From the Department of Molecular Genetics and Biochemistry,
University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
A large number of plasmids have been shown to
replicate by a rolling circle (RC) mechanism. The initiators encoded by
these plasmids have origin-specific, nicking-closing activity that is required for the initiation and termination of RC replication. Since
the initiators of many RC plasmids are rate-limiting for replication,
these proteins are usually inactivated after supporting one round of
replication. In the case of the pT181 plasmid, inactivation of the
initiator RepC protein occurs by the attachment of an oligonucleotide to its active tyrosine residue. We have generated the inactivated form
of RepC, termed RepC*, in vitro and investigated the
effects of attachment of the oligonucleotide on its various biochemical activities. Our results demonstrate that while RepC* is inactive in
nicking-closing and replication activities due to the blockage of its
active tyrosine residue, it is competent in origin DNA binding and DNA
religation activities. We have investigated the oligomeric state of
RepC and RepC* and found that RepC exists as a dimer in solution and
can oligomerize on the DNA. We have generated heterodimers in
vitro between the wild-type and epitope-tagged RepC proteins. In
electrophoretic mobility shift experiments, the initiator heterodimers
generated a novel DNA-protein complex, demonstrating that it binds to
DNA as a dimer. We have shown that a DNA binding mutant of RepC can be
targeted to the origin in the presence of the wild-type protein
primarily through a protein-protein interaction. Interestingly, RepC*
is defective in its ability to oligomerize on the DNA. RepC* inhibited
the DNA binding and replication activity of wild-type RepC to only a
very limited extent, suggesting that it may play only a minor
regulatory role in replication in vivo. Based on these and
earlier results, we propose a model for the role of RepC during the
initiation and termination of pT181 RC replication.
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INTRODUCTION |
Bacterial plasmids provide useful model systems to study the
mechanism and regulation of DNA replication. A large group of plasmids
in Gram-positive bacteria and many in Gram-negative bacteria replicate
by a rolling circle (RC)1 mechanism (for
reviews, see Refs. 1-3). Replication of
RC plasmids is mechanistically similar to those of single-stranded (SS)
DNA bacteriophages of Escherichia coli (4-6). Replication
of RC plasmids is initiated by the generation of a single strand nick
at the origin of replication by the plasmid-encoded initiator proteins. Plasmids of the pT181 family in Staphylococcus aureus encode
initiators that have origin-specific DNA binding and topoisomerase-like
activities (7-9). The origin of replication of pT181 consists of three
sets of inverted repeat (IR) elements, termed IRI, IRII, and IRIII (Ref. 10; Fig. 1). We have previously shown that the pT181 origin contains a bend that is enhanced by binding of RepC (11). The pT181
origin also contains a cruciform region centered around IRII, and RepC
has been shown to enhance cruciform extrusion (12). It has also been
shown that while both IRII and IRIII are required for initiation of
replication, IRII is sufficient for the termination step (13,
14).
The RepC protein consists of 314 amino acids with a molecular weight of
38,000 (15, 16). Footprinting studies have shown that the initiator
encoded by pT181, RepC, binds to IRIII and the proximal arm of IRII
(Ref. 8; Fig. 1). The RepC nick site is located between nt 70 and 71 within the loop of IRII (7). After nicking, RepC becomes covalently
attached to the 5'-phosphate of the DNA through the active site
tyrosine residue at position 191 (17, 18). The sequence-specific DNA
binding domain of RepC has been identified, and amino acids 267-270 in
RepC were shown to be important for this activity (19).
Plasmid pT181 is maintained at an approximate copy number of 22 (20).
Previous studies have shown that RepC is rate-limiting for replication
and that the primary mode of regulation of pT181 copy number involves a
transcription attenuation mechanism of RepC mRNA by an antisense
RNA (21, 22). It has also been shown that overexpression of RepC
in vivo can inhibit pT181 replication, probably due to the
titration of the rate-limiting PcrA helicase (23-25). To prevent the
reutilization of an initiator molecule that has been used for a round
of replication, RepC is inactivated by the attachment of an
approximately 11-nt-long oligonucleotide corresponding to pT181
positions 70-60 located immediately downstream of the initiator nick
site (26, 27). This probably results from the passage of the
replication fork past the RepC nick site to the end of IRII (nt 60)
after one round of replication and the subsequent cleavage and
religation of the DNA by RepC (3, 14). Interestingly, preparations of
the initiator protein from cells containing either wild-type pT181 or
its copy mutants contain an approximately 1:1 mixture of the RepC and
RepC* forms (27, 28). Based on this and other findings, it has been
proposed that RepC exists as a dimer in solution and that attachment of the oligonucleotide to one subunit results in the inactivation of the
other, wild-type subunit by an as yet unknown mechanism (25, 27).
Recently, the RepC/RepC* heterodimer isolated from pT181-containing
cells was found to retain its DNA binding activity but was defective in
its ability to induce cruciform extrusion at the origin (25, 28, 29).
Since preparations of RepC* from pT181-containing cells always contain
a 1:1 mixture of RepC and RepC* forms, we have generated RepC* in
vitro. This preparation consists of approximately 95% of the
molecules in the RepC* form and is suitable for investigation of its
biochemical activities. We have studied the oligomeric state of RepC
and found that RepC exists as a dimer in solution and can oligomerize
upon binding to the DNA. We also report here the finding that while
RepC* also exists as a dimer in solution, attachment of an
oligonucleotide to the initiator protein blocks its oligomerization at
the origin. Our results also demonstrate that while RepC* is defective
in its DNA nicking and replication activities, it retains origin binding and DNA religation activities.
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EXPERIMENTAL PROCEDURES |
Purification of RepC and MBP-RepC Proteins--
Wild-type RepC
protein was purified using an overexpression system as described
earlier (16). For the production of the maltose binding protein
(MBP)-RepC fusion protein (MBP-RepC), the repC gene was
cloned in frame with the MBP coding sequence utilizing the vector
pMAL-c, which contains the
isopropyl-1-thio-
-D-galactopyranoside-inducible tac promoter (30). This resulted in the generation of a
MBP-RepC fusion protein (80-kDa) in which MBP was present at the
amino-terminal end of RepC. Synthesis of the fusion protein was induced
by the addition of isopropyl-1-thio--D-galactopyranoside
to exponentially growing cultures, and the MBP-RepC fusion protein was
purified by affinity chromatography on amylose-resin columns (30). The MBP-RepC fusion protein was eluted with a buffer containing 20 mM Tris-HCl (pH 8), 200 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 10 mM maltose, and the fractions were dialyzed against RepC buffer (20 mM Tris-HCl (pH 8.0), 200 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, and 10% ethylene
glycol). The RepC and MBP-RepC proteins were generally more than 95%
pure as determined by SDS-PAGE and staining with Coomassie Brilliant
Blue. Heterodimers between the RepC and MBP-RepC proteins were
generated by denaturation in vitro. Equimolar amounts of the
two proteins were mixed and incubated at room temperature for 1 h
in RepC buffer containing 7 M urea. This was followed by
successive dialysis of the protein against RepC buffer containing 3 M urea, 1 M urea, and no urea. As controls, the
RepC and MBP-RepC proteins were treated individually in a similar
manner.
Preparation of RepC*--
Seventy micrograms of RepC protein
were incubated with an approximately 4-fold molar excess of an
oligonucleotide containing the right arm and loop of IRII (pT181 nt
75-60) at 32 °C for 15 min. Substantial amounts of the unreacted
oligonucleotide were removed by centrifugation of the reaction mixture
on a G-50 spin column. However, this RepC* preparation was further
purified by FPLC (described below) to completely remove the unreacted
oligonucleotide. This was necessary, since we found that in DNA
relaxation and in vitro replication experiments involving
competition between RepC* and wild-type RepC, the presence of free
oligonucleotide and magnesium ions resulted in the conversion of RepC
to RepC* and gave artifactual results that suggested that RepC*
severely inhibited the RepC activity (data not shown). The extent of
conversion of RepC to RepC* was determined by SDS-PAGE followed by
staining with Coomassie Brilliant Blue. Typically, RepC* preparations
contained no more than 5% of wild-type RepC. To recover RepC activity
from RepC*, the RepC* preparation was incubated with a 20-fold molar excess of an oligonucleotide (13-mer) representing the region immediately 5' to the RepC nick site (nt 83-71) for 15 min at 32 °C
in TEKEM buffer (10 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 100 mM KCl, 10 mM MgCl2, and
10% ethylene glycol). The reaction product was then directly used to
assay for the recovered RepC activities.
DNA Relaxation Assays--
Nicking-closing assays were done as
described previously (7). One microgram of supercoiled
pT181cop608 DNA was incubated with various amounts of the
RepC or RepC* proteins at 32 °C for 30 min in TEKEM buffer, and the
reaction mixtures were subjected to electrophoresis on 0.7% agarose
gels using TBE (Tris borate-EDTA) buffer containing 1 µg/ml ethidium
bromide. To study the inhibitory effect of RepC* on the relaxation
activity of RepC, the proteins were mixed together prior to the
addition of DNA.
Nicking and Religation of Oligonucleotides--
For nicking, 1 pmol of 5'-end-labeled oligonucleotides (pT181 nt 83-60 containing the
IRII and nt 75-60 representing the right arm and loop of IRII (Fig. 1)
were incubated with 200 ng of the RepC or RepC* proteins for 30 min at
32 °C in TEKEM buffer in a reaction volume of 10 µl. For
religation, reaction mixtures containing 600 ng of RepC* along with 1 pmol of labeled oligonucleotides representing the sequence immediately
5' to the RepC nick site (pT181 nt 83-71 and 75-71; Fig. 1) were
incubated for 15 min at 32 °C in TEKEM buffer. The above reactions
were stopped by the addition of 5 µl of sequencing dye, and the
products were resolved on 20% polyacrylamide-urea DNA sequencing gels
(31).
Electrophoretic Mobility Shift Assays--
The binding of
various proteins to the pT181 origin subregions was investigated by
electrophoretic mobility shift assays (EMSAs). Double-stranded
oligonucleotides containing various subregions of the pT181 origin were
5'-end-labeled with polynucleotide kinase and
[
-32P]ATP (31). Unless otherwise indicated, the 53-bp
oligonucleotide (pT181 positions 37-83) was used as the probe.
Reaction mixtures (20 µl) containing the 32P-labeled
oligonucleotides (2-5 pmol), 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM EDTA, 5 mM
dithiothreitol, 10% ethylene glycol, 100 ng of poly(dI-dC), and the
indicated amounts of the initiator proteins were incubated at room
temperature for 10 min and electrophoresed on native polyacrylamide
gels using TBE buffer. The gels were dried and subjected to
autoradiography.
In Vitro Replication Reactions--
Plasmid DNAs were replicated
in vitro using S. aureus extracts as described
earlier (16, 32). Replication reactions were carried out using 200 ng
of pT181cop608 plasmid DNA, 600 µg of protein extract, and
the indicated amounts of the RepC or RepC* proteins. The reaction
products were labeled with [
-32]dATP and incubated for
60 min at 32 °C. DNA was recovered by treatment with proteinase K,
phenol-chloroform extraction, and alcohol-precipitation, and the
samples were analyzed by electrophoresis on 0.7% agarose gels using
TBE buffer containing 0.5 µg/ml ethidium bromide. The gels were dried
and subjected to autoradiography.
FPLC of RepC and RepC*--
Approximately 500 µg of RepC or
250 µg of RepC* proteins were loaded onto an Amersham Pharmacia
Biotech Superose 12 FPLC column (20 ml) that had been previously
equilibrated with the RepC buffer. The column was eluted with 20 ml of
the RepC buffer, and 0.5-ml fractions were collected. In a separate
experiment, bovine serum albumin (67 kDa), ovalbumin (43 kDa), and
chymotrypsinogen A (25 kDa) were used as molecular weight standards to
generate a calibration curve.
Sucrose Gradient Sedimentation Analysis of the RepC
Protein--
RepC protein was subjected to sedimentation in a 5-20%
sucrose gradient (5 ml) in a SW50.1 rotor at 40,000 rpm for 24 h
at 4 °C. Ten micrograms each of bovine serum albumin, ovalbumin, chymotrysinogen, and ribonuclease A along with 16.5 µg of RepC were
used. Fractions (185 µl each) were collected from the bottom of the
tubes and analyzed by SDS-PAGE and silver staining. Fractions were also
assayed for the topoisomerase activity of RepC as described above.
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RESULTS |
Generation of the Inactivated RepC* Protein in Vitro--
To
investigate the biochemical activities of the modified pT181 initiator
protein, we generated RepC* in vitro. Treatment of an SS
oligonucleotide (nt 75-60 of pT181, Fig.
1) containing the nick site with RepC
generated a protein preparation that contained approximately 95% RepC*
and less than 5% wild-type RepC as determined by SDS-PAGE (Fig.
2). The above results suggested that if
RepC exists as a dimer, both of its subunits must be competent in
nicking of the DNA. Alternatively, RepC may exist as a monomer and can be converted to RepC* almost quantitatively at high concentrations of
the oligonucleotide. Treatment of an SS oligonucleotide containing the
complete IRII (nt 83-60; Fig. 1) with RepC generated a protein preparation that contained approximately 50% each RepC and RepC* (Fig.
2). To confirm that RepC* generated in vitro was
"active," we tested its religation activity utilizing SS
oligonucleotides representing the region immediately 5' to the RepC
nick site (Fig. 1). This experiment showed that RepC* retained its
religation activity, and while nt 83-71 ligated efficiently to RepC*,
ligation of nt 75-71 was very poor (Fig.
3). This result is consistent with the
observation that more RepC* was generated in the presence of nt 75 to
60 than with IRII (Fig. 2) due to the less efficient religation of the
released oligonucleotide (nt 75-71) to RepC*. The RepC* preparation
generated in vitro by the above procedure had an approximate
molecular weight of 42,000 and is similar to that generated in
vivo except that it contains less than 5% residual wild-type RepC
as compared with a 1:1 mixture of RepC and RepC* forms isolated from
pT181-containing cells (27). The RepC* preparation generated in
vitro was used to investigate the various biochemical activities
of the modified initiator protein.
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Fig. 1.
Nucleotide sequence of the optimal pT181
origin and the structure of the IRII region. Only the leading
strand of the DNA is shown, and replication proceeds in a rightward
direction. IR sequences IRI, IRII, and IRIII are shown by
horizontal arrows. The RepC binding sequence is
indicated with a dotted line, and the RepC nick
site is indicated by a vertical arrow. Structure
of the 24-bp IRII region (nt 83-60) is shown on the right.
L and R refer to the left and right arms of IRII,
respectively.
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Fig. 2.
SDS-PAGE analysis of the RepC and RepC*
proteins. RepC protein incubated with IRII (nt 83-60) or the
right arm and loop of IRII (nt 75-60), as well as untreated RepC was
subjected to SDS-PAGE on a 12% gel. The positions of RepC and RepC*
are indicated.
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Fig. 3.
Religation activity of the RepC*
protein. Purified RepC* protein was incubated with SS
oligonucleotides representing the sequence immediately 5' to the RepC
nick site (nt 83-71 or nt 75-71), and the reaction products were
analyzed by electrophoresis on a polyacrylamide-urea gel. The
asterisks indicate the positions of the regenerated IRII (nt
83-60) or the right arm and loop of IRII (nt 75-60) oligonucleotides
by the religation activity of RepC*.
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RepC* Is Defective in Nicking-Closing of the DNA--
We
investigated the DNA relaxation activity of RepC* using supercoiled
(SC) pT181cop608 DNA as the substrate. While low levels of
wild-type RepC efficiently relaxed the SC DNA, the RepC* preparation was essentially inactive (Fig.
4A). The weak relaxation
activity in the presence of high levels of RepC* may be due to the
residual amounts of RepC present in the preparation. To further rule
out the possibility that RepC* was inactivated during its preparation in vitro, we incubated RepC* with an oligonucleotide
representing the region immediately 5' to the RepC nick site (left arm
and a portion of the loop of IRII, nt 83-71 in Fig. 1). This treatment was expected to result in the religation of the oligonucleotide attached to RepC* to the substrate oligonucleotide and the concomitant regeneration of wild-type RepC. RepC* treated in such a way was found
to be active in DNA relaxation (Fig. 4A). These results showed that RepC* was inactive in nicking-closing due to the attachment of the oligonucleotide to its active tyrosine residue. RepC* did not
detectably inhibit the relaxation activity of wild-type RepC in
competition experiments (Fig. 4A). We also tested the
cleavage of SS DNA by RepC*. As expected, while RepC efficiently
cleaved the SS oligonucleotide (nt 75-60), RepC* had very low cleavage activity (Fig. 4B). This low level activity of RepC* may be
due to the residual levels of RepC present in the RepC* preparation. The above results indicate that RepC* is defective in SS DNA cleavage and DNA relaxation activities and does not significantly inhibit the
relaxation activity of RepC.
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Fig. 4.
Analysis of the DNA relaxation and SS DNA
cleavage activities of RepC*. A, supercoiled
pT181cop608 DNA was treated with the indicated amounts of
either RepC or RepC* or both. In the lanes labeled LA at the
bottom, RepC* was preincubated with a 13-nt-long
oligonucleotide (positions 83-71) containing the left arm and a
portion of the loop of IRII to regenerate RepC prior to its addition to
the relaxation reactions. The positions of the SC, open circular
(OC), and covalently closed relaxed circular (R)
DNA are indicated. B, a 32P-labeled
oligonucleotide representing the right arm and loop of IRII (nt 75-60)
was treated with 200 ng of RepC or RepC*, and the cleavage products
were analyzed on a 20% polyacrylamide-urea DNA sequencing gel. The
positions of the uncut and cleaved oligonucleotides are
indicated.
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RepC* Is Inactive in in Vitro Replication--
Since RepC* lacked
DNA nicking-closing activity, it was expected to be inactive in
replication. We wished to determine if this was the case and if RepC*
played a regulatory role in DNA replication by inhibiting the activity
of wild-type RepC. As shown in Fig. 5,
wild-type RepC was active in in vitro replication even at
low levels, while RepC* was totally inactive in replication. In
competition experiments, RepC* inhibited the replication activity of
wild-type RepC only to a limited extent (Fig. 5), with an approximately 2-fold inhibition at a 10:1 molar ratio of RepC* to RepC as determined by quantifying the radioactivity using an Ambis 100 radioanalytic detector.
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Fig. 5.
Analysis of the in vitro
replication activity of RepC and RepC*. Plasmid
pT181cop608 DNA was replicated in vitro in the
presence of the indicated amounts of the RepC and RepC* proteins. The
reaction products were analyzed by agarose gel electrophoresis and
autoradiography. SS, single-stranded DNA; RI,
replicative intermediates; SC, supercoiled DNA;
OC, open circular DNA.
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DNA Binding and Oligomerization of RepC and RepC*--
We have
previously shown by footprinting studies that RepC binds to
double-stranded pT181 origin DNA (nt 37-68) that includes IRIII and
the right arm of IRII (8). The DNA binding activity of RepC has also
been shown to be critical for its replication activity (17, 19). We
wished to determine whether RepC* generated in vitro is able
to bind to the pT181 origin of replication. EMSA were carried out using
the following four synthetic, double-stranded oligonucleotides
representing the various subregions of the pT181 origin: a 53-bp DNA
(containing pT181 nt 37-83; Fig. 1) that included both IRIII and IRII;
a 46-bp fragment (pT181 nt 46-83); a 30-bp fragment containing only
IRIII (pT181 nt 37-60); and a 26-bp fragment containing only IRII
(pT181 nt 60-83). The sizes of the various oligonucleotides used were
slightly larger than the respective pT181 sequence they contained due
to the presence of linker sequences at their ends. With the 53- and
46-bp oligonucleotides and low levels of RepC, the C1 DNA-protein
complex was present at much higher levels than the larger C2 complex,
and the levels of the C2 complex increased substantially at higher
levels of RepC (Fig. 6A).
Interestingly, only a single DNA-protein complex (C1) was observed,
with the 30-bp region containing only the IRIII region even at high
levels of RepC (Fig. 6A). Similarly, only a single complex
was observed with the 26-bp IRII region, although the levels of this
complex were much lower than those seen with all the other origin
regions (Fig. 6A). It should be noted that while the sizes
of the four DNA probes used were different, the C1 and C2 complexes
migrated to the same position regardless of the probe used. This may be
due to the small sizes of the probes used where the electrophoretic
mobility was predominantly determined by the size, shape, and charge of
the RepC protein. The above results suggested that two RepC-DNA
complexes were formed at the origin, one due to the binding of RepC to
IRIII, and the other upon binding to both the IRIII and IRII regions.
The RepC* protein retained significant DNA binding activity as tested
by EMSA using the 53-bp fragment as the probe (Fig. 6B).
Interestingly, in the presence of RepC* only the C1 complex was
obtained even at high concentrations of the protein. The C1 complex
observed with RepC* (RC*-C1) migrated slightly slower than the C1
complex observed with RepC, consistent with the fact that the molecular
weight of monomeric form of RepC* is approximately 42,000 as compared
with 38,000 for RepC (26). RepC* competed with RepC for binding to the
DNA to a limited extent when present at higher levels (Fig.
6B). RepC* also generated a single complex with the 46-, 38-, and 23-bp oligonucleotides (data not shown). The above results
showed that RepC* retains significant DNA binding activity and that
while RepC is capable of oligomerization at the origin, RepC* is
inactive in this function.
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Fig. 6.
Binding of the RepC and RepC* proteins to the
pT181 origin. 32P-Labeled synthetic oligonucleotides
containing various portions of the pT181 origin were incubated with the
indicated amounts of the RepC or RepC* proteins in the presence of 100 ng of poly[d(I-C)]. The DNA-protein complexes were electrophoresed
through 6% native polyacrylamide gels. A, binding of the
RepC protein to the various origin regions. RC-C1 and
RC-C2 indicate the two RepC-DNA complexes. The probes used
were 53 bp (nt 37-83); 46 bp (nt 46-83); 30 bp (nt 37-60), and 23 bp
(nt 60-83). B, binding of the 53-bp oligonucleotide to RepC
and RepC* or both. RC*-C1 indicates the complex formed between RepC*
and DNA. Other complexes are as shown in A.
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We further investigated oligomerization of RepC and RepC* when bound to
the DNA utilizing a MBP-RepC fusion. As seen with RepC, the MBP-RepC
protein generated two DNA-protein complexes when the 53-bp
oligonucleotide was used as the probe (Fig.
7). The binding of MBP-RepC to the DNA
was weaker than that of unfused RepC, indicating that the epitope tag
may interfere with the DNA binding activity of RepC to a moderate
degree. The migration of the C1 and C2 complexes obtained with MBP-RepC
(MBRC-C1 and MBRC-C2) was consistent with its monomeric molecular
weight of 80,000, which is slightly more than twice that of wild-type
RepC. When RepC and MBP-RepC were incubated together with the DNA,
their respective C1 and C2 complexes were obtained (Fig. 7).
Interestingly, a novel intermediate complex (IC) was also observed
(Fig. 7). The position of the intermediate complex (between the MBRC-C1 and MBRC-C2 complexes) is consistent with it containing one molecule each of RepC and MBP-RepC, further suggesting that RepC may oligomerize on the DNA. Interestingly, very little or no intermediate complex was
present when RepC* and MBP-RepC were incubated with the DNA (Fig. 7).
The faint IC in these lanes may be due to the residual RepC present in
the RepC* preparations. RepC* also inhibited the formation of the
MBRC-2 complex to a small extent. These results further suggest that
RepC* is defective in oligomerization on the DNA and can inhibit the
DNA binding activity of RepC (and MBP-RepC) to a limited degree. Since
RepC* contains an oligonucleotide attached to the active
Tyr191 residue, it was possible that a free tyrosine at
this position is required for oligomerization of RepC. Alternatively,
the attached oligonucleotide may directly interfere with the
oligomerization of RepC* with itself as well as with wild-type RepC on
the DNA. To distinguish between these two possibilities, we used a
Tyr191 
Ser topoisomerase mutant of RepC (17) in DNA
binding experiments. As shown in Fig. 7, the Tyr Ser mutant
generated both the C1 and C2 complexes and also gave rise to the
intermediate complex in the presence of MBP-RepC. These results clearly
demonstrate that Tyr191 is not required for oligomerization
of RepC on the DNA and that the oligonucleotide attached to
Tyr191 in RepC* is directly responsible for the inhibition
of oligomerization.
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Fig. 7.
Dimerization of RepC and its derivatives on
the DNA. 32P-Labeled 53-bp oligonucleotide (position
37-83) was incubated with the indicated amounts of RepC and its
various derivatives, and the DNA-protein complexes were analyzed by
polyacrylamide gel electrophoresis. N D,
Asn267 to Asp mutant of RepC; Y S, Tyr191 to Ser mutant; MBRC-C1 and MBRC-C2,
DNA-protein complexes obtained in the presence of MBP-RepC.
RC-C1 and RC-C2 indicate the DNA-protein
complexes formed with RepC, Asn Asp, and Tyr Ser.
IC, intermediate DNA-protein complex containing one molecule
each of MBP-RepC and RepC, Asn Asp, or Tyr Ser proteins.
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Since the DNA binding experiments suggested that RepC binds strongly to
IRIII but only weakly to IRII (Fig. 6A), we wished to
determine whether RepC bound to IRIII promotes the binding of another
RepC molecule to IRII. For this, we made use of the Asn267
Asp DNA binding mutant of RepC (17). As shown in Fig. 7, the N267D
mutant bound poorly to the DNA and generated only a faint C1 complex
but no C2 complex. However, when MBP-RepC and the N267D mutant were
incubated together with the 53-bp origin region, the intermediate
complex characteristic of RepC oligomerization on the DNA was clearly
visible (Fig. 7). These results indicate that a second molecule of RepC
is targeted to the DNA primarily through a protein-protein interaction.
However, IRII may be important for stabilization of the oligomer on the
DNA, since no oligomerization occurs in the presence of DNA containing
only the IRIII sequence.
FPLC and Sucrose Gradient Sedimentation Analysis of the RepC and
RepC* Proteins--
We subjected RepC and RepC* to FPLC analysis to
determine their oligomeric state. FPLC of RepC on a Superose 12 column
(Fig. 8A) showed that it
eluted at a position corresponding to a protein slightly smaller than
bovine serum albumin (67 kDa). The peak fractions contained the RepC
protein as determined by SDS-PAGE (not shown). The protein peak also
corresponded to the DNA relaxation (Fig. 8B), in
vitro replication (Fig. 8C), and DNA binding (Fig. 8D) activities of RepC. FPLC analysis of RepC* followed by
SDS-PAGE of column fractions showed that it essentially eluted at the
same position as RepC (Fig. 9,
A and B), demonstrating that the attachment of an
oligonucleotide did not alter the oligomeric state of RepC in solution.
The DNA binding activity of RepC* was present in the protein peak
fractions (Fig. 9C). As expected, fractions containing RepC*
lacked detectable DNA relaxation and in vitro DNA
replication activities (data not shown). The calculated Stokes' radius
for RepC from FPLC was 36 Å. To further investigate the oligomeric state of RepC in solution, sucrose density gradient sedimentation analysis was carried out as described under "Experimental
Procedures." The RepC protein sedimented slightly faster than bovine
serum albumin and had a sedimentation coefficient of 4.8 S (data not shown). Based on the Stokes' radius and sedimentation coefficient, the
molecular weight of RepC in solution was calculated to be approximately
68,400. This is close to the predicted size of a RepC dimer (76,000).
These results suggested that RepC exists as a dimer in solution. To
further investigate the oligomeric nature of RepC in solution, we
generated initiator heterodimers by denaturing RepC and MBP-RepC fusion
proteins together in urea followed by renaturation. The RepC and
MBP-RepC proteins were also individually denatured in urea and
renatured as controls. The above protein preparations were used in EMSA
experiments. A 30-bp probe containing pT181 nt 37-60 that generates
only the C1 complex was used in this experiment. Both urea-treated and untreated RepC and MBP-RepC samples formed their respective C1 complexes (Fig. 10). This experiment
showed that urea treatment did not significantly affect the DNA binding
activity of RepC. If RepC was present as a dimer in solution, urea
treatment of a mixture of RepC and MBP-RepC proteins should generate
significant levels of a RepC/MBP-RepC heterodimer. Binding of such a
heterodimer to the DNA is expected to generate a novel band that should
migrate between the C1 DNA-protein complexes of RepC and MBP-RepC.
Significant levels of such a complex (RC-MBRC) were clearly detectable
in the presence of heterodimer preparations (Fig. 10). It should be noted that the protein preparation will contain both RepC and MBP-RepC
homodimers along with the RepC/MBP-RepC heterodimers. While significant
levels of DNA-protein complexes corresponding to binding by RepC
homodimer and RepC/MBP-RepC heterodimer were present, only low levels
of the MBRC-C1 complex corresponding to binding of the MBP-RepC
homodimer were observed (Fig. 10). This may be due to the fact that the
MBP-RepC protein does not bind as well to the DNA as the RepC homodimer
(and possibly the RepC/MBP-RepC heterodimer). This prediction is
consistent with data shown in Fig. 7. Faint bands corresponding to the
RC-MBRC complex were also visible in urea-treated MBP-RepC samples
(Fig. 10). This band was also observed with the untreated MBP-RepC
sample upon overexposure of the autoradiogram. This weak band may be
generated by the presence of trace amounts of free RepC in MBP-RepC
preparation due to cleavage at the fusion point followed by the
generation of RepC/MBP-RepC heterodimers. The low-level cleavage of MBP
epitope from MBP-RepC protein may increase slightly during the urea
denaturation-renaturation steps. In any event, the presence of such a
band is consistent with a dimeric state of RepC in solution.
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Fig. 8.
FPLC analysis of the RepC protein.
A, elution profile of the RepC protein on a Superose 12 FPLC
column. The positions of the standard protein markers are shown in
A. The DNA relaxation (B), in vitro
replication (C), and DNA binding (D) activities
of the column fractions are shown. Abbreviations are as shown in the
legends for Fig. 4A, 5, and 6A. RC indicates
wild-type RepC used as a control in these experiments.
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Fig. 9.
FPLC analysis of the RepC* protein.
A, elution profile of the RepC* protein on a Superose 12 FPLC column. The positions of the standard protein markers and the
excess oligonucleotide present in the RepC* preparation are shown.
B, SDS-PAGE analysis; C, EMSA of column
fractions. RC and RC* indicate RepC and RepC*
proteins used as positive controls in these assays. Other abbreviations
are as defined in the legend for Fig. 6B.
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Fig. 10.
DNA binding activity of urea-treated RepC
and MBP-RepC proteins. RepC and MBP-RepC proteins were denatured
either individually or together in urea followed by renaturation. EMSA
were then carried out with a probe containing pT181 nt 37-60 that
includes the IRIII region. RC-MBRC, DNA-protein complex
formed in the presence of the RepC/MBP-RepC heterodimer. Other
abbreviations are as defined in the legend for Fig. 7.
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DISCUSSION |
Previous studies have shown that in pT181-containing cells, RepC
and RepC* are present in an approximately 1:1 ratio regardless of the
plasmid copy number (27, 28). Using partially purified initiator
preparations from such cells, RepC* was found to be defective in its
ability to extrude cruciform at the pT181 origin and therefore
defective in its replication activity in vitro (25, 28).
RepC* was also found to inhibit the replication and nicking-closing activities of wild-type RepC (25, 28). Previous studies have also shown
that the RepC/RepC* heterodimer is present in great excess over RepC
in vivo but does not appear to have any regulatory role in
replication (28). Thus, some of the above in vitro results are not consistent with the in vivo results. Also, since
these protein preparations contained an equal amount of RepC and RepC*, it was not possible to unequivocally determine the various biochemical activities of the modified RepC* protein.
We have used a different approach to study the biochemical activities
of RepC* and its possible role in the regulation of pT181 replication.
We have generated RepC* in vitro by incubating purified RepC
protein with an excess of an SS oligonucleotide corresponding to the
right arm and a portion of the IRII loop (Fig. 1). This treatment
generated approximately 95% RepC*, with less than 5% of residual
unmodified RepC. RepC* showed very little or no DNA relaxation activity
(Fig. 3A). This was expected, since the active tyrosine
residue of RepC* is blocked due to the attachment of an
oligonucleotide. Similarly, RepC* lacked any in vitro
replication activity and inhibited the DNA replication activity of
wild-type RepC to only a very limited extent (Fig. 5). In
vivo studies have shown that the inactive initiator is present in
the form of the RepC/RepC* heterodimer (26). The limited replication
inhibitory activity of dimeric RepC* in our experiments may result from
binding of RepC* to the DNA, which may inhibit RepC binding and/or
oligomerization at the origin. Similarly, it is possible that RepC*
present in vivo may have a minor role in the regulation of
replication. These results are consistent with the in vivo
results, where very little inhibition of replication by the RepC*
protein is observed (28).
Electrophoretic mobility shift assays demonstrated that RepC forms two
complexes at the origin containing one (C1) or two (C2) molecules of
RepC, respectively. The C1 complex is likely to be due to the binding
of RepC to IRIII. This assumption is based on the observations that
much higher levels of the C1 complex are observed with the 30-bp IRIII
oligonucleotide than with the 26-bp IRII oligonucleotide (Fig.
6A). Interestingly, RepC* generates only the C1 complex with
both the 53-bp (IRII and IRIII) and 30-bp (IRIII) regions and does not
bind to the 26-bp IRII region (Figs. 6B and 7 and data not
shown). The C1 complex generated by RepC* migrates more slowly than the
C1 complex observed with RepC (Fig. 6B). This is consistent
with RepC* having a molecular weight of 42,000 as compared with 38,000 for RepC (26). When RepC/RepC* isolated from pT181-containing cells was
used in EMSA experiments, it generated a single DNA-protein complex
that comigrated with the complex obtained with wild-type RepC (28).
Therefore, it was not possible to distinguish between the binding of
RepC and RepC* to the DNA in those studies. We have also found that
while an intermediate complex is formed when RepC and MBP-RepC are
incubated together with the 53-bp origin region, such a complex is not
observed in the presence of the RepC* and MBP-RepC proteins (Fig. 7).
These results demonstrate that the oligonucleotide blocks
oligomerization of RepC* molecules at the origin and also interferes
with the assembly of an oligomer on the DNA involving one dimer each of RepC* and wild-type RepC (or MBP-RepC) that lacks the attached oligonucleotide. Furthermore, the Tyr191 Ser
topoisomerase mutant was able to dimerize on the DNA (Fig. 7). The
above results are consistent with the previous observation that RepC
binds to both the IRIII and IRII regions in supercoiled DNA, whereas
RepC/RepC* binds only to the IRIII region (29). The inability of
RepC/RepC* to bind to IRII and oligomerize on the DNA may result in a
lack of melting of the pT181 origin involving IRII, which is critical
for nicking by the initiator protein. How does the attached
oligonucleotide interfere with oligomerization of RepC? The location of
the RepC domain(s) involved in its dimerization or oligomerization on
the DNA is not known. It is possible that this domain is located near
the active tyrosine residue and that the oligonucleotide may inhibit
this interaction. Alternatively, it is possible that the attached
oligonucleotide interferes with the interaction of RepC* with the IRII
DNA and destabilizes the C2 complex. The Asn267 Asp DNA
binding mutant generated the C2 complex in the presence of MBP-RepC
(Fig. 7), suggesting that oligomerization of RepC on the DNA occurs
primarily through a protein-protein interaction. This prediction is
consistent with the observation that RepC binds efficiently to IRIII
but only weakly to IRII (Fig. 6A).
FPLC analysis of RepC on a Superose 12 column and sedimentation
analysis in a sucrose gradient showed that RepC has an approximate molecular weight of 68,400 (Fig. 8 and data not shown). RepC* showed a
pattern similar to that of RepC upon FPLC analysis (Fig. 9). The
calculated molecular weight of RepC based on SDS-PAGE and DNA sequence
analysis of the repC gene is 38,000 (15, 16). Our results
confirm the previous observations that RepC exists as a dimer in
solution (9, 27) and further indicate that the RepC* derivative is also
dimeric in nature. An experiment in which RepC was first denatured in 8 M urea and then diluted to a final concentration of 1 M urea, followed by FPLC in the presence of 1 M
urea, gave a pattern identical to that obtained with untreated RepC
(data not shown). We have also observed that RepC retains its
nicking-closing activity in the presence of 1 M urea in
reactions (not shown). The above results suggest that the two monomers
of RepC are tightly associated in a dimeric molecule and are resistant
to dissociation under mild denaturing conditions. We have also
generated initiator heterodimers in vitro by mixing RepC and
MBP-RepC, followed by denaturation in urea and renaturation by dialysis
in the absence of urea. The above protein preparations generated an
intermediate DNA-protein complex migrating between the C1 complexes
observed with RepC and MBP-RepC (Fig. 10). These studies further
support our conclusion that RepC binds to the origin as a dimer and
subsequently oligomerizes on the DNA.
Based on our results as well as those published by other laboratories,
we propose the following model for the assembly and role of RepC during
rolling-circle replication of pT181 (Fig. 11). A RepC dimer binds to the IRIII
region of the pT181 origin due to a strong sequence-specific
interaction. This promotes targeting of another RepC dimer to IRII
primarily through a protein-protein interaction that results in the
assembly of an initiator oligomer at the pT181 origin. The RepC
oligomer then bends the DNA and melts the origin that exposes the RepC
nick site in IRII in an SS form. One monomer of a RepC dimer nicks the
DNA at the origin and becomes covalently attached to the 5'-end of the
nick through its Tyr191 residue. Following this event, one
dimer of the oligomerized RepC may be released from the origin after
replication has initiated. After extension synthesis utilizing host
replication proteins such as DNA helicase, single-stranded DNA-binding
protein, and DNA polymerase III, the replication fork proceeds around
the circle to approximately 10 nucleotides beyond the RepC nick site
such that the hairpin structure surrounding the nick site is
regenerated. At this stage, interaction of the RepC protein with the
regenerated origin sequence presumably stalls the replication fork.
This step could involve a termination protein that may facilitate
pausing of the replication fork. The RepC protein then cleaves at the regenerated nick site, and a series of concerted cleavage/joining reactions occur that result in the release of a circular, SS leading strand DNA and a nicked open circular DNA containing the newly replicated leading strand. The nick is then sealed by the host DNA
ligase, and the DNA is subsequently converted to the supercoiled form
by DNA gyrase. The newly replicated SC DNA can then rejoin the plasmid
pool undergoing replication. The RepC protein is inactivated by the
attachment of an approximately 10-nt oligonucleotide located immediately downstream of the RepC nick site to the active tyrosine residue of one RepC monomer. The inactivated protein may then be
released as an inactive RepC/RepC* heterodimer. The leading strand DNA
released by displacement synthesis is converted to the double-stranded
form utilizing the single-strand origin. This step involves synthesis
of RNA primers by the host RNA polymerase followed by short extension
synthesis by DNA polymerase I and subsequent replication by DNA
polymerase III. The double-stranded DNA is then sealed by DNA ligase
and supercoiled by DNA gyrase.
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Fig. 11.
A model for the role of RepC protein during
the initiation and termination of rolling circle replication of plasmid
pT181. Details are described under "Discussion."
ORI, origin of replication; SSO, single strand
origin; SSB, SS DNA-binding protein.
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We thank Serban Iordanescu, Neal
DeLuca, and members of our laboratory for helpful discussions. We also
thank Christine Sikora for technical assistance.
Top
Abstract
Introduction
To whom correspondence should be addressed: Dept. Of Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine,
Room East 1240 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.:
412-648-9025; Fax: 412-624-1401; E-mail: Khan{at}med.pitt.edu.
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