Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites

Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species.     

RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus

The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations. Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains. In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.     

Requirement for RecFOR-mediated recombination in priA mutant

Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR-mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V-mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.     

Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1

Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.     

Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli

In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus-Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus-Ter complex without formation of the Tus-Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus-Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA-E and G, moderately to TerH-J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus-Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus-Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009).     

Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle

To investigate the co-ordination between DNA replication and cell division, we have disrupted the DNA replication cycle of Escherichia coli by inserting inverted Ter sites into the terminus region to delay completion of the chromosome. The inverted Ter sites (designated Inv Ter :: spc ^r) were initially inserted into the chromosome of a Δ tus strain to allow unrestrained chromosomal replication. We then introduced a functional tus gene by transforming the Inv Ter :: spc ^r strain with a plasmid carrying the tus gene under control of an arabinose-inducible promoter. In the presence of 0.2% arabinose, the cells formed long filaments, suggesting that activation of the inverted Ter sites by Tus arrested DNA replication and delayed the onset of cell division. Induction of sfiA , a gene in the SOS regulon, was observed following arrest of DNA replication; however, when a sfiB114 allele was introduced into Inv Ter :: spc ^r strain, long filaments were still formed, suggesting that the sfi -independent pathway also caused filamentation. Either recA :: cam ^r or lexA3 alleles suppressed filamentation when introduced in the Inv Ter strain. Interestingly, in both the recA :: cam ^r and lexA3 mutants, virtually all cells had a nucleoid, suggesting that cell division was proceeding even though DNA replication was not complete. These results suggest that DNA replication and cell division are uncoupled when recA is inactivated or when genes repressed by LexA cannot be induced.     

Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex.

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.     

Cells defective for replication restart undergo replication fork reversal

We have studied the fate of blocked replication forks with the use of the Escherichia coli priA mutant, in which spontaneously arrested replication forks persist owing to the lack of the major replication restart pathway. Such blocked forks undergo a specific reaction named replication fork reversal, in which newly synthesized strands anneal to form a DNA double-strand end adjacent to a four-way junction. Indeed, (i) priA recB mutant chromosomes are linearized by a reaction that requires the presence of the Holliday junction resolvase RuvABC, and (ii) RuvABC-dependent linearization is prevented by the presence of RecBC. Replication fork reversal in a priA mutant occurs independently of the recombination proteins RecA and RecR. recBC inactivation does not affect priA mutant viability but prevents priA chronic SOS induction. We propose that, in the absence of PriA, RecBC action at reversed forks does not allow replication restart, which leads to the accumulation of SOS-inducing RecA filaments. Our results suggest that types of replication blockage that cause replication fork reversal occur spontaneously.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 相似文献   

RecA-dependent replication in the nrdA101(Ts) mutant of Escherichia coli under restrictive conditions

Cells carrying the thermosensitive nrdA101 allele are able to replicate entire chromosomes at 42°C when new DNA initiation events are inhibited. We investigated the role of the recombination enzymes on the progression of the DNA replication forks in the nrdA101 mutant at 42°C in the presence of rifampin. Using pulsed-field gel electrophoresis (PFGE), we demonstrated that the replication forks stalled and reversed during the replication progression under this restrictive condition. DNA labeling and flow cytometry experiments supported this finding as the deleterious effects found in the RecB-deficient background were suppressed specifically by the absence of RuvABC; however, this did not occur in a RecG-deficient background. Furthermore, we show that the RecA protein is absolutely required for DNA replication in the nrdA101 mutant at restrictive temperature when the replication forks are reversed. The detrimental effect of the recA deletion is not related to the chromosomal degradation caused by the absence of RecA. The inhibition of DNA replication observed in the nrdA101 recA mutant at 42°C in the presence of rifampin was reverted by the presence of the wild-type RecA protein expressed ectopically but only partially suppressed by the RecA protein with an S25P mutation [RecA(S25P)], deficient in the rescue of the stalled replication forks. We propose that RecA is required to maintain the integrity of the reversed forks in the nrdA101 mutant under certain restrictive conditions, supporting the relationship between DNA replication and recombination enzymes through the stabilization and repair of the stalled replication forks.     

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

The Escherichia coli replication fork arrest complex Tus/Ter mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative “short tract” gene conversion (STGC) and error-prone “long tract” gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/Ter-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/Ter-arrested forks. Here, we report the impact of Ter array length on Tus/Ter-induced HR, comparing HR reporters containing arrays of 6, 9, 15 or 21 Ter sites—each targeted to the ROSA26 locus of mouse embryonic stem (ES) cells. Increasing Ter copy number within the array beyond 6 did not affect the magnitude of Tus/Ter-induced HR but biased HR in favor of LTGC. A “lock”-defective Tus mutant, F140A, known to exhibit higher affinity than wild type (wt)Tus for duplex Ter, reproduced these effects. In contrast, increasing Ter copy number within the array reduced HR induced by the I-SceI homing endonuclease, but produced no consistent bias toward LTGC. Thus, the mechanisms governing HR at Tus/Ter-arrested replication forks are distinct from those governing HR at an enzyme-induced chromosomal double strand break (DSB). We propose that increased spatial separation of the 2 arrested forks encountering an extended Tus/Ter barrier impairs the coordination of DNA ends generated by the processing of the stalled forks, thereby favoring aberrant LTGC over conservative STGC.     

Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.     

Replication termination: mechanism of polar arrest revealed

The Tus-Ter protein-DNA complex of Escherichia coli blocks progression of DNA replication from only one direction at the replication terminus. As the replication fork helicase unwinds one side of Ter, a conserved cytosine flips out of the duplex and binds to Tus, thereby creating a locked complex that blocks the advancing helicase.     

Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.

Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.     

A fork-clearing role for UvrD

The inactivation of a replication protein causes the disassembly of the replication machinery and creates a need for replication reactivation. In several replication mutants, restart occurs after the fork has been isomerized into a four-armed junction, a reaction called replication fork reversal. The repair helicase UvrD is essential for replication fork reversal upon inactivation of the polymerase (DnaE) or the beta-clamp (DnaN) subunits of the Escherichia coli polymerase III, and for the viability of dnaEts and dnaNts mutants at semi-permissive temperature. We show here that the inactivation of recA, recFOR, recJ or recQ recombination genes suppresses the requirement for UvrD for replication fork reversal and suppresses the lethality conferred by uvrD inactivation to Pol IIIts mutants at semi-permissive temperature. We propose that RecA binds inappropriately to blocked replication forks in the dnaEts and dnaNts mutants in a RecQ- RecJ- RecFOR-dependent way and that UvrD acts by removing RecA or a RecA-made structure, allowing replication fork reversal. This work thus reveals the existence of a futile reaction of RecA binding to blocked replication forks, that requires the action of UvrD for fork-clearing and proper replication restart.     

RecA, Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

RecA,Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

Localization of RecA in Escherichia coli K-12 using RecA-GFP

RecA is important in recombination, DNA repair and repair of replication forks. It functions through the production of a protein-DNA filament. To study the localization of RecA in live Escherichia coli cells, the RecA protein was fused to the green fluorescence protein (GFP). Strains with this gene have recombination/DNA repair activities three- to tenfold below wild type (or about 1000-fold above that of a recA null mutant). RecA-GFP cells have a background of green fluorescence punctuated with up to five foci per cell. Two types of foci have been defined: 4,6-diamidino-2-phenylindole (DAPI)-sensitive foci that are bound to DNA and DAPI-insensitive foci that are DNA-less aggregates/storage structures. In log phase cells, foci were not localized to any particular region. After UV irradiation, the number of foci increased and they localized to the cell centre. This suggested colocalization with the DNA replication factory. recA, recB and recF strains showed phenotypes and distributions of foci consistent with the predicted effects of these mutations.