed between the narrow host
range plasmids, P1 and R6K, and RK2 at least partially account for
their different host range.
It was surprising to us that DnaA boxes 1 and 2 can be deleted without
affecting the efficiency of transformation of E. coli (Table
I) or in vitro replication activity (Fig. 2), especially because the four boxes potentially can form a cruciform structure (11).
However, in view of the broad host range replicating activity of RK2,
the experiments performed here with E. coli do not rule out
the possibility that a cruciform structure and/or all four DnaA boxes
are required in bacterial hosts other than E. coli for
optimal replication. Prior to the discovery of all four DnaA boxes,
mutagenesis studies of the RK2 origin region demonstrated a
differential requirement of DnaA boxes between E. coli and
P. aeruginosa (42-44). Thus, it is of interest to test the
DnaA box mutants constructed in this study in other bacterial hosts, as different DnaA and other host replication proteins may act differently at the RK2 origin.
The finding that boxes 4 and 3 are sufficient for oriV
activity indicates that the DnaA-oriV complex is a much more
compact structure than is found for oriC, where the
nucleoprotein complex extends over approximately 200 base pairs. Do the
structural differences reflect the differences in DnaA function for the
two replicons? In the case of oriC, the DnaA protein does
not bind cooperatively to the five boxes, yet it directs the wrapping
of DNA around a core of DnaA protein monomers and is subsequently
responsible for both the opening of the 13-mer region and the
recruitment of the DnaB-DnaC complex. For oriV, the DnaA
protein plays a more limited role in that although it recruits the
DnaB-DnaC complex to the origin region, it cannot by itself form an
open complex. Our results have further demonstrated through mutational
analysis and protein binding experiments that one box, box 4, effectively acts as the organizer for DnaA cooperative binding at
oriV. This binding then initiates a nucleoprotein structure
that is functional for the formation of a prepriming complex. This may
represent a novel type of DnaA binding at a prokaryotic origin. It will be of interest to determine whether this type of DnaA interaction is
found for other plasmid systems and whether it is a critical factor for
the broad host range properties of the RK2 plasmid.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pir
(CC118 (
pir)), (31); the latter two strains were kindly
provided by Dr. Ken Timmis. Analysis of in vivo and in
vitro replication activity of mutated plasmid constructs was
performed as described previously (30). The E. coli DnaA
protein was provided by Dr. Alessandra Blasina. The TrfA protein,
His6-TrfA G254D/S267L, was purified as described previously
(32). In the in vitro experiments performed here and
described previously (30, 33) the histidine-tagged version of the
largely monomeric (34) 33-kDa TrfA mutant protein
(His6-TrfA G254D/S267L), which exhibits elevated iteron
binding (32) and is fully functional in vivo and in
vitro for replication was used. Commercially available proteins
were as follows: HU from Enzyco; bovine serum albumin (fraction V) from
Sigma; Pfu DNA polymerase and DpnI restriction endonuclease
from Stratagene, Inc.; and other restriction endonucleases and T4 DNA
ligase from Promega.
-32P]dATP (6000 Ci/mmol) and the Klenow
fragment of E. coli DNA polymerase (NEN Life Science
Products). Unincorporated ATP was removed using nucleotide removal spin
columns (Qiagen). Binding of DnaA protein to origin DNA containing
mutated DnaA box sequences was performed essentially as described
previously (11), except that DnaA protein was incubated with DNA at
37 °C for 15 min and DNase I (New England Biolabs, Inc.), 0.67 units/reaction, at 37 °C for 30 s.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) plasmid that replicates by the R6K
origin in E. coli strain CC118 (
pir) because this strain encodes the R6K initiator protein. In the parental strain, CC118, only the RK2 origin
may be utilized. Thus, the ratio of transformation frequencies obtained
in CC118 versus CC118(
pir) was used to quantitate
oriV function in vivo. Ratios obtained for
altered origin constructs were normalized to those obtained for pKD19L1
(wild-type oriV) (Table I).
Origins that contained a scrambled sequence in DnaA box 1 (oriV A1) or box 2 (oriV A2) or a deletion of
both boxes 1 and 2 (oriV A
1-2) did not negatively effect
origin function and actually resulted in a somewhat higher level of
plasmid transformation frequencies. However, a scrambled sequence in
DnaA box 3 (oriV A3) or box 4 (oriV A4) reduced
or completely abolished plasmid transformation, respectively.
Similarly, an origin containing a deletion of all four DnaA boxes
(oriV A
1-4) rendered the plasmid inactive. An origin
containing only DnaA box 4 (oriV A
1-3) was only 74%
active compared with wild-type (Table I). The oriV A
1-3 mutant is highly variable; in another set of experiments, replication activity was as low as 32% of the activity of a wild-type origin (data
not shown). Additionally, the transformants that were obtained with the
oriV A
1-3 mutant were lost fairly rapidly compared with the wild-type plasmid in the absence of antibiotic
selection.1 Thus, whereas
both DnaA boxes 3 and 4 are required for replication activity in
vivo, the requirement for DnaA box 4 is the more stringent.
View larger version (21K):
[in a new window]
Fig. 1.
The sequence of the DnaA boxes contained in
the minimal origin of RK2. The four DnaA boxes present at the
leftward end of the origin are arranged as inverted pairs directly
upstream of the TrfA binding sites (iterons) and the four 13-mers in
the A+T-rich region that serve as the site of helix destabilization.
Replication proceeds toward the G+C-rich region.
Transformation frequencies with RK2 replication origins containing DnaA
box mutations
1-3) was slightly less than wild-type except at
the highest amount of TrfA protein.
View larger version (20K):
[in a new window]
Fig. 2.
In vitro replication of
oriV templates containing DnaA box mutations.
In vitro replication was performed using an E. coli C600 extract (fraction II) active for RK2 replication
described under "Experimental Procedures." Reactions contained 0.3 µg of supercoiled wild-type and mutant pKD19L1 template and the
indicated amount of TrfA protein. A, the DnaA box scramble
mutants: filled circle, wild-type oriV;
open circle, oriV A1; open square,
oriV A2; filled square, oriV A3; ×,
oriV A4. B, the DnaA box deletion mutants:
filled circle, wild-type oriV; open
circle, oriV A 1-2; open square,
oriV A
1-3; filled square, oriV
A
1-4.
View larger version (110K):
[in a new window]
Fig. 3.
DNase I footprint of DnaA box scramble
mutations. Restriction fragments (0.6 ng) containing the
oriV region with wild-type or mutated DnaA boxes were
incubated with DnaA protein for 15 min at 37 °C followed by DNase I
treatment for 30 s. The left lane in each template set
( ) is the reaction without protein followed by reactions incubated
with 625 and 2500 ng of DnaA protein (+ and ++, respectively).
Protection from DNase I corresponding to each DnaA box is indicated by
brackets. Mutant templates did not display full DNase I
protection in the region of the box that contained the scrambled
sequence.
View larger version (41K):
[in a new window]
Fig. 4.
DnaA binding to oriV
fragments using gel mobility shift assay. Restriction
fragments (0.15 ng) containing wild-type oriV
(A), a scramble mutation in box 3 (B), and a
scramble mutation in box 4 (C) were incubated with
increasing amounts of DnaA protein for 20 min at 34 °C. The
first lane in each template set ( ) is without protein,
followed by 31, 62, 124, and 248 ng of DnaA protein (lanes from
left to right). The positions of the four
complexes (I-IV) previously described (11) are indicated. The
shaded arrows indicate that a strong or weak band is
present, and the open arrows indicate that no band is
present at the expected position.
View larger version (37K):
[in a new window]
Fig. 5.
DNase I footprinting of specific
nucleoprotein complexes. Linear template (2.5 ng) containing the
wild-type oriV region was incubated with 31 ng of DnaA
protein followed by DNase I treatment as described under
"Experimental Procedures." Complexes I and II were separated by gel
electrophoresis, isolated from the acrylamide gel, and analyzed on a
10% denaturing gel.
Transformation frequencies of additional DnaA box mutants
View larger version (48K):
[in a new window]
Fig. 6.
DnaA binding to the oriV
region containing mutations relating to DnaA box 4. Template
DNA (0.25 ng) was incubated with 0, 31, 62, 124, and 248 ng of DnaA
protein (lanes from left to right) and analyzed
for complex formation by gel electrophoresis. Mutant templates,
described in Table II, used were oriV 4-1(A),
oriV 3-4 (B), oriV 4-Flip
(C), and oriV 4-FlipX (D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Blasina for the generous gift of purified DnaA protein and Drs. E. P. Geiduschek and A. Grove for helpful advice during the course of these experiments.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Research Grant AI-07194.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a National Science Foundation Graduate Fellowship.
¶ To whom correspondence should be addressed: Dept. of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093-0322. Tel.: 619-534-3638; Fax: 619-534-7073; E-mail: dhelinski{at}ucsd.edu.
** Supported by Polish State Committee for Scientific Research Grant 6P04A01115.
1 Doran, K. S., Helinski, D. R., and Konieczny, I. (1999) manuscript in press.
2 K. S. Doran and D. R. Helinski, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|