ABSTRACT
Fis protein participates in the normal control of chromosomal replication in
Escherichia coli
. However, the mechanism by which it executes its effect is largely unknown. We
demonstrate an inhibitory influence of purified Fis protein on replication from
oriC in vitro
. Fis inhibits DNA synthesis equally well in replication systems either
dependent upon or independent of RNA polymerase, even when the latter is
stimulated by the presence of HU or IHF. The extent of inhibition by Fis is
modulated by the concentrations of DnaA protein and RNA polymerase; the more
limiting the amounts of these, the more severe the inhibition by Fis. Thus, the
level of inhibition seems to depend on the ease with which the open complex can
be formed. Fis-mediated inhibition of DNA replication does not depend on a functional
primary Fis binding site between DnaA boxes R2 and R3 in
oriC
, as mutations that cause reduced binding of Fis to this site do not affect the
degree of inhibition. The data presented suggest that Fis prevents formation of
an initiation-proficient structure at
oriC
by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the
regulation of replication initiation.
Initiation of chromosomal replication is a crucial regulatory event in the cell
cycle and must be precisely timed in response to varying external conditions.
In
Escherichia coli
, replication is initiated at the unique origin, termed
oriC.
Within
oriC
,
DnaA protein binds to its binding sites (DnaA boxes), and then promotes
separation of the DNA strands in the AT-rich region (Fig.
1
;
1
,
2
). This duplex melting step is termed open complex formation. DNA structure and
architectural proteins are important at this stage; the opening depends on
negative supercoiling in the origin region, and the presence of the histone-like proteins HU or IHF (
3
-
6
). IHF binds specifically to one site in
oriC
(Fig.
1
;
7
), and presumably facilitates open complex formation by building a proper
nucleoprotein structure. HU protein also facilitates bending of
oriC
, though binding without sequence specificity, and makes an equally efficient
initiator structure as IHF
in vitro
(
5
).
The Fis protein shares certain properties with HU and IHF in being a small,
abundant DNA-bending protein involved in formation of higher order nucleoprotein
complexes. It was first identified as a
Fis binds to
oriC
between DnaA boxes R2 and R3 with high affinity (
16
-
18
). DNase I footprinting experiments indicate also an additional high affinity
Fis binding site to the right of DnaA box R4, as well as sites with less
affinity in the left and central region of
oriC
(Fig
1
;
17
). Footprinting data indicate that binding of Fis between R2 and R3, and binding
of DnaA to R2 and R3 are mutually exclusive (
16
).
Several lines of evidence suggest a role for Fis in DNA replication
in vivo
: (i)
oriC
-dependent plasmids cannot transform
fis
mutant strains efficiently (
16
-
18
); (ii)
fis
null mutants form filamentous cells, show aberrant nucleoid segregation, and
have inhibited DNA synthesis at high temperatures (
17
); (iii) cells carrying a deletion of DnaA box R4 need Fis protein to be viable
(
19
); and (iv) the synchrony of initiation appears to be dramatically reduced in
fis
mutants (
20
; U. von Freiesleben and K. V. Rasmussen, personal communication). Furthermore,
in vivo
footprinting studies indicate that Fis remains bound to
oriC
through most of the cell cycle, but is released at the time of initiation of
replication (
21
).
In contrast, no positive effect of Fis protein on replication
in vitro
has been observed. Hiasa and Marians (
22
) found that Fis is unable to stimulate replication from
oriC in vitro
. Instead, high concentrations of Fis inhibited replication in the absence of HU
and IHF protein. However, this inhibition was relieved when either of these two
proteins was present (
22
).
We describe here further studies on the effect of Fis protein on replication
in vitro
using two reconstituted enzyme systems for replication of supercoiled
oriC
plasmids: one that requires transcriptional activation by RNA polymerase (RNAP)
and one that does not. Inhibition by Fis was found to occur both in the
presence and absence of IHF or HU, with the extent of inhibition governed by
the level of DnaA protein and the amount of transcriptional activation.
Ribonucleoside triphosphates, deoxyribonucleoside triphosphates, poly(dI-dC)[middot]poly(dI-dC) and Sephadex G-50 Nick columns were from Pharmacia; [[gamma]-
32
P]ATP (>5000 Ci/mmol) and [[alpha]-
32
P]dTTP (800 Ci/mmol) from Amersham; cellulose phosphate P11 from Whatman; polyvinyl alcohol (MW 30-70.000) from Sigma.
Plasmid pBSoriC (3640 bp), also called pTB101 (
4
) contains a 678 bp
Hin
cII-
Pst
I fragment spanning
oriC
(-189 to +489 bp) cloned into the pBluescript vector. pBSoriC-fis1 is a pBSoriC derivative containing six base substitutions in Fis site
I in
oriC
[Fis ->
Bam
HI; (
23
)]. Plasmid pOC170 (3853 bp) contains the
oriC
region -176 to +1497 (
18
). pOC170-fis2 (pOC170
oriC
131), contains six base substitutions (different from the fis1 mutation) in Fis
site I (
18
).
The plasmids were purified by two successive equilibrium centrifugations in CsCl-ethidium bromide density gradients as described (
24
), followed by desalting over Sephadex G-50 Nick columns.
Replication proteins gyrase B subunit, SSB, primase, [beta]-subunit of DNA polymerase III holoenzyme and HU [purified as
described in (
25
,
26
)] were a gift from A. Kornberg. DnaA protein, DNA polymerase III* and gyrase A
subunit were purified as described by Sekimizu
et al
. (
27
), Maki
et al
. (
28
) and Kruklitis and Nakai (
29
), respectively. DnaB-DnaC in equimolar complex
(N. P. J. Stamford,
et al.
, manuscript in preparation) was provided by N.Dixon. IHF protein was a gift
from H. E. Nash.
Escherichia coli
RNA polymerase was bought from Pharmacia.
Fis protein was overproduced in the
E.coli
strain JM83 harboring a T7 expression system: plasmid pGP1-2 (
30
) expressing T7 RNAP under control of the temperature sensitive [lambda]P
L
promoter, and plasmid pCF351 (
31
) having expression of the
fis
gene regulated by the T7 RNAP-inducible promoter [Phi]10. Strong overproduction of Fis protein is possible in this
transformed strain. An overnight culture grown at 28oC in Terrific Broth [17 mM KH
2
PO
4
, 72 mM K
2
HPO
4
, 2.4% (w/v) Bacto-yeast extract and 0.4% (v/v) glycerol] containing ampicillin (40 [mu]g/ml) and kanamycin (20 [mu]g/ml) was diluted 1:1000 into fresh medium (6 l), and grown to an
optical density (OD
600
) of 1.4-1.5. Expression of Fis protein was then induced by shifting the culture
to 42oC. After 40 min, the cells were harvested by centrifugation (4200
g
; 0oC), resuspended in 50 mM Tris-HCl/10% sucrose and frozen in liquid nitrogen. Thawed cells were
resuspended in buffer A (25 mM HEPES-KOH pH 7.6, 0.1 mM EDTA, 2 mM DTT, 15% glycerol), and sonicated before
addition of lysozyme, as this increased the yield of Fis protein. The lysis was
performed according to the procedure by Dixon and Kornberg (
3
), except NaCl (1 M) was included in the lysis reaction. The lysate was
clarified by centrifugation (25 400
g
; 0oC), and the supernatant was diluted with buffer A to a conductivity equal
to that of buffer A containing 200 mM NaCl. The diluted lysate (445 ml) was
applied to a cellulose phosphate column (bed volume 150 ml) equilibrated in
buffer A containing 200 mM NaCl. Bound proteins were eluted with a linear
gradient of NaCl (800 ml, 200 mM-1 M, in buffer A). Fractions were tested for
oriC
binding activity in an electrophoretic mobility shift assay (see below); active
fractions eluted at ~750 mM NaCl. These were pooled (Fr II), diluted in buffer A to a
conductivity equal to that of buffer A containing 200 mM NaCl, and applied to a
heparin-agarose column (bed volume 120 ml) equilibrated in buffer A containing
200 mM NaCl. Bound proteins were eluted with a linear gradient of NaCl in
buffer A (1.2 l, 200 mM-1 M). Fis protein (Fr III; 77 ml; 1.22 mg/ml) eluted as
a single peak (550 mM NaCl) of high purity (>99% as judged by silver stained
SDS-PAGE). Contaminating DNase activity was not detectable in the preparation
of purified Fis protein.
The purified Fis protein was sensitive to dilution; a >10-fold dilution into the same buffer (buffer A containing 550 mM NaCl)
reduced the specific activity for binding to
oriC
, possibly due to precipitation of the protein. Inclusion of polyvinyl alcohol
at 5% (w/v) in the dilution buffer reduced the loss of binding activity, and
was therefore included in all dilutions.
The RNAP-independent reaction (25 [mu]l) contained 30 mM Tricine-KOH (pH 8.2); 12 mM magnesium acetate; 2 mM ATP; 0.04%
polyvinyl alcohol; 200 ng supercoiled DNA template (pBSoriC unless otherwise
stated; equal to 600 pmol nucleotides, or 84 fmol molecules); 125 ng DnaB-DnaC in equimolar complex; 180 ng gyrase A subunit; 180 ng gyrase B
subunit; 450 ng SSB; 23 ng primase; 112 ng DNA polymerase III* (Pol III); 26 ng [beta] subunit of Pol III; 8 ng HU and 32 ng DnaA unless otherwise stated; dATP,
dTTP, dCTP and dGTP each at 0.1 mM with [[alpha]-
32
P]dTTP at 30-200 c.p.m./pmol of deoxynucleotides.
The RNAP-dependent reaction (25 [mu]l) additionally contained UTP, GTP and CTP each at 0.5 mM, 100 ng HU,
and RNAP at the indicated amounts. DnaA protein was added at the indicated
amounts. In this replication system, RNaseH is sometimes added to inhibit DnaA-independent initiations originating outside
oriC
. However, the amount of DnaA-independent replication in our assay was <10% of the total DNA synthesis. RNaseH was therefore omitted.
Mixtures were assembled at 0oC and incubated at 29oC for 20 min, and then precipitated with 500 [mu]l cold 10% trichloroacetic acid. Total nucleotide incorporation
was measured by liquid scintillation counting after filtration onto GF/C glass-fiber filters.
During purification of Fis we used a 270 bp
Sau
96-
Cla
I
restriction fragment (R-ori; Fig.
1
) containing DnaA boxes R2, R3 and R4, and part of the
mioC
open reading frame (
32
). The fragments were dephosphorylated with calf intestinal alkaline phosphatase
and 5'-end-labeled with [[gamma]-
32
P]ATP using T4 polynucleotide kinase. For testing the affinity of Fis for the
mutated pBSoriC-fis1
oriC
, we used 350 bp PCR-amplified DNA fragments containing the whole
oriC
region (coordinates -20 to +330; T-ori, Fig.
1
). The fragments were purified with Spin-bind columns (MedProbe) before 5'-end-labeling with [[gamma]-
32
P]ATP. The mobility shift reaction mix (20 [mu]l) contained 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 75 mM KCl, 2 mM DTT, 10% glycerol, 2 [mu]g poly(dI-dC)[middot]poly(dI-dC), 1.5 fmol labeled DNA fragments and the
indicated amounts of corresponding, unlabeled DNA. Protein was added to the
mixture at the indicated amounts. Reactions were incubated at 30oC for 20 min and subjected to electrophoresis through 5% polyacrylamide
gels. The gels were dried and subjected to autoradiography. The relative
intensities of the bands were quantified by scanning densitometry (Molecular
Dynamics Computing Densitometer, Model 300A).
Replication of
oriC
plasmids
in vitro
can be reconstituted with purified replication proteins, among which DnaA is
the key initiator protein. As long as the plasmid is sufficiently negatively
supercoiled, DnaA is able to separate the two strands of the double helix.
However, this DNA opening does not occur readily with DnaA as the sole actor.
IHF and HU proteins both facilitate bending of
oriC
, and their presence strongly stimulates the opening reaction (
3
,
5
,
6
). We first examined the effect of purified Fis protein in a replication
reaction independent of RNA polymerase [also known as the solo primase system (
33
,
34
)] where separation of the strands was facilitated by the presence of either HU
(8 ng; Fig.
2
A) or IHF (8 ng; Fig.
2
B). Fis protein significantly inhibited replication, and the effect was equally
strong whether HU or IHF was used. Replication reactions containing both HU and
IHF (8 ng of each) were inhibited to the same extent by Fis as the reactions
containing either HU or IHF (data not shown). Omitting HU or IHF reduced the
activity by >95%. Fis protein had the same inhibitory effect in such
`unstimulated' reactions (data not shown). Low concentrations of Fis were not
able to stimulate the existing feeble activity in systems lacking HU or IHF
(see Discussion).
The negative effect of Fis suggests that it does not contribute to the formation
of an initiation-proficient nucleoprotein structure. Rather, it prevents its formation,
possibly by forming an alternative structure at
oriC
. If so, such an inhibitory structure might predominate when positively acting
initiation factors are at low levels. To investigate this, we tested the effect
of Fis at limiting amounts of (i) DnaA protein and (ii) RNA polymerase. First,
we varied the amount of DnaA protein in the RNAP-dependent reaction in the absence or presence of a moderate level of Fis
(72 ng; 35 dimers per oriC-plasmid) (Fig.
4
A). A reaction containing 16 ng DnaA (~four molecules of DnaA per plasmid) was inhibited >90% by 72 ng Fis
(considering a basal level of ~30 pmol nucleotides of dNTPs incorporated; Fig.
4
A). With an increase of DnaA to its optimal concentration (64 ng), the
inhibition by Fis decreased to ~30%. Excessive levels of DnaA partially inhibited replication even without
Fis, and alleviated the Fis-mediated inhibition. This lessening of inhibition by Fis at high DnaA
concentrations was clearly demonstrated in a similar experiment in the RNAP-independent reaction (containing 8 ng HU; Fig.
4
B). Here, even the complete inhibition at optimal DnaA concentrations by a high
level of Fis (190 ng) could be partly overcome with excessive amounts (200 ng)
of DnaA (Fig.
4
B).
Plasmid pBSoriC-fis1 is a derivative of an
oriC
plasmid and contains base substitutions in the primary Fis binding site between
DnaA boxes R2 and R3 (Fis site I; Fig.
1
) (
23
). The ability of this
oriC
sequence to bind Fis protein was examined using an electrophoretic mobility
shift assay with Fis protein and DNA fragments containing mutated or wild-type
oriC
(Fig.
6
A). Quantification of
the autoradiogram by scanning densitometry demonstrated a considerably reduced
binding efficiency of Fis to the mutated
oriC
(Fig.
6
B). The complex containing mutated
oriC
migrated more slowly than the complex with wild-type
oriC
, indicating possible structural differences.
We do not know whether this is due to altered protein-DNA contact at site I, or to Fis binding at another site.
We have shown that Fis protein inhibits replication from
oriC in vitro
. The level of inhibition depended on conditions that affect open complex
formation, indicating that Fis inhibits the strand separation reaction. An
interesting feature of the inhibition by Fis was the independence of a
functional primary Fis binding site I between DnaA box R2 and R3. Fis was able
to form inhibitory complexes with
oriC
in spite of alterations to this site and thus reduced binding to the origin. In electrophoretic mobility shift assays with
oriC
fragments containing the fis1 mutation at Fis site I, an amount of Fis equal to six dimers per DNA fragment gave a strong primary
shift and a faint secondary shift. The higher order shifts are probably a
result of Fis binding to several sites on the fragment. Increasing the
concentration of Fis to 35 dimers per
oriC
, a ratio that significantly inhibited the replication reactions,
resulted in a pattern with multiple shifted bands (not shown). Thus, at the
ratios of Fis to
oriC
used in the replication reactions (7-90 dimers per
oriC
plasmid), Fis probably binds at multiple sites, both within the normal and
mutated origins, as well as outside
oriC
. Also, Fis protein is shown to be capable of inducing conformational changes
even in DNA apparently lacking Fis binding sites (
38
). It is thus possible that several Fis dimers contribute to the formation of an
initiation-deficient protein-
oriC
structure, and that the primary Fis binding site is dispensable for building
this complex. This may explain why Fis inhibited replication of templates
having mutated Fis I sites in a manner indistinguishable from that of wild-type plasmids.
Hiasa and Marians (
22
) reported that high Fis concentrations inhibit
in vitro
replication in the absence of HU or IHF, but that stimulatory amounts of HU or
IHF can overcome this inhibitory effect. This contrasts with our findings that
Fis is an efficient inhibitor of initiation in HU- and IHF-stimulated replication assays. However, we found that the inhibition
by Fis was dependent on the levels of RNA polymerase and DnaA protein, being
more pronounced the more limiting these factors were. The inhibition was
alleviated when the levels of DnaA or RNA polymerase exceeded the
concentrations optimal for replication. The level of DnaA used by Hiasa and
Marians corresponds to >100 molecules per
oriC
-plasmid, whereas the optimal concentration in our assay is much lower, ~15 DnaA molecules per
oriC
-plasmid. This may explain the discrepancy. However, as our replication
systems differ somewhat in total composition, it is difficult to draw
conclusions based solely upon a comparison of this single parameter.
In the above mentioned work, it was found that, in contrast to HU and IHF, low
amounts of Fis do not stimulate replication
in vitro
(
22
). We also investigated this issue. No stimulation was detectable; the only
effect of Fis was to reduce the amount of replication. It has been suggested (
18
) that some DnaA protein preparations are contaminated by Fis protein. The lack
of stimulation by adding Fis to
in vitro
replication reactions could thus be due to residual Fis being present in the
replication mixture, already exerting a positive effect. However, immunoblot
analysis with a detection limit of 0.1 ng Fis failed to detect Fis in a sample
containing 30 times a normal complement of replication proteins (not shown).
Hence, the purified replication proteins were, for all practical purposes, free
from Fis contamination.
In vivo
, Fis is needed for efficient transformation of
oriC
plasmids, as lack of Fis or Fis binding site I on the plasmid lead to feeble
transformation (
16
-
18
). Also, introduction of a DnaA box R4 deletion into a
fis
mutant renders the mutant cells inviable (
19
). These seemingly positive effects of Fis on replication
in vivo
, contrasting its demonstrated negative effect on replication
in vitro
, raise interesting questions about this protein's role in control of
replication initiation. Evidently, Fis has a negative effect; whether it also
has a directly positive effect remains unclear.
Cassler
et al
. (
21
) have shown that a Fis-
oriC
complex exists throughout the cell cycle, but is replaced by an IHF-
oriC
complex as cells initiate replication. We suggest that Fis protein contributes
to the formation of a structure at
oriC
that is incapable of promoting strand opening. In order for initiation of
replication to occur, the preinitiation structure containing Fis protein must
give way to a replicatively active initiation complex. Our data indicate that,
in vitro
, this may be achieved simply by providing more replicatively active DnaA
protein or more transcriptional activity near
oriC
.
An important aspect of Fis being part of an inactive complex at
oriC
, is that with varying growth conditions, the cell experiences large
fluctuations in Fis concentration (
39
). Inasmuch as a large increase in the level of Fis protein would permit the
inactive complex to persist longer, and thus cause a delay in initiation, Fis
protein may also be involved in adjusting the initiation frequency in response
to changes in growth rate.
We thank our collaborators Hiroshi Nakai and Nicholas Dixon, with whom we
maintain and share our supplies of
E.coli
replication proteins. We are grateful to Arthur Kornberg and Howard E. Nash for
the kind gifts of pure proteins, to Walter Messer for plasmids pOC170 and
pOC170
oriC
131, and to Erik Boye for helpful discussions throughout this work. The
technical assistance of Anne Wahl is gratefully acknowledged. This work was
supported by grants from the Research Council of Norway (to K.S. and S.W.), and
from the National Institutes of Health (GM 49700) and National Science
Foundation (MCB 9408830) (to E.C.)
REFERENCES
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