A Replication Terminus Located at or Near a Replication Checkpoint of Bacillus subtilis Functions Independently of Stringent Control*

Ashish GautamDagger and Deepak Bastia§

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have examined a replication terminus (psi 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 psi 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 psi 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 (psi 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 psi 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta  subunit of RNA polymerase, causing turning off of transcription from certain promoters (3).

The effect of stringent response on DNA replication has been investigated in bacteria and in bacteriophage lambda . 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 psi L and psi 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 lambda , 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 lambda  ori. lambda  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).

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 psi 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 psi 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

Bacterial Strains and Plasmid Constructs-- The E. coli strains JM109 (sup E44, rel A1, recA, endA1, gyrA96,hsdR17D, Delta [lac, proAB],[F'tra D36,lacIq Delta  (lacZ)M15,proA+,rk-,mk-B+) was used for making M13 DNA, DH5alpha [F'sup E44, lacU169 (phi 80 lacZ Delta  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.

RNA Mapping-- Total RNA was prepared from wild type B. subtilis IS 58 RelA and IS 56 rel A- 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.

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 [gamma -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.

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 [gamma -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.

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-beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of the psi 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 psi 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 psi  sites. Top, genomic map of the chromosome of B. subtilis showing the locations of the two replication checkpoints (psi L and psi 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 psi L. The psi 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.

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 psi 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).

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).


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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.

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- 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 Delta relA strain grown in relaxed and stringent conditions. Autoradiograms of the Northern blots show: in the Delta 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.

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 pAMbeta 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.

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.


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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).

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.


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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

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: ppGpp, guanosine tetraphosphate; RTP, replication terminator protein; bp, base pair(s); PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.