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

Sharma, Bela; Hill, Thomas M.

doi:10.1111/j.1365-2958.1995.mmi_18010045.x

Cited by 83 publications

(77 citation statements)

References 39 publications

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“…Insight to the problem has been acquired through the examination of the consequences of stalling replication forks in vivo by various means. Placement of Ter sequences outside of the usual configuration at the terminus region of the chromosome generated strains that required RecA and RecBCD for survival if the replication-fork arrest protein Tus also was present (7,8). The models developed to explain these observations suggested that DSB formation was occurring at the stalled replication fork.…”

mentioning

confidence: 95%

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

McGlynn

Lloyd

Marians

2001

Proc. Natl. Acad. Sci. U.S.A.

147

116

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Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression. T he picture of how DNA replication proceeds around the bacterial chromosome has changed over the last decade as a result of research in many laboratories (1). Even though the two replication forks that form at oriC have a sufficiently high enough inherent processivity to complete replication of the chromosome, it is clear that this is generally not what happens. Instead, the replication forks formed at the origin become inactivated at high frequency as a result of an encounter with roadblocks either in or on the template strands. These roadblocks can take many forms: a nick in one of the template strands, a DNA adduct formed as a result of endogenous damage, secondary structure in the template, and frozen proteins on the DNA. Survival then depends on both correction of the damage and reactivation of DNA replication.Although there is a large body of both genetic and biochemical data informing the mechanisms that act to repair damaged nucleotides in DNA, except in one instance, the mechanisms of replication-fork restart are less well defined. Replication-fork restart after an encounter with a template nick, leading to double-strand break (DSB) generation and detachment from the growing fork of one of the nascent sister duplexes-sometimes termed replication fork collapse (2)-is effected by a marriage of homologous recombination and DNA replication proteins (3). Here, the DSB generated is processed by RecBCD to generate a recombinogenic 3Ј single-stranded tail that is used for RecAcatalyzed strand invasion with the intact sister duplex, creating a D loop. This structure is recognized by PriA (4, 5), which then directs the a...

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mentioning

confidence: 95%

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

McGlynn

Lloyd

Marians

2001

Proc. Natl. Acad. Sci. U.S.A.

147

116

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“…If a newly initiated replication fork were to reach a stalled fork and copy nascent DNA at the stalled fork, this would create double-stranded ends, leading to a collapsed replication fork (6,32,47). Strains carrying DNA replication termination sites (Ter) at new positions in the chromosome depend on RecA, RecBC, and RuvABC (all proteins involved in recombinational repair) for viability (6,21, 0.47) Fork breakage was not evident in these cells, although linear DNA was created when a new fork reached a fork blocked at Ter.…”

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confidence: 99%

“…The nature of the stalled fork determines which proteins are involved in this process of fork reversal and replication restart. For example, RecA is required for RuvABC action when replication is arrested by inactivation of DnaB helicase (46), but RecA is not required when fork arrest is caused by the lack of Rep helicase (46) or a defect in the HolD subunit of the Pol III clamp loader (15) or in a dnaEts mutant (18).If a newly initiated replication fork were to reach a stalled fork and copy nascent DNA at the stalled fork, this would create double-stranded ends, leading to a collapsed replication fork (6,32,47). Strains carrying DNA replication termination sites (Ter) at new positions in the chromosome depend on RecA, RecBC, and RuvABC (all proteins involved in recombinational repair) for viability (6, 21, 0.47) Fork breakage was not evident in these cells, although linear DNA was created when a new fork reached a fork blocked at Ter.…”

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confidence: 99%

Escherichia coliCells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

Grigorian¹,

Lustig²,

Guzmán

et al. 2003

J Bacteriol

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The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, ␤ clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, ␤ clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the ␤ clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ϳ100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.The dnaA operon of Escherichia coli (Fig. 1A) contains the genes dnaA, dnaN, and recF, which encode the DNA replication initiator protein, DnaA; the ␤ subunit of DNA polymerase III holoenzyme (Pol III); and RecF, respectively. The concentration of DnaA protein is a critical factor in determining the timing of initiation of DNA replication from oriC within the cell cycle, and it is likely that additional DnaA protein must be synthesized between rounds of replication (55). Although DnaA levels control the timing of initiation during growth and new DnaA synthesis appears to be necessary prior to new initiation events, the cell does not tolerate well increases in the amount of DnaA protein. When the concentration of DnaA is raised 1.5-to 3-fold, the number of replication forks increases, but the rate of replication decreases, and many of these additional forks appear to terminate replication prematurely (3). Katayama (24) called this an attenuation type of response to excessive initiation events, where forks stall before reaching the terminus.Such stalled replication forks in cells with increased levels of DnaA could lead to double-strand breaks (DSBs) which, if not repaired, would cause cell death. There are now many examples in which cells with arrested replication forks depend on some form of recombinational repair for survival: e.g., rep mutants (40); dnaBts, dnaEts, and dnaNts mutants ...

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“…Similar dependence on RecA has been reported for E. coli strains with large chromosomal inversions (33) or ectopic termination blocks (18,49) or in cases of integrative suppression of oriC (36). Among all these RecA-dependent strains with diverse replication mechanisms from different origins (oriC, oripBR322, oriP2sig5, and oriR1), the only shared characteristic seems to be the prolonged arrest of replication forks at the Ter sites due to asymmetry in the overall chromosome replication.…”

Section: Discussionmentioning

confidence: 75%

“…It differs from the RuvAB-, RecBCD-, RecF-, or RecG-mediated mechanisms activated in E. coli when the replication forks are blocked because cells are deficient for the nonessential helicase Rep (47) or due to DNA lesions (6,38). It is also different from the recombinational rescue of strains with replication forks blocked at a Ter-Tus complex (17,18,49), which were described in terms of iSDR-type replication restart from a RecBCD-mediated D-loop structure (20). However, the TerTus systems used in these studies were not the natural arrest sites but rather a Ter site within the lac gene and an inverted Ter duplex cassette in the terminus region.…”

Section: Discussionmentioning

confidence: 97%

RecA-Mediated Rescue ofEscherichia coliStrains with Replication Forks Arrested at the Terminus

2001

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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.Recombination and replication are intimately coupled processes with a large number of shared gene functions. The involvement of the replication mechanism in recombination and repair is well established (11,43,46). The extensive and vital role of recombination in chromosome replication was not evident until recently, although its key role in the replication of several bacteriophages has been recognized (25,29,42). Early evidence for the involvement of recombination in Escherichia coli chromosome replication came from special situations in which the initiation factor-dependent, oriC-specific assembly of replisomes could be bypassed through homologous-recombination-dependent replication processes called SDRs (for stable DNA replication) (21,22,30). SDR is constitutive (cSDR) in rnh mutants when initiation from oriC is inhibited and stable R loops can act as cryptic initiation sites. SDR can also be induced (iSDR) in response to DNA damage in UV-irradiated, thymidine-starved, or drug-treated cells in which recombinogenic invasion at double-strand-break (DSB) sites leads to the formation of branched structures and/or D loops, which act as the assembly sites for a new replisome. RecA is believed to be critical for replication initiation for both SDRs (51, 52), but each has its own distinctive recombination-repair pathway (20). DSBs have also been shown to act as hot spots for recombination-mediated initiation of replication (1,12,25).Even normal replication, initiated from oriC in wild-type E. coli, relies on recombination-repair pathways for the apparently unhindered progression of replication forks over the full length of the bacterial chromosome (40, 47). The importance of the recombination-repair pathways in maintaining genome stability during replication in E. coli and other organisms is now...

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

Cited by 83 publications

References 39 publications

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Escherichia coliCells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

RecA-Mediated Rescue ofEscherichia coliStrains with Replication Forks Arrested at the Terminus

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