From the Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, October 18, 2000, and in revised form, November 27, 2000
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ABSTRACT |
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We have examined a replication terminus ( Escherichia coli, Bacillus subtilis, and possibly other
bacteria respond to adverse metabolic conditions, such as amino acid starvation by synthesizing and accumulating the alarmone, guanosine tetraphosphate (ppGpp),1 the
so called magic spot 1. The alarmone shuts down the synthesis of
several RNAs including rRNA. It is an adaptive response to metabolic
stress and it promotes energy conservation by turning off some of the
synthetic processes (1). In E. coli, ppGpp synthesis is
mediated by two genes called relA and spoT that
encode ppGpp synthetase I and a gene product that catalyzes both the synthesis and degradation of ppGpp, respectively (2). In B. subtilis, a relA homolog has been found but no homolog
for spoT has been discovered so far (see Ref. 1).
Apparently, inhibition of protein synthesis by amino acid
analogs, such as arginine hydroxamate causes intracellular accumulation of uncharged tRNA, that in turn activates the relA gene.
Synthesis of ppGpp is thus induced and the alarmone binds to the The effect of stringent response on DNA replication has been
investigated in bacteria and in bacteriophage A recent article reported the existence of a short sequence of 17 bp
that was located at or near the left checkpoint and had some homology
with the core sequence of the Ter sites of B. subtilis. The sequence bound to a single dimer of RTP and was
reported to have arrested replication in a polar fashion under
stringent but not under relaxed conditions (8). This result was
surprising because two interacting dimers of RTP, bound to the core and
auxiliary site are known to be essential for replication termination
(9-11) and in fact polarity is generated from symmetrical dimers of
RTP (12) by differential interaction with the core and auxiliary sites
of Ter (13).
In view of these apparently puzzling differences between the behavior
of a normal Ter and the one present at or near the left checkpoint of B. subtilis, we decided to reinvestigate the
characteristics of the site in vivo and in vitro.
We identified a 28-bp long terminator site that we call Bacterial Strains and Plasmid Constructs--
The E. coli strains JM109 (sup E44, rel A1,
recA, endA1,
gyrA96,hsdR17D, RNA Mapping--
Total RNA was prepared from wild type B. subtilis IS 58 RelA and IS 56 rel A Gel Mobility Shift Analysis--
The assays were carried out as
described (10). Briefly, PCR products of Ter 1, core, L1 and
L1 core-binding sites were amplified from pUC18BS3 (Ter1
RTP-binding site cloned as EcoRI-HindIII
fragment), pUC18core (Ter1 core-binding site cloned as
EcoRI-HindIII fragment), pBAG 62 (LI RTP-binding
site cloned as EcoRI-BamHI fragment in pWS64-1
vector (20) and pBAG60 (L1 core RTP-binding site cloned as
EcoRI-BamHI fragment in pWS 64-1) plasmids,
respectively, using universal M13 forward and reverse primers. These
fragments were end-labeled with [ Dissociation Rate Analysis--
Dissociation rate analysis was
performed using 10 fmol of end-labeled Ter1 and L1 PCR
products and 100 pmol of respective oligo pairs having either
Ter1- or L1-binding sites. L1 and Ter1 were incubated at room temperature for 15 min with 400 fmol and 40 pmol
of wild-type RTP, respectively, in a 20-µl reaction volume containing
40 mM Tris-Cl (pH 7.5), 4 mM MgCl2,
50 µg/ml bovine serum albumin, 50 mM potassium glutamate.
10,000-fold excess of unlabeled annealed homologous oligonucleotide was
added for increasing time intervals from 0 to 3 h. The reaction
was stopped with 4 µl of 6 × DNA loading dye and samples
resolved on 8% native polyacrylamide gels.
Helicase Assays--
Helicase assays were performed as described
(14). Briefly, 10 pmol of complementary oligonucleotides having
Ter1, L1, or L1 core-binding sites were 5'-end-labeled using
[ Preparation of Replication Intermediates--
Ter1
and L1 sequences were cloned as EcoRI-BamHI
fragments in both orientations in the shuttle vector pWS 64-1 (20) and transformed into RTP overproducer B. subtilis strain SU230.
These clones were grown in 50 ml of SM minimal medium (0.2% ammonium sulfate, 1.4% potassium phosphate dibasic, 0.6% potassium phosphate monobasic, 0.1% sodium citrate, 0.02% magnesium sulfate, 0.02% casein hydrolysate, 0.6% glucose, 0.05% tryptophan) or in rich LB
medium having 20 µg/ml erythromycin at 30 °C 240 rpm to 0.5 OD590 and RTP was induced by adding 0.4 µM
isopropyl-1-thio- One-dimensional Gel Analysis--
The
HindIII-digested samples were resolved on a 25-cm 1%
agarose gel and used for Southern transfer to nitrocellulose filters. The blots were baked under vacuum at 80 °C for 2 h and
prehybridized for 4 h at 42 °C in 50% formamide, 6 × SSC, 0.4% fat-free milk. PWS 64-1 shuttle vector DNA was radiolabeled
with random priming using the NEBlot kit (New England Biolabs) and used
as a probe for hybridizations at 42 °C overnight. The blots
were washed in 1-0.2 × SSC, 0.1% SDS, and exposed to x-ray film.
Two-dimensional Gel Analysis--
The HindIII- and
PstI-digested samples were used for two-dimensional analysis
and was performed according to published methods (15). The
PstI- and HindIII-digested replication
intermediate samples were subjected to first dimension electrophoresis
on 25-cm long 0.6% agarose gel without ethidium bromide at 30 V for
16 h and stained the gel in 1 mg/ml ethidium bromide. The sample lanes were excised and run in 1.5% agarose, 1 µg/ml ethidium bromide for the second dimension at 150 volts for 8 h. The resolved
samples were transferred to a nitrocellulose sheet and probed with
labeled vector (pWS 64-1) DNA, washed, and exposed to an x-ray film.
Identification of the
We were guided by a wealth of prior information on RTP-Ter
interaction that had established that a single dimer of RTP bound to
DNA was not competent to arrest replication forks and that two
interacting dimers bound to the appropriate sequence were essential for
replication fork arrest (11, 17). Our expectation was that barring
surprises, the approach mentioned above might identify a terminus
sequence in this region of B. subtilis. By examining
sequences in the gnt-sacXY region we found a
28-nucleotide long sequence that yielded the expected double shift when
bound to RTP as described below. The sequences of Interaction of RTP with L1 Sequence--
Ter1 (complete
RTP-binding site), core (TerI site with only the core
sequence), L1 (complete, 28-bp long RTP-binding site), and L1 core
(17-bp long putative L1 core site) were used for gel shift analysis. It
should be noted that the DNA sequences were first cloned into a plasmid
vector and then the DNA fragments (~200 bp long) containing the
specific sequences and flanking vector sequences were amplified by PCR
and end labeled. Increasing concentrations of RTP were added to these
DNA fragments and analyzed on 8% polyacrylamide gels.
Ter1 shows filling of first and the second site
represented by the two gel shifts and conversion of all of the free DNA
into the second shifted band required 40-fold excess of RTP (Fig.
2A). Core sequence showed only one shift because it bound
only one dimer of RTP (Fig. 2B). L1 DNA also showed a single
shift when lower amounts of RTP were used. But at higher levels
(160-4000 fmol) of RTP, a second shift indicative of binding of two
dimers was visible (Fig. 2C). There were major differences between the gel shift pattern of Ter1 and L1 sequences in
that L1 was not only a very weak binder but showed minimal
cooperativity. In fact we could not elicit complete binding and
shifting of a majority of the free L1 DNA to the position of the second
shift under the experimental condition that included some carrier DNA. The binding of L1 to RTP was quantitated and is shown in Fig. 2E.
L1 core DNA showed a single gel shift over the entire range of RTP
concentrations, indicating that only a single dimer of RTP bound to the
DNA (Fig. 2D). A comparison of the binding profiles of L1
with L1 core supports the conclusion that the complete L1 sequence
bound to two dimers RTP. The binding was rather weak and showed little
or no cooperativity in comparison with the Ter1 sequence.
Measurements of the Stability of RTP-DNA Complexes--
We
followed up the binding experiments described above with competition
experiments with an excess of unlabeled double stranded, homologous
oligonucleotides. First, Ter1 and L1 were fully complexed with an excess of RTP and then challenged with a 10,000-fold molar excess of the unlabeled DNA and aliquots were run on nondenaturing gels
to resolve and measure the released free DNA from the DNA-RTP complex.
Typical autoradiograms of the competition experiments for the
Ter1 and L1 DNAs are shown in Fig.
3, A and B,
respectively. The quantitative analysis was performed from data
averaged from three sets of gels and are shown in Fig. 3C.
It is clear from the data that whereas the Ter1-RTP complex
dissociated to a half-maximal value in over 180 min, the L1-RTP complex
dissociated fully in a few seconds after the addition of the competing
unlabeled DNA. In the absence of the competing DNA, both complexes were
stable past 180 min (Fig. 3, A and B, lane C2).
The yxcC Gene Is under Stringent Control--
The L1 sequence is
located within the yxcC gene that is transcribed in a
direction opposite to that of replication fork progression. We wished
to determine whether this gene was under the stringent control for two
reasons. First, because the L1 site is located within the reading frame
of the gene, we were interested in investigating if ppGpp-mediated
modulation of transcription might have some effect on replication
termination at this site and second, we wished to use the
transcriptional status of this gene upon addition of arginine
hydroxamate as a reporter that the cells were truly under stringent conditions.
Stringent response deficient relA In Vivo Analysis of Replication Termination by L1
Sequence--
The shuttle vector pWS 64-1 was used for cloning L1 and
Ter1 sequences is a chimera of pGEM3Zf(+) (Promega)
and the unidirectionally replicating B. subtilis plasmid
pAM
As expected, Ter1 yielded a replication intermediate (a band
with slower mobility than the linear DNA band) that were indicative of
an arrested fork. The arrested fork was seen in the blocking but not in
the nonblocking orientation, even though by design we had loaded
4-5-fold more of the nonblocking orientation DNA in the gels (Fig.
6A, compare the first
two lanes; arrow). In contrast, L1 was able to block replication
fork in both relaxed and stringent conditions in both orientations,
although the level of blockage was less in comparison to that of
Ter1 (lanes 3-6 of Fig. 1A, arrow).
Thus, L1 appeared to be bipolar and did not really have a blocking or
nonblocking orientation.
We wanted to make sure that the bands whose position is marked by an
arrow in Fig. 6A were authentic replication
intermediates rather than artifacts (of perhaps incomplete cleavage or
due to residual protein-bound DNA), by examining them in Brewer-Fangman two-dimensional gels (15). If the band represented authentic Y-shaped
stalled forks, they would form a spot on the replication arc and at the
correct expected position in the two-dimensional gels. The blocking
orientation of Ter1 (predigested with PstI) but
not the nonblocking orientation, had an abundance of stalled intermediates (Fig. 6B, arrow). No arrested forks
were seen in the nonblocking orientation. The arrest of the fork is so
strong that a replication arc is not visible at this level of exposure and the monomer spot and the arrested intermediate predominate at this
level of exposure of the autoradiogram. Thus the "bubble arc"
leading upto the Ter1 and the Y and double Y (Xs) arc
generated by the few forks that move past the terminus are not visible
in the autoradiogram. We have previously reported the expected pattern of an unidirectional ori in two-dimensional gels when the forks are
arrested at a Ter site (14). The present pattern is
consistent with that result.
The L1 DNA, in the so-called blocking orientation (forks approaching
the core site), after PstI digestion generated a replication arc that was visible leading up to and just beyond the spot
corresponding to a stalled replication fork (see arrow in
Fig. 6D). The generated correspond to the pattern expected
from a bubble arc (14). However, the parts of the arc that would
be generated by the few forks that go past the L1 site forming single
and double Y-shaped molecules are not visible even in this moderately
long exposure. Similar analysis was performed in the so-called
nonblocking (forks approaching the auxiliary site) orientation of L1
with the same results (not shown).
We cut the DNA samples at both HindIII sites (Fig. 5) to
examine if the termination spot still survived the digestion. If the
spot were an artifact produced by partial digestion of circular plasmid
DNA by a single cutter, it would not fall on the replication arc and
would not be visible after cutting of the DNA at two sites. HindIII digested DNA. L1(blocking orientation) from cells
kept under both relaxed and stringent conditions yielded the
termination spot (Fig. 6C, arrow). It should be kept in mind
that L1 has no nonblocking orientation. The "blocking" and
"nonblocking" simply mean whether the core or the auxiliary site
first encounters the approaching forks. Once again it should be noted
that the monmer spot and the stalled intermediate predominated and the
bubble arc was not visible at this level of exposure. The arc can be seen in very long exposure of the autoradiogram that obliterates the
termination spot due to overexposure (not shown). It should be noted
that the pattern of replication intermediates shown here agrees with
those reported previously for that generated from an unidirectional
replicon bearing a Ter site, when the replication intermediates are cut within the bubble near the Ter site
(10, 14). We have also examined the replication intermediates from the
same plasmid containing the L1 core sequence by one-dimensional gels
and found no evidence of replication arrest, consistent with earlier
published results (Ref. 8 and data not shown).
In Vitro Analysis Helicase Arrest by L1-RTP Complex--
The
rationale was to authenticate the bipolar arrest of L1 forks seen
in vivo by an in vitro approach. We expected that
L1 should arrest DnaB helicase in both orientations whereas,
Ter1, used as a control should show polar arrest of helicase
under identical conditions. The results confirmed these expectations.
The autoradiograms of typical helicase assays showed that while the
Ter1 partial duplex substrate arrested DnaB in an
orientation-dependent fashion, L1 arrested the helicase in
an orientation-independent fashion (Fig.
7, A and B). The
quantitative analysis of the data from three separate assays showed
that both the core site of L1 and the nonblocking orientation of
Ter1, as expected, elicited very low levels of helicase
arrest at high RTP concentrations. Both orientations of L1 were more
efficient in helicase arrest than both the preceding controls but
clearly less efficient than the blocking orientation of Ter1
(Fig. 7C). Thus the in vitro helicase arrest data
appear to be consistent with the in vivo replication arrest
data.
The work reported here makes a mechanistically significant point,
i.e. despite very weak binding of RTP to a variant
Ter site (L1) and despite the fact that the L1-RTP complex
was unstable and dissociated almost completely in a few seconds after
being challenged with an excess of competitor DNA, the complex was
still able to arrest replication forks in vivo and DnaB
helicase in vitro.
The bacterial chromosome probably does not exist as naked DNA but is
coated with proteins (18), some of which are strong DNA binders
(e.g. lac repressor), but the replication forks apparently are able to navigate through the bound proteins without detectable arrest until reaching the TerC region where they are
terminated. This consideration would suggest that any protein bound to
DNA, much less a weak DNA-protein complex such as the L1-RTP complex, by itself, would not be expected to be able to arrest replication forks
unless the complex had some special attributes such as an ability to
interact with the helicase and abrogate its activity. The observation
reported here, along with published evidence (11), taken together,
would suggest that in addition to RTP-Ter (L1) interaction,
protein-protein interaction between RTP and the helicase is probably
involved in fork arrest.
It is surprising that L1 was able to arrest forks from both directions
unlike the unipolar arrest that characterizes most Ter sites
with the exception of a bipolar Ter in a plasmid of B. subtilis (19). However, the plasmid-based bipolar Ter,
unlike the L1 site, has a symmetrical sequence and it makes symmetrical base to amino acid contacts with
RTP.2 At this time, we do not
have a reasonable molecular explanation for the observed bipolar nature
of L1 with respect to replication fork arrest. One would speculate that
the L1-RTP complex creates a conformation that promotes helicase-RTP
contacts with the helicase coming from either direction and results in
abrogation of DNA unwinding.
It might be worthwhile to discuss as to what might have caused another
laboratory to conclude that the L1 sequence bound a single dimer of
RTP. Perhaps the explanation for the difference is that only the 17-bp
core sequence of L1 was used by them for RTP binding experiments and
the binding behavior of the complete core and auxiliary sequence was
not examined (8). It is, however, more difficult to explain as to why
stringent response-dependent conditional arrest of
replication was observed by the other group in a genomic DNA fragment
that included L1 (8). We saw no evidence that replication arrest at L1
was modulated by stringent response.
Is L1 involved at all in stringent control dependent fork arrest? It is
possible that it might be one component of the conditional fork
arresting system and that other RTP-binding sites, that cooperate with
L1 to create an efficient conditional barrier to fork movement, may
exist downstream of this site in the chromosome. It should be kept in
mind that L1 by itself was not as strong an arrester of replication
forks as the Ter1 terminus. In the future, genomic knock out of the L1 site, more precise mapping of the site of arrest of
the forks in cells under stringent conditions, and more novel
approaches to actually isolate the stalled fork from the bacterial
chromosome are likely to be needed to solve the interesting mechanism.
L1)
located on the left arm of the chromosome of Bacillus
subtilis and within the yxcC gene and at or near the
left replication checkpoint that is activated under stringent
conditions. The
L1 sequence appears to bind to two dimers of the
replication terminator protein (RTP) rather weakly and seems to possess
overlapping core and auxiliary sites that have some sequence
similarities with normal Ter sites. Surprisingly, the
asymmetrical, isolated
L1 site arrested replication forks in
vivo in both orientations and independent of stringent control.
In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1
terminus. The key points of mechanistic interest that emerge from the
present work are: (i) strong binding of a Ter (
L1)
sequence to RTP did not appear to be essential for fork arrest and (ii)
polarity of fork arrest could not be correlated in this case with just
symmetrical protein-DNA interaction at the core and auxiliary sites of
L1. On the basis of the result it would appear that the weak
RTP-L1Ter interaction cannot by itself account for fork
arrest, thus suggesting a role for DnaB-RTP interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of RNA polymerase, causing turning off of transcription from
certain promoters (3).
. In B. subtilis, induction of stringent response by addition of arginine
hydroxamate causes replication forks initiated from oriC to
be arrested at check points, that we have labeled as
L and
R
(Fig. 1), that are located ~200 kilobases on either sides of
oriC (4, 5). Apparently, arrest at both of the checkpoints
requires the replication terminator protein (RTP), suggesting the
existence of Ter-like sites about the checkpoints. Upon
return to relaxed conditions, the forks are released from the
checkpoints and continue the duplication of the chromosome until
arrested and terminated at the terminus region called TerC
(Fig. 1, top; see Ref. 16). In bacteriophage
, stringent response
causes the turning off of the ori-O-P transcript, thereby shutting down
the synthesis of O and P proteins and possibly also abrogating the
transcriptional activation of the
ori.
Replication
is thereby turned off at the initiation step (6). Initiation from oriC
of E. coli appears to be terminated at the origin under
stringent conditions (5). Interaction of ppGpp with RNA polymerase is
believed to promote fork passage by dislodging the RNA polymerase
stalled at damaged sites on DNA and thus, in this context, stringent
conditions promote rather than block fork progression (7).
L1 that
bound to two dimers of RTP and arrested replication in a bipolar mode
in vivo, when present in a plasmid context. The 17-bp
sequence reported by Autret et al. (8) is included in the
28-bp sequence. The isolated 17-bp core site of
L1 (L1), on the
other hand, neither arrested replication in vivo nor DnaB
helicase in vitro. The RTP-L1 complex had a half-maximal stability of just a few seconds whereas the Ter1-RTP complex
under identical conditions had a half-life of ~180 min. The
observation that a very weak terminator-RTP complex still showed
appreciable replication arrest in vivo tends to support the
conclusion that strong protein-DNA interaction by itself is not
obligatory for replication fork arrest at the Ter sites of
B. subtilis.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
[lac,
proAB],[F'tra D36,lacIq
(lacZ)M15,proA+,rk
,mk
B+)
was used for making M13 DNA, DH5
[F'sup E44,
lacU169 (
80 lacZ
M15), hsdR17,
recA1, endA1, gyrA96,
thi-1, relA1} was used for cloning purposes.
Wild type B. subtilis IS58 (trpC2
lys3), isogenic relA
mutant of
Bacillus IS 56 were used for RNA mapping and genomic DNA
preparation. The RTP overproducer strain SU230 of B. subtilis (20) was used for transforming shuttle vector clones and
preparation of replication intermediates.
strains which
were grown in relaxed and stringent conditions. To 25 ml of cell
culture pellet, 1 ml of TRIZOL reagent (Life Technologies) was added,
mixed, and incubated at 30 °C for 5 min to lyse the cells.
Chloroform (200 µl) was added, mixed, and centrifuged at 12,000 × g for 15 min at 4 °C. The upper aqueous layer having RNA was precipitated using equal volume of isopropyl alcohol. The RNA
pellet was washed with 70% ethanol and dissolved in RNase-free water.
The RNA samples were resolved on formaldehyde-agarose gels and blotted
onto nitrocellulose filters. YxcC gene of B. subtilis was amplified from genomic DNA using the primer set
5'-GATCAGGTTAGCCGCCGATAACACCAAAGTCG-3' and
5'-AAATGTCTTAATCAAACCTTACTCCGCGCGGG-3'. The PCR product was radiolabeled using the random priming NEBlot kit (New England Biolabs)
and used for Northern hybridization.
-32P]ATP and T4
polynucleotide kinase. 10 Fmol of labeled DNA was used in each
reaction, that were carried out in 20-µl volumes of 40 mM
Tris-Cl (pH 7.5), 4 mM MgCl2, 50 µg/ml
bovine serum albumin, 50 mM potassium glutamate, 5 µg of
calf thymus DNA, and increasing amounts of RTP protein (0, 20, 30, 40, 80, 120, 160, 200, 300, 400, 800, 1200, 1600, 2000, and 4000 fmol). The
reactions were carried out at room temperature for 30 min and resolved
on 8% native polyacrylamide gels.
-32P]ATP and T4 polynucleotide kinase and annealed to
1 pmol of M13mp18BS3REV (Ter1 RTP-binding site cloned in
nonblocking orientation as EcoRI-HindIII fragment) or M13 mp19BS3 (Ter1 RTP-binding site cloned in
blocking orientation as EcoRI-HindIII fragment),
M13 mp18 L1 (L1 RTP-binding site cloned in nonblocking orientation as
EcoRI-BamHI fragment), M13 mp19 L1 (L1
RTP-binding site cloned in blocking orientation as
EcoRI-BamHI fragment) and M13 mp19 L1 core (L1
core RTP-binding site cloned in blocking orientation as
EcoRI-BamHI fragment) single-stranded DNA,
respectively. These helicase substrates was purified through CL
4B-Sepharose spin column and 10 fmol of the substrate used in each
reaction of 20 µl volumes containing 40 mM Tris-Cl (pH 7.5), 4 mM MgCl2, 50 µg/ml bovine serum
albumin, 2 mM ATP, 50 mM potassium glutamate, 5 mM dithiothreitol, and increasing amounts of RTP (0, 0.4, 0.8, 1.6, 2.4, 3.2, and 4.0 pmol), after 15 min incubation at room
temperature 100 ng of DnaB helicase was added to the reaction mixture
and continued incubation for 30 min at 37 °C. The reaction was
stopped with SDS-EDTA-bromphenol blue dye and resolved on 8%
native polyacrylamide gels.
-D-galactopyranoside for 2 h and
250 µg/ml arginine hydroxamate added for 2 h to induce stringent
response. 10 mM sodium azide was added and cells kept on
ice, sedimented at 6,000 × g and the cell pellet was
processed as follows. Briefly, the cells were suspended in 4 ml of 50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 25% sucrose,
and 200 µg/ml lysozyme and kept on ice for 15 min. 1 ml of 10% SDS
was very gently mixed and incubated on ice for 15 min and then 4.2 ml
of 5 M NaCl was added and left on ice overnight. The
supernatant was collected by centrifuging at 25,000 rpm at 4 °C for
20 min and plasmid DNA precipitated with 0.8 volume of isopropyl
alcohol. The pellet washed in 70% ethanol was dissolved in 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (TE). The
samples were digested with pancreatic RNase (2 mg/ml), phenol
extracted, precipitated with 2 volumes of ethanol, air dried, and
dissolved in TE. The samples were quantitated and used for digestion
with PstI or HindIII and subjected to
one-dimensional and two-dimensional gel analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
L1 Site Near the Replication
Checkpoint--
Previous work (16) had placed the left replication
checkpoint of B. subtilis between the gnt and
sacXY markers (Fig. 1, top). We endeavored to search for sequences that had
homology with the consensus core sequence of Ter and came up
with a sequence marked as
L1 (Fig. 2,
A and B). Our strategy was to look for sequences,
initially identified by a computerized search of the genome (for
homology with consensus core sequence of Ter sites), that
yielded a double gel mobility shifts upon binding to purified RTP.
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Fig. 1.
B. subtilis genome showing the
position of origin of replication (oriC), termination
(terC) and the probable position of stringent
response sites. Top, genomic
map of the chromosome of B. subtilis showing the locations
of the two replication checkpoints (
L and
R) with respect to the
other genetic markers. Bottom: A, map showing the direction
of DNA replication, the direction of transcription of the genes located
in the region of the left checkpoint
L. The
L1 (L1) site that
binds to two dimers of RTP, is located in the reading frame of the
yxcC gene. Note that the gene is transcribed in a direction
opposite to that of DNA replication. B,
nucleotide sequences of the Ter1, Ter1 core, L1,
and L1 core. The G sites that contact RTP are underlined in
the Ter1 sequence. The presumptive contacts of the L1 are
also underlined.
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Fig. 2.
Interaction of RTP with Ter1
and L1, Ter1 core, and L1 core DNAs.
Autoradiograms of 8% nondenaturing polyacrylamide gels showing
A, interaction of Ter1 DNA with RTP.
The binding is manifested in two shifts of the free DNA, the first due
to filling of one site by one dimer and the second shift due to the
binding of a two dimers; B, Ter1 core sequence shows a
single shift due to filling of the core by one RTP dimer;
C, L1 sequence showing a single shifted band at
the lower concentrations of RTP and in addition, a double shifted band
at higher concentrations of RTP. Note that the binding to L1 is
qualitatively much weaker than that to Ter1 as indicated by
the appearance of the second shift at the higher range of
concentrations of RTP (400-4000-fold excess) in comparison to
Ter1. There seemed to be very poor cooperativity in
comparison with binding to Ter1. Under the experimental
conditions, significant amounts of free L1 DNA remained even at the
highest concentration of RTP used. D, binding of L1 core to
RTP shows only a single shift, due to the binding of only a single
dimer of RTP to the 17-bp long L1 core. Lanes 1-15 show
binding elicited by 0, 20, 30, 40, 80, 120, 160, 200, 300, 400, 800,
1200, 1600, 2000, and 4000 fmol of RTP, respectively.
L1 (called L1
hereafter) is shown in Fig. 1, bottom panel,
panel B. A comparison with the Ter1,
Ter1 core, with L1 and the presumptive core sequence of L1
are shown (Fig. 1, bottom, panels A and B). The G
residues that were identified by methylation protection analysis of
Ter1 are underlined (10). The G residues of L1 that might
correspond to the contact points on Ter1 are also underlined
(Fig. 1, bottom, panel B).
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Fig. 3.
Measurement of the off-rate of RTP from
Ter1 and L1 DNAs. Autoradiograms of 8% nondenaturing
acrylamide gels showing the release of free DNA from fully formed
RTP-DNA complex by a 10,000-fold excess of unlabeled, homologous
competitor DNA. A, autoradiogram showing the release of free
DNA from the RTP-Ter1 complex as a function of time.
Lane F, free Ter1 DNA; lane C1,
Ter 1-RTP complex without competitor DNA near time zero;
lanes 0-180 show the dissociation of RTP at 0-, 10-, 20-, 30-, 60-, 120-, and 180-min time intervals, respectively. Lane
C2 shows the stability of Ter1-RTP complex after 180 min of incubation without any competitor DNA. B, same as in
A except that the complex was formed by saturation binding
of RTP to L1 DNA. Note that competing DNA elicited almost complete
release of L1 DNA from the protein-DNA complex at the earliest time
point (lane 0, B). C, quantitative
analysis of the competition data derived from 3 sets of gels such as
shown in A and B. Note that whereas
Ter1 shows 50% release in ~180 min, L1 DNA is
released from the complex almost immediately after addition of the
competing DNA.
and isogenic
wild type B. subtilis strains were analyzed for the levels
of yxcC mRNA in vivo during normal and
stringent conditions. The Northern blots showed that the transcript
that hybridized to the yxcC DNA probe was present in
relA cells regardless of arginine hydroxamate treatment (compare the lanes RelA
stringent
with the one marked RelA
relaxed,
Fig. 4). In contrast, in isogenic wild
type cells treatment with arginine hydroxamate caused the transcript to
be greatly reduced in comparison with the nontreated sample (compare
the lane marked RelA+ relaxed with
RelA+ stringent in Fig. 4). Thus, the
data support the conclusion that the transcription of the
yxcC gene was under stringent control and the arginine
hydroxamate treatment as used in our experiments appeared to induce
stringent control.
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Fig. 4.
Mapping of yxcC RNA under
relaxed and stringent conditions. Total RNA was prepared from wild
type B. subtilis and isogenic relA strain
grown in relaxed and stringent conditions. Autoradiograms of the
Northern blots show: in the
relA strain, the presence of
yxcC transcript in both relaxed and stringent conditions,
whereas in wild type strain, the RNA was present in relaxed but not in
stringent conditions. Equal amounts of total RNA were loaded in each
lane.
1 (20). The vector had ampicillin and erythromycin resistance
markers that were expressed in E. coli and B. subtilis, respectively. The L1 and Ter1
sequences were cloned between the EcoRI and BamHI
sites in both orientations and transformed into the RTP overproducer
strain SU230 of B. subtilis. The replication intermediates
from cells grown under stringent and relaxed conditions were purified
and analyzed by one- and two-dimensional agarose gel electrophoresis,
after digestion with either PstI or HindIII. The
plasmid DNA was cut once by PstI but twice by
HindIII (Fig. 5).
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Fig. 5.
The recombinant DNA clone containing L1 (or
Ter1) in a shuttle vector that was used for in
vivo analysis of the ability of L1 to promote fork arrest
in vivo. Note the location of the functional,
unidirectionally initiating oriB, that functions in B. subtilis. The arrow shows the direction of replication.
The L1 or the Ter1 sequences were cloned between the
EcoRI (E) and the BamHI (B)
sites of the polylinker. Note the location of the L1-ter
with respect to oriB. The replication intermediates were
either cleaved at the single PstI (P) or the two
HindIII (H) sites before one- or two-dimensional
gel analysis.
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Fig. 6.
In vivo analysis of L1 template
for its ability to promote RTP-mediated replication arrest in
vivo. A, autoradiograms of one- and
two-dimensional agarose gel analysis of PstI (single cut) or
HindIII (2 cuts) replication intermediates of
Ter1 and L1. Ter1 (HindIII digested)
in the blocking orientation (BLK) is able to arrest
replication forks as indicated by the band (arrow)
corresponding to Y-shaped, arrested intermediates. Note that even
though 4-5 times more DNA from the Ter1 nonblocking
orientation (N.Blk) had been loaded, no stalled intermediate
band is visible. L1 (digested with HindIII) shows blockage
in both orientations. Note that samples from both relaxed and stringent
conditions show the arrested intermediate. The labeling of the lanes is
self-explanatory. Note that the level of replication intermediate band
of L1 samples are significantly lower in comparison with that present
in Ter1. B, two-dimensional analysis
of PstI-digested samples shows the replication
intermediate-specific spot in Ter1 blk but not in
Ter1 nonblk sample. C, two-dimensional analysis
of HindIII-digested samples shows the replication
intermediate specific spot in both L1blk relaxed and L1 blk stringent
samples (arrow). D, two-dimensional gel analysis
of PstI-digested samples the termination-specific spot in
L1blk relaxed and L1 blk stringent samples. Note that the termination
specific spot is right on the arc of replication intermediates
(arrow).
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Fig. 7.
Helicase assays show bipolar nature of L1
sequence. A, autoradiograms of the helicase assay on
Ter1 (also called IR1) partial duplex substrate showing the
polar nature of DnaB helicase arrest. The wedge shaped marks
indicate increasing range of RTP concentrations. Lane 1 shows the helicase substrate, lane 2 shows the release of
oligonucleotide by DnaB helicase in absence of RTP, and lanes
3-7 show the effect of increasing concentrations of RTP on
helicase activity. Note the polarity of RTP mediated block by comparing
IR1 Blk versus N.Blk. B,
autoradiogram showing that both orientation of L1 show arrest of
helicase activity by RTP. C, quantitative analysis of
helicase arrest on IR1, L1, IR1-core, and L1 core substrates. Note that
both orientations of L1 arrest helicase almost equally, albeit less
effectively than the blocking orientation of Ter1 (IR1). The
core of L1 is much less effective a substrate in promoting RTP-mediated
helicase arrest.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health, NIGMS, and a Merit Award from the National Institutes of Health, NIAID, (to D. B.).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.
Present address: Dept. of Cancer Genetics, Roswell Park
Cancer Institute, Buffalo, NY 14263.
§ To whom correspondence should be addressed. Tel.: 919-684-3521; Fax: 919-684-8735; E-mail: bastia@abacus.mc.duke.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009538200
2 B. K. Mohanty, D. E. Bussiére, T. Sahoo, K. S. Pai, W. J. J. Meijer, S. Brow, and D. Bastia. (2001) J. Biol. Chem. in press.
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ABBREVIATIONS |
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The abbreviations used are: ppGpp, guanosine tetraphosphate; RTP, replication terminator protein; bp, base pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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