From the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Received for publication, October 13, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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Stable maintenance of P1 plasmids in
Escherichia coli is mediated by a high affinity
nucleoprotein complex called the partition complex, which consists of
ParB and the E. coli integration host factor (IHF) bound
specifically to the P1 parS site. IHF strongly stimulates
ParB binding to parS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the
partition complex, we have investigated the DNA binding activity of
various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for
the formation of the minimal, high affinity partition complex. A
fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no
longer stimulated by IHF. The ability of IHF to stimulate ParB binding
to parS correlated with the ability of ParB to dimerize via
its C terminus. Using full and partial parS sites, we show
that two regions of ParB, one in the center and the other near the C
terminus of the protein, interact with distinct sequences within
parS. Based on these data, we have proposed a model of how
the ParB dimer binds parS to form the minimal partition complex.
The P1 prophage exists as a low copy number plasmid in
Escherichia coli, and its active partition system ensures
that P1 is stably maintained in a bacterial population. The P1
par operon encodes two trans-acting factors, ParA
and ParB, and a cis-acting centromere-like site,
parS. Analogous systems exist in a number of low copy number
plasmids, and the genes for homologues of both ParA and ParB have been
identified in the chromosomes of several bacterial species (1-7).
Both ParA and ParB are required for and have multiple functions in
partition. ParB binds specifically to the parS site (8, 9),
along with E. coli integration host factor
(IHF).1 Together, these two
proteins bend and wrap the DNA into a structure called the partition
complex (10-13). Bright, localized foci of ParB bound to parS in
vivo have been visualized by immunofluorescence (14). ParA is an
ATPase and binds the par operator region, repressing parA and parB expression (15). ParA also
interacts directly with ParB bound at parS in an
ATP-dependent manner (16). ParA is required for proper
localization of the ParB-parS complexes (14), although it is
not known how ParA promotes positioning of these complexes.
The plasmid centromere, parS, is a multipartite DNA site. An
IHF binding site is flanked by two types of distinct sequences, called
box A and box B, which are specifically recognized by ParB (11, 13)
(Fig. 1A). Previous mutational and deletion analyses have
shown that the presence and relative spacing of boxes A2, A3, B1, and
B2 are essential for wild-type parS function in
vivo, while boxes A1 and A4 are dispensable (11, 13, 17). ParB and
IHF enhance each other's affinity for parS (12, 17). IHF binding creates a large bend in parS (12), and an inherently bent DNA sequence can partially substitute for the IHF site in parS (18). One dimer of ParB binds across the IHF-directed
bend in parS (19), forming a structure in which the DNA is
wrapped around the protein core (10, 12, 19). Subsequent dimers load
onto the partition complex with increasing ParB concentrations via a
series of protein-protein interactions and specific and nonspecific
protein-DNA interactions that tether increasing amounts of ParB to the
partition complex.
Two independent self-association domains have been identified within
ParB (Fig. 2A) (20). One domain is located within the last
59 amino acids of ParB and is probably the domain that mediates dimerization in solution. The second domain is located near the N
terminus of the protein. Our observations suggested that this domain is
involved in oligomerization (i.e. dimer-dimer interactions) but that some type of conformational change in ParB is required for
oligomers to form (20). First, the N terminus of ParB is relatively
susceptible to proteolysis, suggesting that it is not very stably
folded in solution. Second, self-association of the N terminus,
measured by chemical cross-linking experiments in vitro, was
observed only when the C-terminal half of ParB was removed. One
possibility is that DNA binding elicits a conformational change that
removes the inhibitory effect of the C terminus on N-terminal
self-association, promoting oligomerization (20). The N-terminal
oligomerization domain either overlaps or is adjacent to a region of
ParB that is required for an interaction with ParA (Fig. 2A)
(20, 21).
Previously we began to define the structural domains of ParB by
identifying stable fragments generated by limited exposure to trypsin
(20). Tryptic digestion resulted in fragments with increasingly
C-terminal start sites (Fig. 2A, arrows) that
extended to, or close to, the C terminus of ParB. The two longest lived fragments started at residues 142 and 185, respectively (Fig. 2A, arrows C and D). These
results suggested that the C-terminal dimerization domain forms the
core of a very stably folded C-terminal half of ParB (20).
Two different regions of ParB appear to have a role in its specific DNA
binding activity, one near the C terminus of ParB and the second in the
middle of the protein (Fig. 2A). Point mutations near the C
terminus disrupt DNA binding activity (22). Proteins with these
mutations also fail to dimerize, and it has been suggested that lack of
dimerization prevents DNA binding. These mutations all fall within the
last 59 amino acids of ParB, a region that encompasses the C-terminal
dimerization domain (20). This region also includes the
"discriminator recognition sequence" (DRS), defined by
domain-swapping experiments (23). When residues 281-302 of P1 ParB
were swapped with the equivalent region of the ParB protein of P7, the
resulting hybrid protein recognized P7 parS in an in
vivo partition assay (23). The P7 parS site is very similar to P1 parS. The P1 and P7 box A sequences are
interchangeable, but the box B sequences are not (24). Thus, the
swapping experiment defined the DRS as a region that contacts box B
sequences directly or promotes a fold that allows box B binding.
Analysis of the ParB sequence identified a putative helix-turn-helix
(HTH) motif, from residue 166 to 189, that has also been implicated in
ParB's DNA binding activities (25). Point mutations located on either side of the putative HTH domain disrupt DNA binding activity (22). It
has been proposed that this region of the protein binds the box A
sequences in parS (23).
In this study, we have examined the interaction of ParB with its
specific sequences in parS by asking how different regions of the protein contribute to its DNA binding activities. Our results suggest a model of how protein-DNA (ParB-box A and ParB-box B) and
protein-protein (dimerization) interactions contribute to the
architecture of the partition complex.
Bacterial Strains and Media--
E. coli DH5
(F- endA1 hsdR17
(rK Reagents and Buffers--
Sources for reagents were as follows:
dithiobis(succinimidyl propionate) (DSP), bovine serum albumin, and
guanidine hydrochloride (Sigma); imidazole and Bradford reagent
(Bio-Rad). Enzymes for cloning were purchased from New England Biolabs
or Roche Molecular Biochemicals.
Plasmid Construction--
The new constructs created for this
work were generated by PCR and cloned into pET15b (Novagen), which
encodes a hexahistidine tag. pJS10 (20) was used as the substrate for
PCR amplification. To create the 142-333 ParB fragment, the region of
parB encoding this portion was amplified by PCR. The
upstream primer (5'-GCGCCATATGGACGTTCAGACAGCATTG) added a single Met
residue upstream of residue 142. M13 forward primer was the downstream
primer. This PCR fragment was gel-purified (QIAEX II), digested with
NdeI and BamHI, and ligated into pET15b, creating
pJS208. For 1-330, 1-325, and 1-317 ParB, the upstream primer
(5'-GCGCATATGTCAAAGAAAAACAGACCAAC) incorporated an NdeI site
at the start site. The downstream primers,
5'CGCGGGATCCTTACTTTTTATCGAGGCTC, 5'CGCGGGATCCTTACTTTCTAAGGATATGCCC, and
5'CGCGGGATCCTTACCTGTCGAGTTCTTCCTG, created stop codons in place of
residues 331, 326, and 318, respectively, and created a
BamHI site downstream of the stop codons. The
NdeI-BamHI fragments were then cloned into
pET15b, generating pJS207, pJS212, and pJS213.
To create the internal deletions of ParB, the region of parB
encoding residues 185-333 was amplified using an upstream primer (5'-CGCGCTCGAGTGCTCTCCAGGCAGCGAGTG) that placed a XhoI site
just upstream of codon 185 and the M13 forward primer. The resulting XhoI-BamHI fragment was cloned into pET15b to
create pJS206. The regions encoding residues 1-142 and residues 1-165
were amplified with an upstream primer,
5'-GCGCATATGTCAAAGAAAAACAGACCAAC (placing an NdeI site at
the start site), and a downstream primer, either 5'-CGCGCTCGAGTCTTTCGCTAAATTTTGCGC (creating an XhoI site
just downstream of codon 142) or 5'-CGCGCTCGAGCCATCATTTTTCATTCGCAT (creating an XhoI site just downstream of codon 165). The
NdeI-XhoI fragments were cloned into pJS206 to
generate pJS210, encoding DNA Substrates--
The plasmids pBEF165 and pBEF166 contain the
P1 parS sequence in opposite orientations in a modified
pBluescript vector (12). For gel mobility shift assays with the intact
parS site, pBEF165 was digested with either
BamHI, yielding a 252-bp parS fragment, or
XbaI, yielding a 132-bp parS fragment. The DNA
fragments were labeled at their 3'-ends with
[
Competitor fragments for the gel mobility shift assays were 270 bp in length and were generated by PCR from pALA207 (28). The
parS-containing fragment was amplified using the primers
5'-CACTTGTGTGAATCCCTTTTC and 5'-CAGGAAGAACTCGACAGGATG. The nonspecific
fragment was amplified with the primers 5'-GCCTCTTGCGGGATATCGTC and
5'-CTGTCGCAGCAGAGATGATG. The fragments were then purified from agarose gels.
The single site oligonucleotide substrates used for gel mobility shift
assays are illustrated in Fig. 1B. The lower strand of each
pair was labeled at the 5'-end with [ DSP Cross-linking--
Protein samples were diluted to between 8 and 20 µg/ml in 50 mM HEPES-KOH (pH 7.5), 150 nM NaCl, 0.1 mM EDTA. DSP (20 mg/ml in
dimethylformamide) was added to 0.1 mg/ml, and the mixtures were
incubated at room temperature. To stop the cross-linking and
precipitate the proteins, samples were mixed with an equal volume of
30% trichloroacetic acid, incubated on ice for 20 min, and centrifuged
at 4 °C. The pellets were washed with acetone and resuspended in 20 µl of 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol,
0.025% bromphenol blue. The samples were incubated at 90 °C for 3 min and were then analyzed by SDS-polyacrylamide gel electrophoresis.
Electrophoretic Mobility Shift Assays--
The standard reaction
mixture (10 µl) contained 0.1 nM 32P-labeled
parS substrate in 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 10%
glycerol, 100 µg/ml bovine serum albumin, 100 µg/ml sonicated
salmon sperm DNA, and 5 mM DNase I Protection Assays--
Standard 20-µl reaction
mixtures contained 10-50 fmol of 32P-labeled substrate, 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 10%
glycerol, 100 µg/ml sonicated salmon sperm DNA, 100 µg/ml of bovine
serum albumin, 10 mM MgCl2, 2 mM
CaCl2, and 7 mM In this work, we have examined the architecture of the partition
complex by determining the DNA binding properties of various regions of
ParB. ParB is 333 amino acids in length, and we previously examined its
domain structure by partial proteolysis mapping and by the
self-association activity of various ParB protein fragments (20). Here
we have used several of those fragments to examine their interaction
with the P1 parS site. All fragments contained a
polyhistidine tag to facilitate purification. In addition, we have
constructed six new versions of ParB (Fig. 2B). Our previous partial proteolysis experiments identified two tryptic fragments that
were relatively resistant to further digestion.
His-ParB-(142-333) (constructed here) and His-ParB-(187-333)
(constructed previously (20)) correspond to these two tryptic fragments
(Fig. 2A; Ref. 20). To examine the function of the extreme C
terminus of ParB, we removed 3, 8, and 16 residues from its C terminus,
generating His-ParB-(1-330), His-ParB-(1-325), and His-ParB-(1-317),
respectively. Finally, we made two internal deletions of ParB that
removed the HTH motif. The first removed only this predicted motif
(residues 166-184), and the second removed residues 143-184, the
region between the start sites of the two stable tryptic fragments
mentioned above (Fig. 2B).
We first tested the dimerization activity of the new protein fragments
in a cross-linking assay (Fig. 3). His-ParB-(142-333) and both
proteins with internal deletions (His-ParB-( The Region between Residues 142 and 325 Contains All Information
Required for Binding to the Full parS Site--
In the presence of
IHF, one dimer of ParB binds to parS in a very high affinity
protein-DNA complex (12, 19). This interaction requires that ParB
contact its specific recognition sequences on either side of the
IHF-directed bend simultaneously (13). In the absence of IHF, ParB
affinity for parS is much lower, and binding is primarily dependent on
the box A and box B sequences on the right side of the IHF site (Fig.
1). Therefore, we can distinguish two
types of ParB binding: the latter (low affinity), requiring specific
contacts but not IHF, and IHF-stimulated DNA binding (high affinity),
requiring both specific contacts and the IHF-directed bend in the DNA.
In this work, we address the question of what regions of ParB
contribute to the different aspects of ParB's DNA binding
activities.
We first examined several C-terminal fragments of ParB (i.e.
truncated from the N terminus) for their parS binding
activity. Full-length ParB (or His-ParB in our assays), and three
fragments, His-ParB-(47-333), His-ParB-(67-333), and
His-ParB-(142-333), bound to parS, and this binding was
stimulated by IHF (Fig. 4, A-C). The affinity of the
different ParB fragments for parS differed, but the relative
affinity of each ParB fragment for parS was much higher with
IHF than without IHF. Therefore, the first 141 residues of ParB are not
required for IHF-stimulated parS binding.
Next, we assessed the importance of the central portion of ParB in DNA
binding. His-ParB-(187-333) and His-ParB-(275-333) contain both the
C-terminal dimerization domains and the DRS but are missing the
putative HTH domain located near the center of ParB (Fig.
2). Neither fragment had any
parS binding activity, with or without IHF (Figs.
4D and 8 and data not shown). Therefore, a region N-terminal
to residue 187 is required for stable parS binding in this
assay. Since the first 141 residues were not required for binding (Fig.
4C), the region between amino acids 142 and 187, which
includes the putative HTH motif, contains information required for DNA
binding. We next examined the activity of ParB fragments lacking only
this region, His-ParB-(
Finally, we were interested in the role of the C terminus of ParB in
DNA binding. We deleted 3 and 8 residues from the C terminus of ParB
and tested the resulting fragments of ParB for their ability to bind
parS in the gel mobility shift assay. His-ParB-(1-330) and
His-ParB-(1-325) both exhibited IHF-stimulated DNA binding (Fig. 4,
G and H), and both were able to dimerize as
judged by cross-linking assays (Fig.
3).
DNase I footprinting experiments showed that His-ParB,
His-ParB-(47-333), His-ParB-(67-333), and His-ParB-(142-333)
exhibited very similar patterns of protection of the parS
sequence (Fig. 5A and data not shown). As predicted from the
gel mobility shift experiments, these results indicate that the first
141 amino acids of ParB are not required for minimal partition complex
formation. Neither His-ParB-(187-333) nor His-ParB-(275-333) provided
any protection of parS from DNase I cleavage, also
consistent with the results from the gel mobility shift assays (data
not shown). Similarly, His-ParB-(
Footprinting experiments with His-ParB-(1-330) produced a pattern of
protection similar to that of His-ParB but with additional enhancements
(Fig. 5, B and C). First, on the upper strand of parS, one band in box B1 was strongly enhanced. On the lower
strand, one band near the center and one near the right boundary of the IHF binding site were strongly enhanced. These differences indicate changes in the geometry of the complex that make the minor groove more
accessible to DNase I in these regions. These enhancements were not
observed in the presence of His-ParB-(1-325), whose protection pattern
was very similar to that of His-ParB.
C-terminal Dimerization Is Required for High Affinity parS
Binding--
In contrast to the fragments with small C-terminal
deletions, His-ParB-(1-317), His-ParB-(1-312), His-ParB-(1-293), and
His-ParB-(1-274) bound parS, but a high protein
concentration was required to observe binding (500-1000
nM), and little or no stimulatory effect by IHF was
observed (Fig. 4, I and
J, and data not shown). The IHF-insensitive binding is
relatively weak, as is full-length ParB binding to parS in
the absence of IHF (12). Therefore, removal of only 16 amino acids from
the C terminus removed the ability of IHF to stimulate ParB's specific
DNA binding activity. The appearance of IHF-insensitive binding
correlated with the loss of dimerization activity (20) (Figs. 2 and 3).
Note that the N-terminal fragments with IHF-insensitive binding
activity retain the putative HTH domain. His-ParB-(1-317) and
His-ParB-(1-312) also retain the DRS.
ParB fragments that exhibited IHF-insensitive DNA binding activity in a
gel mobility shift assay showed no detectable protection patterns in
parS in DNase I footprinting experiments (Fig.
5D and data not shown). This
was unexpected, because we had expected to observe binding to at least
the box A motifs, since these fragments retained the HTH motif. We
concluded that the parS binding activity of these
IHF-insensitive ParB fragments was too weak to be detectable in a DNase
I footprinting assay. However, His-ParB-(1-317) did show evidence of a
specific interaction with parS. The same enhancements observed in the presence of His-ParB-(1-330) were also observed with
His-ParB-(1-317) (Fig. 5D).
We tested whether the IHF-insensitive binding activity was still
specific for parS, using competition assays (Fig.
6). The ability of an N-terminal fragment
of ParB to bind to a labeled parS substrate was challenged
by a 270-bp unlabeled DNA fragment containing either the
parS sequence or a nonspecific sequence. With both
His-ParB-(1-317) and His-ParB-(1-312), the parS DNA was a
better competitor than the nonspecific DNA, indicating that binding was
specific (Fig. 6 and data not shown). All ParB fragments that bound
parS also contained some nonspecific DNA binding activity, a
property of the full-length protein (12).
N-terminal fragments smaller than His-ParB-(1-274) had no DNA binding
activity (data not shown). His-ParB-(1-234) is not cross-linked by
DSP, and His-ParB-(1-189) has some self-association activity through the N-terminal oligomerization domain (20). Neither fragment
interacted with parS. Therefore, a region C-terminal to
residue 234 is required for binding, and N-terminal self-association of
the protein is not sufficient to promote DNA binding.
Binding to Individual Box A or Box B Sequences--
While the
different DNA binding activities of the ParB fragments used in this
study supported the hypothesis that the putative HTH and the DRS
regions interact with box A and box B sequences, respectively, we asked
whether we could demonstrate direct interactions to isolated box A
and/or box B sequences. We designed synthetic oligomer (25-bp)
substrates that contained only the box A inverted repeat
(i.e. box A2-box A3), only a single box B sequence, or box
A2 + A3 combined with box B1 ("box A/B"). The last substrate essentially represents the minimal parS site, from box A2 to
B1, that is functional in vivo (17, 18, 29). As controls, we designed two nonspecific oligomer substrates. One contained the same
sequence as the box B oligomer, except that the box B sequence itself
was scrambled, and the second contained an unrelated 25-bp sequence
(Fig. 1B). Binding to these oligomeric substrates by His-ParB or His-ParB fragments was weak and often produced smeary complexes. Nonetheless, we could compare binding of the box A/B substrate to that of the single site substrates, which provided additional insight into ParB's DNA binding activities.
ParB binds to the minimal parS site weakly but specifically,
and this site can promote partition in vivo, although it is
less efficient than the full wild-type site in the presence of IHF (10-12). We expected, and found, that His-ParB binding to isolated box
A and box B sequences would be weaker than to the box A/B sequence
(Fig. 7A). Nevertheless, it
looked specific, since it was better than to either nonspecific
oligonucleotide.
We examined the binding of several ParB protein fragments to these
oligonucleotide substrates. His-ParB-(142-333) exhibited much weaker
binding to the single-site substrates than to the box A/B substrate
(Fig. 7B). This result, consistent with the DNase I
footprinting data (Fig. 5A), illustrates that
His-ParB-(142-333) recognizes box A and box B sequences but that both
are required for significant DNA binding activity. In contrast,
His-ParB-(
Both His-ParB-(1-330) and His-ParB-(1-317) bound to the box A/B
substrate better than to either box A or box B substrate alone (Fig. 7,
D and E). As shown also by gel mobility shift
assays and DNase I footprinting assays (Figs. 4G and
5C), the removal of 3 amino acids from the C terminus of
ParB did not affect its specific DNA binding activities, and
His-ParB-(1-330) must contain both the box A and box B recognition
domains of the protein. While His-ParB-(1-317) appeared to bind the
box A substrate better than the box B substrate, the DNase I
footprinting experiment shows an interaction of His-ParB-(1-317) with
box B (the enhancements in Fig. 5D). We suggest that removal
of 16 residues from the C terminus does not remove but it does
destabilize the domain of ParB that recognizes box B. Without proper
box B recognition, ParB binding is specific but weak, and it is
insensitive to IHF (Figs. 4I and 6).
The Putative Helix-Turn-Helix Domain Can Function as a
Monomer--
Our experiments indicate that a dimerized C terminus of
ParB is necessary but not sufficient for IHF-stimulated DNA binding activity; ParB must contain both the C terminus (including the DRS) and
the HTH for this activity. Since HTH domains often function as dimers
(30), we asked whether this region in ParB must be dimerized via the
C-terminal domain for ParB to bind parS. We formed
heterodimers between native ParB (no histidine tag) and His-ParB-(275-333), at a 10:1 molar ratio, by denaturing and then slowly renaturing the protein mixture. This small C-terminal fragment of ParB is able to dimerize but has no DNA binding activity (Figs. 2B and 8A). To
confirm that heterodimers were formed, a portion of the
ParB/His-ParB-(275-333) mixture was purified by nickel affinity
chromatography. The majority of the mixture was retained on the column
(data not shown). ParB dimers that bound the column must contain at
least one histidine tag and therefore must be present in the form of
heterodimers.
The renatured proteins were used in gel mobility shift assays (Fig. 8).
In the presence of IHF (Fig. 8A), the
ParB/His-ParB-(275-333) heterodimer mix formed a complex that migrated
between the parS + IHF (I) complex and the
parS + IHF + ParB (I + B1) complex
(Fig. 8A). This intermediate complex (I + (B/275)1), represents heterodimers binding to
parS. Therefore, a single putative HTH domain, in the presence of a dimerized C terminus is sufficient to allow
IHF-stimulated parS binding. Native ParB dimer complexes
were also produced from the mixture, even when the heterodimers had
been repurified over a nickel affinity column. This suggested that
homodimerization of native ParB occurred over the course of the
experiment. Finally, no intermediate complexes were observed in the
absence of IHF (Fig. 8B); only complexes of the size
expected from full-length ParB were seen.
Assembly of the P1 partition complex first requires the binding of
one dimer of ParB and one Fig. 9 illustrates how we think one dimer
of ParB interacts with and might be positioned on parS in
the presence of IHF. His-ParB-(142-333) interacts with both the box A
and box B sequences, and this interaction is stimulated by IHF.
Therefore, His-ParB-(142-333) contains all of the information required
for assembly of the minimal partition complex that contains one dimer
of ParB. The putative HTH motif of ParB is required for stable binding
to the intact parS site (Fig. 4, E and
F) and is necessary for an interaction with a box A inverted
repeat on an oligonucleotide substrate (Fig. 7C). In our
model, the HTH region of each monomer interacts with a box A sequence
in the A2-A3 inverted repeat, similar to a typical HTH protein such as
the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mK+) supE44
thi-1 recA1 gyrA96 relA1) was used for all plasmid
constructions. E. coli BL21 (F- ompT
hsdSB
(rB
mB+) dcm
gal) (
DE3, pLysS) (26) was used for fusion protein expression and purification. All bacterial cells were grown in LB medium or on LB
plates (27). Ampicillin and chloramphenicol, when used, were present at
100 and 25 µg/ml, respectively.
142-185 ParB, and pJS211, encoding
166-185 ParB. This cloning strategy replaced the region between
residues 143 and 185 or between residues 165 and 185 of ParB with two
serine residues.
-32P]dATP and DNA polymerase I large fragment and
purified with phenol/chloroform extraction and ethanol precipitation
(27). For DNase I footprinting reactions, DNA substrates were generated
by digesting either pBEF165 or pBEF166 with BamHI and
SmaI or BamHI and BglII, generating a
211- or 240-bp parS fragment, respectively. They were
32P-labeled at the 3'-end of the BamHI site. The
fragments labeled on the upper strand (as drawn in Fig. 1A)
were generated from pBEF165, and fragments labeled on the lower
strand were generated from pBEF166.
-32P]ATP and T4
polynucleotide kinase. The unincorporated nucleotide was removed by gel
filtration or by ethanol precipitation. The labeled oligomers were then
mixed with an equal amount of the appropriate upper strand oligomer in
100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA. The mixture was heated to 70 °C and was then
allowed to cool slowly to room temperature, to allow the two strands to anneal.
-mercaptoethanol. The
mixtures were assembled on ice, incubated at 30 °C for 20 min, and
analyzed by electrophoresis on 5% polyacrylamide gels in 90 mM Tris borate, 1 mM EDTA at 180 V for 3 h
in a water-cooled gel apparatus. The gels were dried on Whatman DE81
paper and exposed either to film or to a PhosphorImager screen
(Molecular Dynamics, Inc., Sunnyvale, CA). When the single site
oligomers were used as substrates, 1 nM labeled substrates
were used per assay, and the final salt concentration of each reaction
was ~140 nM NaCl.
-mercaptoethanol. The
mixtures were assembled on ice and incubated at 30 °C for 15 min.
Bovine pancreatic DNase I was then added (1 µl of a 1 µg/µl
solution). After 2 min at 30 °C, the reaction was stopped by the
addition of 80 µl of 1.6 M ammonium acetate, 400 µg/ml sonicated salmon sperm DNA, and 0.1 M EDTA. After
phenol/chloroform extractions of the mixtures, the DNA was precipitated
with ethanol and resuspended in 4 µl of formamide dye (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
166-184) and
His-ParB-(
143-184)) were cross-linked to dimer-sized smears in the
presence of DSP, a cross-linking reagent that reacts primarily with
lysines. His-ParB-(1-330) and His-ParB-(1-325) were also able to
dimerize, as determined by this assay (Fig. 3). His-ParB-(1-317), however, was not cross-linked, indicating that it is predominantly monomeric in solution. It behaves as His-ParB-(1-312) (Fig. 3), which
we previously reported to not be cross-linked by DSP (20). Therefore,
the region between residues 317 and 325 of ParB defines the C-terminal
boundary of its dimerization domain. The abilities of previously
isolated ParB fragments to dimerize are summarized in Fig.
2B (20).
View larger version (22K):
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Fig. 1.
The P1 parS site.
A, tall and short
boxes outline the box A and box B sequences, respectively,
and the black line shows the IHF binding site
(12). B, synthetic 25-base pair oligomers used as substrates
in gel mobility shift assays.
166-184) and His-ParB-(
143-184). Neither
protein bound parS with or without IHF (Fig. 4, E
and F), although some apparently nonspecific or unstable
binding activity was observed at the highest protein concentrations.
These results indicated that the central region of ParB is required for
parS binding and the C-terminal DRS is not sufficient for
stable parS binding.
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Fig. 2.
The structural and functional domains of
ParB. A, schematic diagram of ParB domains. The
black regions represent the regions of ParB
involved in protein-protein and protein-DNA interactions.
HTH, a predicted HTH motif; DRS, a second region
implicated in DNA binding (see the introduction). The
dimerization and oligomerization domains are at the C and N termini,
respectively. The N terminus also contains a region required for
interactions with ParA. The arrows indicate the N terminus
of fragments generated by tryptic cleavage of ParB. B,
diagram of ParB fragments used in this study. The black
regions of His-ParB are as in A. Dimerization
activity, as determined by DSP cross-linking is indicated in the
first column (this study and Ref. 20). The
second column summarizes the ability of the ParB
fragments to bind parS, and the third column indicates
whether binding was stimulated by IHF. N/A, not
applicable.
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Fig. 3.
Cross-linking of His-ParB and of ParB
fragments. His-tagged proteins were incubated with DSP at room
temperature for 20 min and analyzed by electrophoresis through a 12%
polyacrylamide gel. Lane M, size markers.
143-184) and
His-ParB-(
166-184) afforded no protection from DNase I cleavage
(data not shown).
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Fig. 4.
DNA binding activity of several ParB
fragments in the presence and absence of IHF, measured in gel mobility
shift assays. 32P-Labeled parS fragments
were incubated with the ParB proteins and/or IHF, and the mixtures were
analyzed by electrophoresis through a 5% polyacrylamide gel. The
parS fragments were generated from BamHI
(A, B, D, E, F,
G, H, and J) or XbaI
(C and I) digests of plasmid pBEF165, yielding
252- and 132-base pair substrates, respectively. The BamHI
digest also generated a 120- and a 3040-base pair vector fragment. The
XbaI digest created only one 3280-base pair vector fragment.
IHF, when present, was 400 nM. The positions of free DNA
(parS) and the IHF-parS (I) complexes
are indicated. The concentrations of His-ParB and of all ParB
truncations are shown as nM monomers. Note that the large
vector fragment contains an IHF binding site. A,
His-ParB and His-ParB-(47-333) in the presence or absence of IHF.
B, His-ParB-(67-333) in the presence or absence of IHF. The
same substrate was used in both panels, but the gel on the
right was run further, and the small nonspecific fragment
ran off the bottom of the gel. C, His-ParB-(142-333) in the
presence and absence of IHF. D, His-ParB-(187-333) in the
presence of IHF. E, His-ParB-( 166-184) in the presence
and absence of IHF. F, His-ParB-(
143-184) in the
presence and absence of IHF. G, His-ParB-(1-330) in the
presence and absence of IHF. H, His-ParB-(1-325) in the
presence and absence of IHF. I, His-ParB-(1-317) in the
presence and absence of IHF. J, His-ParB-(1-312) in the
presence and absence of IHF.
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Fig. 5.
DNase I footprinting of His-ParB and ParB
fragments at parS. The upper and
lower strands correspond to the upper and lower
strands shown in Fig. 1. The DNA substrates were 211-bp (A
and D) or 240-bp (B and C) DNA
fragments that were 32P-labeled at the 3'-end of either the
upper or lower strand. Protein-DNA complexes were treated briefly with
DNase I (see "Experimental Procedures") and analyzed on 6%
sequencing gels. IHF, when present, was at 400 nM. ParB
concentrations are reported for monomers. On each gel, Maxam-Gilbert
G > A sequencing reactions were included as markers. The
box A, box B and IHF sites are shown beside each gel.
A, protection of the upper strand of parS from
DNase I digestion by His-ParB-(142-333) or His-ParB. B,
protection of the upper strand of parS from DNase I by
His-ParB-(1-325), His-ParB-(1-330), or His-ParB. C,
protection of the lower strand of parS from DNase I by
His-ParB or His-ParB-(1-330). The arrows indicate
enhancements created by His-ParB-(1-330). D, protection of
the upper strand of parS from DNase I digestion by His-ParB,
His-ParB-(1-317), or His-ParB-(1-312). The arrow indicates
the enhancement generated by His-ParB-(1-317).
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Fig. 6.
Competition analysis of His-ParB-(1-317)
binding to parS. The 32P-labeled DNA
substrate was a BamHI restriction digest of pBEF165 and
was present at 10 fmol/lane. The fragment containing parS
was 252 bp (arrow). Increasing amounts of unlabeled 270-bp
DNA fragments containing parS or nonspecific sequence
(indicated above the lanes in fmol) were added to
the reaction mixtures prior to the addition of His-ParB-(1-317) (at 1 µM monomer). No IHF was added, and the mixtures were
analyzed by electrophoresis in a 5% polyacrylamide gel (see
"Experimental Procedures").
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Fig. 7.
DNA binding activity of ParB fragments to
oligomeric DNA substrates. The oligomeric DNA substrates are
described in Fig. 1B. They were prepared as described under
"Experimental Procedures," and the specific activities (cpm/pmol)
of all substrates were the same. Increasing amounts of His-ParB and
ParB fragments were incubated with each of the oligomeric substrates
and analyzed by electrophoresis in a 5% polyacrylamide gel. Protein
concentrations are shown as µM monomer. A,
His-ParB; B, His-ParB-(142-333); C,
His-ParB-( 166-184); D, His-ParB-(1-330); E,
His-ParB-(1-317).
166-184) showed weak but detectable binding to the box B
substrate, but not to the box A substrate (Fig. 7C).
Notably, binding to the box B substrate was about the same as to the
box A/B substrate, implying that only the box B sequence contributes to
its specific DNA binding activity.
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Fig. 8.
ParB/His-ParB-(275-333) heterodimers bind to
parS. A, DNA binding by His-ParB-(275-333), ParB
homodimers, and ParB/His-ParB-(275-333) heterodimers in the presence
of IHF. The proteins were incubated with a 132-bp parS
fragment, and the reaction mixtures were analyzed by electrophoresis
through a 5% polyacrylamide gel. The protein concentrations (as
nM dimers) are indicated above each
lane. The concentration of ParB/His-ParB-(275-333)
heterodimers assumes that all ParB monomers are paired with a
His-ParB-(275-333) fragment. When present, IHF was at 400 nM. To form ParB/His-ParB-(275-333) heterodimers, 25 µg
of ParB and 65 µg of His-ParB-(275-333) (10-fold molar excess) were
mixed, diluted in 100 µl of 6 M guanidine-HCl, 0.1 M NaH2PO4, 0.01 M Tris
(pH 8.0), and then dialyzed against 300 ml of the same buffer for
4 h at 4 °C to denature both proteins. The proteins were
renatured by successive dialysis steps against decreasing
concentrations of guanidine HCl followed by a final dialysis
against 50 mM sodium phosphate, pH 8.0, 300 mM
NaCl, 7 mM -mercaptoethanol, 10% glycerol. Both
His-ParB-(275-333) and ParB were denatured and renatured in separate
dialysis bags as controls. To repurify the ParB/His-ParB-(275-333)
mixture, half of the denatured/renatured mixture was purified over a
20-µl nickel-agarose affinity chromatography column (20). Bound
protein was eluted with 500 mM imidazole. The
arrows indicate the following complexes: IHF complex
(I); IHF plus ParB complex 1 (I + B1);
and IHF plus ParB/His-ParB-(275-333) complex 1 (I + (B/275)1). B, DNA binding by homo- and
heterodimers in the absence of IHF. B1, ParB complex
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
heterodimer of IHF to
parS, forming a very high affinity protein complex. Previous
biochemical experiments have shown that ParB simultaneously contacts
its recognition sequences on both sides of the IHF-induced bend in the
DNA (13). Here, we have used a series of ParB fragments to determine
the role of different domains in parS binding activity and
to position the different regions of ParB within the nucleoprotein
complex. Our results show that dimerization of ParB is essential for
the ability of IHF to stimulate ParB binding to parS.
Therefore, the ParB-ParB interaction across the IHF-directed bend is at
the dimerization interface. In addition, our data support the
prediction that the ParB HTH region recognizes the box A motifs and
that the DRS is part of the domain that recognizes the box B motifs.
While ParB recognizes two distinct sequences with two distinct regions
of the protein, both interactions are important to observe maximal parS binding activity.
repressor. Our results also indicate that the C terminus is
directly involved in box B binding and that the bend induced by IHF is
unable to stimulate ParB binding to parS in the absence of
the C-terminal dimerization domain of ParB (Fig. 4, I and
J). These observations support our previous prediction that
the dimerized C termini of a ParB dimer interact with both box B1 and
B2 simultaneously at or near the dimerization interface (19).
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Fig. 9.
Model of the minimal partition complex.
A, schematic diagram of the regions of ParB that are
involved in binding the box A and box B sequences of parS.
The black bar at the top represents
the minimal region sufficient for parS binding activity.
Below the schematic diagram is the predicted secondary
structure of ParB. The boxes indicate predicted loops, the
arrows show predicted -sheets, and the
cylinders denote predicted
-helices. Only structures
predicted with greater than 82% probability are shown. B,
model of a single ParB dimer in the partition complex. 142-333 ParB
contains all of the information required for DNA binding; therefore,
the N terminus is omitted from this diagram. The dimerized C termini
hold the two box B sites together, effectively wrapping the DNA around
the ParB dimer. The two HTH motifs bind the Box A2-A3 inverted repeat.
The N terminus of ParB (not shown) would be available for dimer-dimer
and ParA-ParB interactions.
Two observations suggest that dimerization and box B binding are at least partially separable functions. First, His-ParB-(1-317), a protein fragment that did not dimerize, was able to recognize the box B sequence, as evidenced by the enhancement observed in its DNase I footprint (Fig. 5C). Second, His-ParB-(1-317) binding to the oligomer substrates was enhanced by the presence of a box B sequence (Fig. 7E), supporting the idea that dimerization and box B binding are separable functions. However, we cannot exclude the possibility that a small amount of dimerization occurs in the presence of the DNA, particularly if this deletion has only partially destabilized the dimerization domain.
The Putative Helix-Turn-Helix Domain-- Based on sequence alignments, ParB has been predicted to contain a HTH domain between residues 166 and 187 (25). Secondary structure prediction of ParB by PHD (31, 32), shown schematically in Fig. 9A, indicates a high probability that a helix is formed between residues 169 and 174 and between residues 180 and 188 of ParB. Between these two helices is a gap that includes a glycine residue, a residue that is highly conserved in the turn region of a classical HTH motif (30). While our biochemistry does not provide any structural evidence for such a domain, we have shown that the region spanning this putative motif is required for native DNA binding activity.
Typically, HTH proteins, such as the repressor, bind their
recognition sites as dimers (30). The arrangement of a ParB dimer
interacting with boxes A2 and A3 resembles that of a typical HTH dimer
interacting with an inverted repeat binding site. The formation of an
IHF-stimulated complex by ParB/His-ParB-(275-333) heterodimers (Fig.
8) shows that the HTH region need not act as a dimer. However, binding
of ParB/His-ParB-(275-333) heterodimers to parS was
completely dependent on IHF. Therefore, in the absence of IHF, both HTH
domains must be present. These results suggest that the relative
importance of box A and box B sequences differs in complexes with and
without IHF. Without IHF, both box A2 and A3 must be filled to promote
binding, whereas with IHF, the box B sequences tethered at the
dimerization interface can compensate for the loss of one HTH-box A interaction.
ParB-like proteins share only limited homology (5, 33, 34), but one thing that many have in common is a predicted HTH motif (25). Similarly, the sequences of the specific sites that have been identified to date are quite different, but all of the known sites contain an inverted repeat (8, 35-37). It will therefore be interesting to see whether these other proteins bind their cognate site in a manner similar to that of P1 ParB.
Higher Order Partition Complexes--
The minimal partition
complex contains one dimer of ParB, but at higher protein
concentrations additional dimers join the complex to form even higher
order complexes (19). We have suggested that additional ParB dimers are
stabilized by interactions between the N-terminal self-association
domains in ParB as well as by protein-DNA interactions. DSP
cross-linking experiments showed that this domain was in the N-terminal
half of ParB, but yeast two-hybrid experiments narrowed this to within
the N-terminal 61 residues (20). Both His-ParB and His-ParB-(47-333)
formed higher order complexes in our experiments, while
His-ParB-(67-333) did not, consistent with our model. However,
His-ParB-(142-333) also appeared to form higher order complexes. We
cannot tell whether there are additional self-association contacts that
are more C-terminal to residue 142 of ParB or whether these higher
order complexes are promoted only by protein-DNA interactions. Another
possibility is that the N-terminal oligomerization domain is mainly
involved in dimer-dimer interactions that mediate pairing of partition complexes. The role of this domain awaits further structural analyses of ParB in these higher order complexes.
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ACKNOWLEDGEMENTS |
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We thank Alan Davidson and Lori Frappier for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a University of Toronto Open Fellowship (to J. A. S.) and Grant MOP-37997 from the Canadian Institutes of Health Research (to B. E. F.).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.
To whom correspondence should be addressed: Dept. of Molecular and
Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8,
Canada. Tel.: 416-978-1665; Fax: 416-978-6885; E-mail: b.funnell@utoronto.ca.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M009370200
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ABBREVIATIONS |
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The abbreviations used are: IHF, integration host factor; HTH, helix-turn-helix; DRS, discriminator recognition sequence; DSP, dithiobis(succinimidyl propionate); bp, base pair(s); PCR, polymerase chain reaction.
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