Replication arrests during a single round of replication of the <i>Escherichia coli</i> chromosome in the absence of DnaC activity

Maisnier‐Patin, Sophie; Nordström, Kurt; Dasgupta, Santanu

doi:10.1046/j.1365-2958.2001.02718.x

Cited by 59 publications

(70 citation statements)

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“…The fact that the number of two-chromosome cells does not reach zero during incubation at a non-permissive temperature in the control could be a result of incomplete replication in some of the cells. It has previously been shown that lack of active DnaC during elongation leads to a failure to complete replication in 18 % of cells, because of the lack of loading of DnaC by the primosomal proteins (Maisnier-Patin et al, 2001).…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli datA site promotes proper regulation of cell division

Flåtten

Skarstad

2014

Microbiology

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In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP-DnaA protein. INTRODUCTIONIn Escherichia coli cell division involves the formation of a septum that separates the cell into two daughter cells. It is crucial that the two daughter nucleoids are segregated into each half of the cell, so that each daughter cell receives a copy of the genetic material and that none of the nucleoids are trapped in the septum. To ensure this, cell division and chromosome segregation are highly regulated processes involving a number of different proteins (see Egan & Vollmer, 2013;Harry et al., 2006;and Weiss, 2004, for reviews), the most studied being FtsZ. This protein polymerizes to form a ring-like structure at the site of division and serves as a scaffold for recruitment of other division proteins (Bi & Lutkenhaus, 1991). How the timing of the formation of the FtsZ ring is regulated is not clear.It is known that the chromosome replication cycle is independent of the cell division cycle, but that cell division is dependent on chromosome replication (Boye & Nordström, 2003; Nordström et al., 1991). That is, a blockage of chromosome replication stops cell division (Helmstetter & Pierucci, 1968;Walker & Pardee, 1968). Thus, a checkpoint mechanism must exist. The mechanism ensures that cell division does not occur in the absence of replication and thus prevents the formation of DNA-free (anucleate) daughter cells (Boye & Nordström, 2003; Nordström et al., 1991). Whether this mechanism is related to other regulatory circuits necessary for coupling the division cycle to cell physiology and to the growth rate of the cells, is not clear (see Haeusser & Levin, 2008, for a review).The DnaA protein is one of the main players in initiation of replica...

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Section: Resultsmentioning

confidence: 99%

The Escherichia coli datA site promotes proper regulation of cell division

Flåtten

Skarstad

2014

Microbiology

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“…As both transcription and the insertion of newly transcribed-translated proteins into membrane (transertion) have been implicated as mechanisms contributing to bacterial chromosome segregation (12,31,45,49,61), we wished to test the consequence of inhibiting transcription (and thereby ongoing transertion) on segregation of ori loci. To do so, we synchronized cells for DNA synthesis using dnaC(Ts) mutation (40) and treated them with rifampin (300 g/ml) to block transcription. dnaC(Ts) mutant cells were grown exponentially at 30°C and then shifted to the nonpermissive temperature (37°C) at an A 600 of ϳ0.1 to block replication initiation but allow completion of ongoing rounds of DNA synthesis.…”

Section: Vol 192 2010 E Coli Origin Segregation 6147mentioning

confidence: 99%

Independent Segregation of the Two Arms of theEscherichia coli oriRegion Requires neither RNA Synthesis nor MreB Dynamics

Wang

Sherratt

2010

J Bacteriol

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The mechanism of Escherichia coli chromosome segregation remains elusive. We present results on the simultaneous tracking of segregation of multiple loci in the ori region of the chromosome in cells growing under conditions in which a single round of replication is initiated and completed in the same generation. Loci segregated as expected for progressive replication-segregation from oriC, with markers placed symmetrically on either side of oriC segregating to opposite cell halves at the same time, showing that sister locus cohesion in the origin region is local rather than extensive. We were unable to observe any influence on segregation of the proposed centromeric site, migS, or indeed any other potential cis-acting element on either replication arm (replichore) in the AB1157 genetic background. Site-specific inhibition of replication close to oriC on one replichore did not prevent segregation of loci on the other replichore. Inhibition of RNA synthesis and inhibition of the dynamic polymerization of the actin homolog MreB did not affect ori and bulk chromosome segregation.The chromosome of the extensively studied bacterium Escherichia coli undergoes simultaneous replication and segregation and has no apparent mitotic apparatus for chromosome segregation, a situation very different from that of eukaryotes, where replication and segregation occur in temporally separate periods of the cell cycle. An unsolved mystery of the bacterial cell cycle is how chromosome segregation takes place. Several mechanisms have been proposed to drive the segregation of origin and bulk DNA after replication. In one model, cell elongation is proposed to be a crucial factor, in which the two newly replicated origins are attached to the inner membrane and separated by cell growth between them along the long axis of the cell (25). However, it is now clear that elongation occurs throughout the cell and the movement of the origins is much faster than the rate of cell elongation, indicating that cell elongation alone is not responsible for segregation (55,60).Active partitioning systems were first found in low-copynumber plasmids, where they are required for stable inheritance by distributing the daughter plasmids to both daughter cells (reviewed in reference 14). These systems fall into two families; one uses the ParM actin and its associated protein and binding sites to drive newly replicated sister plasmids apart during cycles of actin polymerization and depolymerization (4, 19). The second parABS family is less well understood mechanistically, although ATP hydrolysis-dependent cycles of ParA movement appear to play a key role in the segregation process (48).Later, it was found that many bacterial chromosomes also utilize parABS systems for their segregation, for example, Bacillus subtilis (23, 37), Caulobacter crescentus (41), and both chromosomes of Vibrio cholerae (22). The typical chromosomal par locus consists of two genes, parA and parB (soj and spo0J in B. subtilis), and a cis-acting parS DNA element. ParB is a DNA-binding prote...

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“…A substantial fraction of replication forks stall or collapse at sites of DNA damage during bacterial DNA replication (1)(2)(3). Potential blocks to replication include non-coding lesions in the DNA template, DNA strand breaks, or proteins bound to DNA ahead of the replication fork.…”

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Situational Repair of Replication Forks

Robu

Inman

Cox

2004

Journal of Biological Chemistry

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Replication forks often stall or collapse when they encounter a DNA lesion. Fork regression is part of several major paths to the repair of stalled forks, allowing nonmutagenic bypass of the lesion. We have shown previously that Escherichia coli RecA protein can promote extensive regression of a forked DNA substrate that mimics a possible structure of a replication fork stalled at a leading strand lesion. Using electron microscopy and gel electrophoresis, we demonstrate that another protein, E. coli RecG helicase, promotes extensive fork regression in the same system. The RecG-catalyzed fork regression is very efficient and faster than the

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Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity

Cited by 59 publications

References 61 publications

The Escherichia coli datA site promotes proper regulation of cell division

The Escherichia coli datA site promotes proper regulation of cell division

Independent Segregation of the Two Arms of theEscherichia coli oriRegion Requires neither RNA Synthesis nor MreB Dynamics

Situational Repair of Replication Forks

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