Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo

Possoz, Christophe; Filipe, Sérgio R.; Grainge, Ian; Sherratt, David J.

doi:10.1038/sj.emboj.7601155

Cited by 112 publications

(143 citation statements)

References 32 publications

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“…Repressors bound to arrays of operators have been found to block the progression of the replisome in Escherichia coli (Possoz et al 2006). We observed a similar replication roadblock in B. subtilis (Supplemental Fig.…”

Section: Tetr-gfp Is Stripped Off Teto Arrays During Translocationsupporting

confidence: 62%

“…However, some DNA-binding proteins have very high affinity for their cognate binding sites. In fact, in some instances these interactions are sufficient to impede the movement of a translocating complex as strong as the replisome (Possoz et al 2006). The biophysical data presently available for either FtsK or SpoIIIE do not allow us to estimate whether these enzymes could displace proteins tightly bound to DNA.…”

mentioning

confidence: 92%

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SpoIIIE strips proteins off the DNA during chromosome translocation

Marquis¹,

Nollmann²,

Ptacin³

et al. 2008

Genes Dev.

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The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.[Keywords: Chromosome segregation; chromosome translocation; sporulation; FtsK; AAA+ ATPase] Supplemental material is available at http://www.genesdev.org.

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Section: Tetr-gfp Is Stripped Off Teto Arrays During Translocationsupporting

confidence: 62%

mentioning

confidence: 92%

SpoIIIE strips proteins off the DNA during chromosome translocation

Marquis¹,

Nollmann²,

Ptacin³

et al. 2008

Genes Dev.

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“…We have previously shown that tight binding of fluorescent fusions of either TetR or LacI to arrays of their cognate operators can be achieved when they are expressed in the absence of their inducers (AT and IPTG, respectively) and results in replication blockage at the array (46). Furthermore, replication restarts rapidly upon relief of these tight repressorbinding events.…”

Section: Resultsmentioning

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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“…5C). The following contrasting results were reported regarding the stability of E. coli replisomes: the replisome blocked by an array of repressor-operator complexes loses the ability to continue replication with a half-life of 4 -6 min in vitro (55), whereas it retains the ability to resume replication upon removal of the block for several hours in vivo (56). The dissociation of stalled DnaB from DNA observed in the present study accounts at least in part for the inactivation of the replisome in vitro, although the in vitro half-lives of stalled replisome (4 -6 min) and DnaB (36 min) differ considerably.…”

Section: Discussionmentioning

confidence: 97%

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

Nakano

Miyamoto-Matsubara

Shoulkamy

et al. 2013

Journal of Biological Chemistry

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Background: DNA-protein cross-links (DPCs) are formed by DNA-damaging agents. Results: DPCs on the translocating strand but not on the nontranslocating strand block hexameric replicative helicases in a size-dependent manner. Stalled helicases dissociate from DNA with a half-life of 15-36 min. Conclusion: DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. Significance: Reversible and irreversible protein roadblocks may have distinct effects on replisomes.

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Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo

Cited by 112 publications

References 32 publications

SpoIIIE strips proteins off the DNA during chromosome translocation

SpoIIIE strips proteins off the DNA during chromosome translocation

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

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

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