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Journal of Bacteriology, November 1998, p. 5954-5960, Vol. 180, No. 22
Department of Biology,
Received 21 May 1998/Accepted 4 September 1998
In order to form the catalytic nucleoprotein complex called the
invertasome in the Hin-mediated DNA inversion reaction,
interactions of the DNA-binding proteins Hin and Fis are required.
Assays for these protein-protein interactions have been exploited with
protein cross-linkers in vitro. In this study, an in vivo assay
system that probes protein-protein interactions was developed. The
formation of a DNA loop generated by protein interactions resulted in
transcriptional repression of an artificially designed operon, which in
turn increased the chance of survival of Escherichia coli
host cells in a streptomycin-containing medium. Using this system, we
were able to assay the Hin-Hin interaction that results in the pairing
of the two recombination sites and protein interactions that result in
the formation of the invertasome. This assay system also led us to find
that an individual Hin dimer bound on a recombination
site can form a stable complex with Fis bound on the recombinational
enhancer; this finding has never been observed in in vitro studies.
Possible pathways toward the formation of the invertasome are discussed
based on the assay results for a previously reported Hin mutant.
Hin invertase from Salmonella
typhimurium belongs to the recombinase family, which includes Gin
invertase from phage Mu, Cin invertase from phage P1, and resolvases
from Tn3 and the transposon The inversion reaction requires the interaction of DNA-binding proteins
at a distance. It has been generally accepted that after DNA binding, a
two-step pathway is used to assemble a functional synaptic complex
(Fig. 1) (15). The first step
is to bring distant hix sites in close proximity through the
interaction of hix-bound Hin proteins, forming a
"paired-hix" structure (12). Negative supercoiling assists the Hin-Hin interaction to promote the formation of the paired-hix structure (26). The next step
is to assemble the paired-hix structure with the Fis-bound
enhancer to make a nucleoprotein complex called an invertasome
(12). Negative supercoiling is essential for the formation
of the invertasome (12, 26). However, no experimental data
support the notion that the paired-hix structure is required
for the formation of the invertasome.
This nucleoprotein complex has been suggested to form at a branch point
in negatively supercoiled DNA (21). It is in the invertasome
complex that Hin is able to cleave the middle hix sites and
exchange the cleaved DNA ends to bring the intervening DNA to an
inverted configuration (17). A current hypothesis is that
Fis interacting with Hin in the invertasome triggers a conformational
change(s) in the dimer interface to initiate DNA cleavage by Hin
(11, 25). The region of Fis that is responsible for
triggering the change in Hin resides in the N terminus (29) and contains a flexible Protein interactions that result in hix pairing and the
formation of the invertasome have been assayed in vitro. By use of protein cross-linkers, the paired-hix structure and the
invertasome formed on a plasmid DNA were visualized by electron
microscopy (12). The same structures were also detected as
discrete bands by agarose gel electrophoresis (26). In this
study, we devised an in vivo assay system to study protein-protein
interactions on DNA at a distance. We were able to show not only that
the paired-hix structure and the invertasome are formed and
stably maintained in vivo but also that an individual Hin dimer bound
on hix can form a stable complex with Fis bound on the
enhancer; the latter finding has never been observed in in vitro
studies. With the in vivo assay system, the hix-pairing and
invertasome-forming activities of Hin mutants, each with a
single-amino-acid change in the dimer interface, were analyzed. The
consequences of the assay results are discussed in the context of the
protein-protein interactions necessary to assemble the invertasome.
Construction of plasmids.
Plasmids with an artificial operon
were constructed as follows. The rpsL gene isolated on a
0.75-kb HindIII-EcoRI fragment from pHSG664
(9) was cloned into pBluescript II SK+
(Stratagene) digested with HindIII and EcoRI,
creating pHL100. An EcoRI site was created in the middle of
the Pribnow box of the rpsL gene in pHL100 by changing the
adenine residue (base 78 upstream from the ATG start codon of the
rpsL gene) (31) to guanine by a site-directed
mutagenesis method (23), creating pHL101. The small
EcoRI fragment (155 bp, from pHL101) generated as the result
of site-directed mutagenesis was replaced with a 341-bp
EcoRI fragment containing the promoter ant and
the operator hixL-AT (Fig. 2) from pKH37 (K. T. Hughes,
University of Washington), creating pHL102. A BamHI fragment
from pHL102 containing the entire open reading frame of the
rpsL gene, the promoter, and the operator was filled in with
the large fragment of E. coli DNA polymerase I (Klenow
fragment; Bethesda Research Laboratories) and cloned into the filled-in
EcoRI site of low-copy-number plasmid pLG339 (35)
in the orientation opposite that of the tetracycline resistance gene in
pLG339. The plasmid generated was designated pSingle. pDouble1 was
constructed by cloning a synthetic 33-bp DNA containing the
hixL-WT (Fig. 2) and EagI sites on both ends into
the EagI site of pSingle. This cloning placed the
hixL-WT site about 1.1 kb away from hixL-AT and
toward ori of pSC101. pDouble2 was generated by replacing a
300-bp EcoRV DNA fragment of pSingle with the synthetic DNA
of hixL-WT containing blunt ends. pTriple was generated by replacing a 300-bp EcoRV fragment of pDouble2 with a
synthetic 74-bp DNA containing the recombinational enhancer sequence.
DNA sequence analysis showed that the 300-bp EcoRV fragment
removed from pDouble2 does not have any functional determinants that
are essential for the purpose of this study. This cloning placed the enhancer and hixL-WT sites 200 bp away from
hixL-AT. pDouble3 was generated by removing the
hixL-WT sequence from pTriple by EagI digestion
and ligation. To make pDouble3', a synthetic 32-bp DNA fragment
containing the proximal half site of the enhancer (5'-TCGGGTGTCAACAATTGACCAAAATATCGATT-3') was inserted
into the EcoRV site of pSingle. The nucleotide
sequences of the recombined promoter, operator, and rpsL
structural gene regions of these plasmids were confirmed by a
double-stranded DNA sequencing method (28).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vivo Assay of Protein-Protein Interactions in
Hin-Mediated DNA Inversion
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(8). Hin
promotes the inversion of a chromosomal DNA segment of 996 bp that is
flanked by the 26-bp DNA sequences of hixL and hixR (19). Hin-mediated DNA inversion in S. typhimurium leads to the alternative expression of the H1 and H2
flagellin genes known as phase variation. Hin (21 kDa) exists in
solution as a homodimer and binds to hix sites as a dimer
(7). In addition to Hin and the two hix sites, a
cis-acting DNA sequence (recombinational enhancer) and its
binding protein (Fis, 11 kDa) are required for efficient inversion in
vitro (18).
View larger version (11K):
[in a new window]
FIG. 1.
Schematic expression of the Hin-mediated inversion
reaction. Negative supercoiling of the substrate plasmid is required
for efficient inversion. Negative supercoiling is not included in these
drawings to show protein interactions more clearly. Hin binds to the
two hix sites and Fis binds to the enhancer in a substrate
plasmid. Hin pairs the hix sites, creating two DNA loops.
All the necessary proteins and their DNA-binding sites are brought in
close proximity to form the invertasome, creating three DNA loops of
different sizes. In the invertasome, Hin cleaves the middle of each
hix site and exchanges the cleaved DNA ends, followed by
ligation. The intervening DNA between the two hix sites is
now inverted.
-hairpin structure (32).
Recently, it was suggested that Hin needs to separate (melt) the two
DNA strands at the hix sites after DNA cleavage to perform
strand exchange (24). After the strand exchange, the DNA
ends can be religated by Hin.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Measuring SF.
Streptomycin-resistant E. coli
HB101 (supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5
mtl-1) harboring both the rpsL operon-containing
plasmid and the Hin-producing plasmid was grown at 37°C for 12 h
in 2 ml of Luria-Bertani (LB) medium supplemented with ampicillin (100 µg/ml), kanamycin (50 µg/ml), and
isopropyl--D-thiogalactopyranoside (IPTG) (20 µM).
Proper serial dilutions of the culture were made and plated on
two different agar plates. One contained kanamycin and ampicillin, and
the other contained streptomycin (100 µg/ml), kanamycin, and
ampicillin. Both plates contained a 20 µM concentration of IPTG. The
plates were incubated at 37°C for 24 h. Colony numbers were
counted, and the survival frequency (SF) was calculated. SF was defined
as the ratio of Strr Ampr Kanr
colonies per milliliter to Ampr Kanr colonies
per milliliter. Measuring the SF in a Fis-negative Strr
strain, CSH50fis::cat (34),
was performed as described above. CSH50fis::cat is a fis derivative
of CSH50 [ara 
(pro-lac) thi rpsL].
Recombination on pTriple. Even though we used two different hix operators to prevent any recombination, it is possible that a small amount of inversion or deletion between the hix operators in pTriple might have led to the streptomycin-resistant phenotype of HB101/pTriple/pHinWT. Thus, we investigated if any recombination occurred when pTriple (Kanr) coexisted with pHinWT (Ampr) in HB101. First, fresh HB101 was transformed with plasmids isolated from an overnight culture of HB101/pTriple/pHinWT, and transformants were plated on solid media containing kanamycin to select ones that had pTriple. Five hundred Kanr colonies were stabbed on two different solid media, one containing both ampicillin and kanamycin (to look for those transformed with both pTriple and pHinWT) and the other containing kanamycin and streptomycin. All the Kanr but Amps colonies (about 450) were Strs. Thus, this experiment showed that in vivo Hin could not perform either inversion or deletion on pTriple. If either inversion or deletion had occurred on pTriple in HB101/pTriple/pHinWT, fresh HB101 transformed with recombinant pTriple would have been Strr. The same experiment was performed with HB101/pDouble1/pHinWT and HB101/pDouble2/pHinWT, and all the Kanr and Amps colonies were Strs. Again, Hin was not able to carry out any type of recombination on pDouble1 and pDouble2.
Western blot analysis. The amount of Hin produced in HB101 harboring the Hin-producing plasmid with different concentrations of IPTG was measured by Western blot analysis. Cells grown to an optical density at 600 nm of 0.2 in 20 ml of LB medium with the proper antibiotics were pelleted and washed with 1 ml of 50 mM Tris-HCl (pH 7.5). Cells were resuspended in 50 µl of 1× sodium dodecyl sulfate (SDS) gel loading buffer and boiled for 3 min. Samples were centrifuged at 13,000 × g for 5 min, and 20 µl of supernatant was loaded on an SDS-12.5% polyacrylamide gel. Hin protein bands were visualized with horseradish peroxidase-conjugated secondary antibodies (Amersham).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Rationale for the in vivo assay of protein interactions occurring at a distance. The initial idea for our in vivo assay system to study protein interactions in the Hin-mediated inversion reaction was based on the following observations. First, for more efficient repression of a promoter, a prokaryotic gene regulation apparatus frequently uses more than one operator. Having more operators increases the local concentration of repressors through protein-protein interactions (reviewed in references 1 and 33). Such interactions often create a DNA loop. In addition, it has been shown that the DNA loop itself represses a promoter more efficiently than does the mere binding of repressors to operators. Second, Escherichia coli strains, such as HB101, having a mutant allele of rpsL (rpsL20, coding for ribosomal protein S12) in their chromosome are resistant to the antibiotic streptomycin. Since the rpsL20 allele is recessive, a merozygotic strain that harbors the wild-type rpsL gene on a plasmid becomes Strs (6, 30).
We constructed a series of plasmids that bear an artificial rpsL operon on a low-copy-number plasmid, pLG339 (Kanr) (35), derived from pSC101 (5). The artificial operon was built such that hix sites and the recombinational enhancer site served as operators (Fig. 2A). To prevent recombination (deletion or inversion) between the hix sites in the plasmids, we used two hix sites that are different only in the central two bases (hixL-WT for the second operator [O2] and hixL-AT for the first operator [O1]) (Fig. 2B). Inversion between these two hix sites is blocked because the central two bases become noncomplementary after strand exchange (13). We confirmed that, in vivo, Hin cannot perform either inversion or deletion on the substrate plasmids (see Materials and Methods). The original promoter for the rpsL structural gene was removed, and the ant promoter from phage P22 (2, 14) was inserted instead. The Hin protein that served as a repressor in this system was supplied from a high-copy-number plasmid, pHinWT (Ampr) (Fig. 2C), that was derived from pBluescript II SK+. These two plasmids were compatible in E. coli. Another trans-acting protein, Fis, was provided from the fis gene in the chromosome of the host HB101 (16, 22).
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Assay for streptomycin sensitivity.
First of all, the
streptomycin sensitivity of HB101 harboring the artificial
rpsL operon in a plasmid was tested when the hin
gene was not present. A single colony of HB101 having both pBluescript
II SK+ (Ampr) and one of the rpsL
operon-containing plasmids (Kanr) was grown in 2 ml of LB
medium containing ampicillin and kanamycin for 12 h. Cells were
plated on agar plates containing kanamycin and ampicillin and agar
plates containing those antibiotics plus streptomycin, and the SF in
the presence of streptomycin (ratio of Strr
Ampr Kanr colonies per milliliter to
Ampr Kanr colonies per milliliter) was
calculated. The SFs of HB101 cells having both rpsL
operon-containing plasmids and pBluescript were within the range
of 10
7 (Table 1), showing
that HB101, originally Strr, became Strs when
it harbored the wild-type rpsL gene in these plasmids. HB101 carrying the promoterless rpsL gene in a plasmid was
Strr (data not shown). These results indicated that
the inserted ant promoter successfully drives the
transcription of the rpsL structural gene when Hin, the
repressor, is not present.
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Assay for Hin binding to hix sites.
Our in vivo
assay for Hin binding to hix was performed with an
rpsL operon-containing plasmid, pSingle (Fig. 2A), that has a single hix operator (O1) between
the ant promoter and the wild-type rpsL
structural gene. We anticipated that the binding of Hin to hix would result in the repression of transcription
from the ant promoter, which in turn would increase the SF
of the host cells. Because the wild-type hin gene in the
plasmid pHinWT is under the control of the tac promoter, the
SF of HB101 harboring both pHinWT and pSingle (HB101/pHinWT/pSingle)
was investigated with increasing amounts of the gratuitous inducer
IPTG. As the concentration of IPTG increased, the SF of
HB101/pSingle improved up to 102 (Table 1). Western
blot analysis of HB101/pHinWT showed that as the IPTG concentration
increased from 0 to 40 µM, Hin production also increased accordingly
(Fig. 3). These results showed that the
more Hin is induced, the more the ant promoter is repressed; they also showed that the binding of Hin to the hix operator
can increase the SF of the host cells. These results indicated that the
experimental design was valid.
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Assay for hix-pairing activity of Hin. The next step in Hin-mediated inversion is to bring the two hix sites close together (hix pairing). This action was shown in vitro to be accomplished by the interaction between the Hin dimers bound to each hix site without the participation of Fis (12, 26). To assay the hix-pairing activity of Hin, an additional hix site was inserted in pSingle as a second operator (O2) 1.1 kb away from O1, generating pDouble1 (Fig. 2A). In the chromosome of S. typhimurium, the two hix sites are separated by a 993-bp DNA segment. We hypothesized that the ant promoter in pDouble1 would be more efficiently repressed than that in pSingle because of the DNA loop generated by the interaction between Hin dimers bound to hix operators. Thus, a higher SF was expected for HB101 harboring pDouble1. However, with the IPTG concentrations tested, the SFs of HB101/pHinWT/pDouble1 were the same as those of HB101/pHinWT/pSingle (Table 1), suggesting that the expected cooperative effect between the two operators did not occur. Two possibilities were considered. One is that the paired-hix structure in vivo is so transient (so is the DNA loop) that it cannot repress the promoter more than the mere binding of Hin to O1. The other possibility is that the paired-hix structure is stable in vivo but that somehow the DNA loop is not effective in transcriptional repression.
It has been demonstrated that the DNA loop per se in the galactose operon is important for more effective repression (3). Choy et al. (4) showed that as the size of the DNA loop generated by the interactions of repressor proteins bound to distant operators increased from 114 to 614 bp, the gal promoters were relieved from repression. Based on these observations, we contemplated that the size of the DNA loop formed in pDouble1 (1.1 kb) might have been too large. Thus, we reduced the distance between the hix operators from 1.1 kb (in pDouble1) to 200 bases and generated the plasmid pDouble2 (Fig. 2A). Indeed, the SF of HB101/pHinWT/pDouble2 with 20 µM IPTG was 10 times higher than that of HB101/pHinWT/pDouble1 or that of HB101/pHinWT/pSingle (Table 1). This result demonstrated that the paired-hix structure is formed and stably maintained in vivo. More importantly, this result suggested that hix-pairing activity can be measured in vivo and was consistent with the model that the inflexibility of the gal promoters enclosed by the DNA loop impedes transcription initiation by preventing RNA polymerase from unwinding DNA for open complex formation (4).Assay for invertasome formation. Formation of the invertasome requires two proteins, Hin and Fis, and three DNA-binding sites in the same plasmid, two hix sites and one enhancer site. The 65-bp enhancer is composed of two Fis-binding sites separated by 48 bp (12). Presumably, a Fis dimer binds to each binding site. After establishing that the DNA loop formed between cis-acting elements separated by at least 200 bp (as in pDouble2) provides more effective repression of the ant promoter, plasmid pTriple (Fig. 2A) was constructed to measure invertasome formation in vivo. pTriple has the enhancer sequence inserted in pDouble1 200 bp away from the hix operator O1. The relative locations of the three cis-acting DNA sites in pTriple were almost identical to those found in the H region of the Salmonella chromosome. Thus, if the invertasome is formed as shown by electron microscopy (12), we hypothesize that the smaller DNA loop, established between the enhancer and O1, would be the active component for transcriptional repression. The other DNA loop, generated by the enhancer and O2, would not participate in the repression, because the ant promoter is located in the smaller loop.
HB101/pHinWT/pTriple showed the highest SF (Table 1). With 20 µM IPTG, 57% of cells were able to survive in the presence of streptomycin, whereas for HB101/pHinWT/pDouble2, 22% of cells survived. The SF of HB101/pHinWT/pTriple with 20 µM IPTG was identical to that of HB101/pHinWT harboring only the vector pLG339 (Kanr and no rpsL operon), suggesting that the ant promoter in pTriple was so efficiently repressed that hardly any transcripts of the rpsL gene were made.Interaction between the Fis-enhancer complex and individual Hin-hix complexes. The results with pTriple raised the question of whether or not Fis bound on the enhancer can interact with Hin bound on the O1 complex without the presence of the Hin-O2 complex. This question was tested by removing O2 from pTriple. The resulting plasmid, pDouble3, has only two operators, the enhancer and O1 (Fig. 2A). If there is a Fis-Hin interaction, then SF of HB101/pHinWT/pDouble3 will be higher than that of HB101/pHinWT/pSingle. Indeed, with 20 µM IPTG, the SF of the pDouble3-containing strain was 10 times higher than that of the pSingle-containing strain, suggesting that the Fis-enhancer complex can interact with individual Hin-hix complexes without the formation of the paired-hix structure and that the resulting nucleoprotein complex can be as stable as the paired-hix structure in vivo.
Because there are two Fis-binding sites in the enhancer, we tested whether both sites are necessary to make the stable complex between Hin and Fis, as shown for pDouble3. One of the Fis-binding sites in the enhancer was removed from pDouble3, and the resulting plasmid (pDouble3') was assayed. The SF of HB101 harboring pDouble3' and pHinWT with 20 µM IPTG was almost the same as that of HB101/pHinWT/pDouble3, suggesting that a Fis dimer bound on the half enhancer could make a stable complex with a Hin dimer bound on the hix site. As a control experiment, pDouble3, pDouble3', and pTriple were assayed in a Fis-negative Strr strain (CSH50fis::cat). The SFs of CSH50fis::cat/pHinWT harboring each of these plasmids were the same as that for Hin binding on O1, as in HB101/pHinWT/pSingle. These results suggested not only that Fis specifically acts upon the enhancer sequence but also that it is Fis that interacts with Hin.Analysis of dimer interface mutants with the in vivo assay
system.
With the established in vivo assay system, Hin mutants
that have been characterized in vitro were analyzed. Their abilities for hix binding, hix pairing, and invertasome
formation were analyzed with pSingle, pDouble2, and pTriple,
respectively. The interactions between individual Hin-hix
and Fis-enhancer complexes were measured with pDouble3. Previously,
we substituted each amino acid residue from M101 to H107 with cysteine
(25). These residues were shown to exist in a long 
helix
that forms the dimer interface of Hin. Among the mutants, M101C
and H107C had 100 and 40% of the inversion activity of the wild
type, respectively. However, the DNA-binding activity of H107C
could not be measured (25). R103C and F104C were binding
competent but inversion incompetent (25). All the assays
were performed with 20 µM IPTG. The amounts of Hin proteins induced from these mutants by 20 µM IPTG were more or less the same
(data not shown). The results are summarized in Table
2.
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4, which is 2 orders of magnitude lower than that of
the wild type, suggesting that the DNA-binding activity of R103C was
imperfect. So was the hix-pairing activity of R103C. The SF
for invertasome formation by R103C was 0.07, significantly lower
than that of the wild type. Thus, the inversion-incompetent
phenotype of R103C was partly due to a defect in protein
interactions. However, R103C could make a stable contact with the
Fis-enhancer complex. Although the SF for the DNA-binding activity of
F104C was 10% of that of the wild type, invertasome formation by this
mutant was shown to be as efficient as that of the wild type. These
results suggested that F104C was defective in one of the steps after
invertasome formation.
The SF of H107C with pSingle (4 × 107) suggested
that it was completely defective in DNA binding, but the SF for
hix pairing (0.01) indicated that H107C was able to
make the paired-hix structure, albeit less
efficiently. Thus, the defect in DNA binding seemed to be corrected
by hix-pairing activity. H107C was shown to exist as a
homodimer in solution (25). Thus, it is likely that
interactions between H107C dimers during their brief stay on
hix stabilized DNA binding. The SF of H107C with
pDouble3 was 3 × 108, suggesting that the unstable
DNA binding of H107C dimers, however, could not be stabilized by
the Fis-enhancer complex. The SF of H107 with pTriple was 0.09. Taken
together, these results suggested that H107C was able to make the
invertasome with less stability (see Discussion); therefore, the lower
inversion activity of H107C (25) was a result of the
unstable invertasome structure. The difference between R103C and H107C,
both of which showed the same SFs with pTriple, seemed to be that the
invertasome formed by R103C was defective in inversion but that the
invertasome formed by H107C was able to make the inverted product.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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SF measures the relative stabilities of nucleoprotein
structures.
The increase in the SF from 107 to
102 for HB101/pSingle when Hin was induced with 20 µM
IPTG showed that the binding of Hin to O1 was
certainly effective in transcriptional repression of the ant
promoter. The assay results for the four dimer interface mutants with
pSingle were consistent with our previous results (25) and
showed subtle differences in the binding stability of the Hin mutants.
Thus, hix-binding activity can be quantitatively measured
with pSingle. The same idea of using hix as an operator has
been used to measure the hix-binding activity of Hin in an in vivo assay system called challenge phage (14). A
further 10-fold increase in the SF for HB101/pDouble2 indicated
synergy between the two hix operators as a result of a
Hin-Hin interaction. Based on the differences in SF between pDouble1
and pDouble2, which are different only in the distance between the
hix operators, we concluded that the size of the DNA loop is
important in transcriptional repression. Furthermore, the structural
difference between pDouble1 and pDouble2 eliminates the possibility
that the synergistic effect shown for pDouble2 was caused by Hin
binding to quasi-binding sites somewhere in pDouble2.
Transcriptional repression in pTriple. In reactions with substrate plasmids containing hixL-WT, hixL-AT, and the enhancer (as in pTriple), Hin made extensive knots instead of an inverted product (13). It was proposed that in the invertasome formed in these plasmids, multiple rounds of strand exchange, each of which occurred through a 180° clockwise rotation of one set of Hin subunits, resulted in the generation of extensive knots. One might argue that the most efficient repression in pTriple might have been accomplished by the DNA knots generated in the invertasome rather than the invertasome structure itself formed on pTriple. However, the assay results for F104C with pTriple argue against this possibility. Purified F104C protein showed no DNA cleavage activity (data not shown), suggesting that there could not be any strand exchange or formation of DNA knots in the invertasome formed by F104C. The SF of pHinF104C/pTriple was the same as that of pHinWT/pTriple, suggesting that the invertasome formed by F104C was as stable as that formed by the wild type. Therefore, these arguments support the notion that the stable DNA loop formed between O3 and O1 as a result of invertasome formation is solely responsible for the repression in pTriple. It is likely that the formation of an O3-O1 DNA loop of about 200 bp is aided by the HU protein, as shown in vitro (10). These data also suggest, contrary to the proposal by Kanaar et al. (20), that Fis should stay in the invertasome after DNA cleavage and even during the multiple strand exchange in pTriple to maintain repression.
Protein interactions at a distance for assembly of the
invertasome.
It was surprising that individual Hin-hix
complexes could make a stable complex with the Fis-enhancer complex,
because a protein-protein interaction between Hin and Fis has never
been observed. This result raised the possibility that the invertasome
can be formed by assembly of each Hin-hix complex onto the
Fis-enhancer complex, bypassing the formation of the
paired-hix structure. However, the assay results for H107C
argue against this possibility. The SF of H107C with pDouble3 is 3 × 108, suggesting that a DNA loop between
O3 and O1 cannot be
formed. (This result does not necessarily imply that H107C has lost its ability to enter into a stable interaction with Fis but rather that the
interaction is transient due to the unstable hix-binding activity of H107C.) However, considering that the transcriptional repression on pTriple is achieved by the small DNA loop formed between
O3 and O1, the
O3-O1 DNA loop must have been
assembled in the invertasome formed by H107C in order to acquire the SF of 0.09 for pTriple. Otherwise, the SF of pHinH107C/pTriple would have
been that for binding, which is 107 or less. Thus, the
O3-O1 DNA loop, which cannot be made
on pDouble3 in H107C, can be made passively during the process of
invertasome formation. These results strongly suggest that the
invertasome can be assembled without a preformed
O3-O1 DNA loop.
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ACKNOWLEDGMENTS |
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We thank H. E. Choy and C. Park for thorough review of the manuscript.
This research was supported by KOSEF grant 961-0502-015-2 and by grant 97-4431 from the Basic Science Research Program, Ministry of Education, Korea, awarded to H. M. Lim.
S.Y.L. and H.J.L. contributed equally to this research.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, College of Natural Sciences, Chungnam National University, Taejon 305-764, Korea. Phone: 82-42-821-6276. Fax: 82-42-822-9690. E-mail: hmlim{at}hanbat.chungnam.ac.kr.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Bacteriology, November 1998, p. 5954-5960, Vol. 180, No. 22
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