The eclipse period of Escherichia coli

Freiesleben, Ulrik von; Krekling, Martin A.; Hansen, Flemming; Løbner‐Olesen, Anders

doi:10.1093/emboj/19.22.6240

Cited by 83 publications

(112 citation statements)

References 57 publications

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“…Lack of topo IV activity also leads to an increase in the steady-state level of negative supercoiling because of a shift in the balance of topoisomerase activities in the cell (27,28,30,32). SeqA-deficient strains exhibit aberrant nucleoid formation, abnormal segregation of chromosomal DNA, and an increased steady-state level of negative superhelicity (11)(12)(13)(14)17). These until now unexplained parC-and parE-like phenotypes of seqA mutants might be explained by the results we are reporting here, namely that the SeqA protein is required for proper activity of the topo IV enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…Microscopy of immunolabeled or green fluorescent protein-tagged SeqA has revealed that SeqA is predominantly localized in foci situated at the replication forks, presumably forming complexes with the newly replicated DNA and possibly contributing to proper segregation of daughter chromosomes (4,11,15,16). Overproduction of the SeqA protein interferes with the segregation of replicated chromosomes and leads to delayed cell division (14,15). These results indicate that an optimal level of SeqA is required for the segregation of chromosomal DNA.…”

mentioning

confidence: 99%

“…Strains with seqA mutations exhibit aberrant nucleoid distribution, a higher frequency of anucleoid cells, and filamentation (11)(12)(13)(14). Microscopy of immunolabeled or green fluorescent protein-tagged SeqA has revealed that SeqA is predominantly localized in foci situated at the replication forks, presumably forming complexes with the newly replicated DNA and possibly contributing to proper segregation of daughter chromosomes (4,11,15,16).…”

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confidence: 99%

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SeqA Protein Stimulates the Relaxing and Decatenating Activities of Topoisomerase IV

Kang

Han

Park

et al. 2003

Journal of Biological Chemistry

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The SeqA protein, which prevents overinitiation of chromosome replication, has been suggested to also participate in the segregation of chromosomes in Escherichia coli. Using a bacterial two-hybrid system, we found that SeqA interacts with the ParC subunit of topoisomerase IV (topo IV), a type II topoisomerase involved in decatenation of daughter chromosomes and relief of topological constraints generated by replication and transcription. We demonstrated that purified SeqA protein stimulates the activities of topo IV, both in relaxing supercoiled plasmid DNA and converting catenanes to monomers. The same moderate levels of SeqA protein did not affect the activities of DNA gyrase or topoisomerase I. At higher levels of SeqA, topo IV favored the formation of catenanes, caused by intermolecular strand exchange among plasmid DNA aggregates formed by SeqA. Excess SeqA inhibited the activity of all topoisomerases. We also found that stimulation of topo IV was dependent upon the affinity of SeqA for DNA. Our results suggest that this stimulation is mediated by the specific interaction of topo IV with SeqA.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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SeqA Protein Stimulates the Relaxing and Decatenating Activities of Topoisomerase IV

Kang

Han

Park

et al. 2003

Journal of Biological Chemistry

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“…Thyminelimited E. coli cells have 100% viability, show normal growth rates, and divide on time, but their CRC increases to compensate for the slower-moving replication forks (Zaritsky et al 2006). The system that regulates these extra initiations is proposed to be the one that determines the "eclipse period," a cell cycle phase of enforced origin inactivity following each replication initiation, lasting 60% of the generation time (von Freiesleben et al 2000;Olsson et al 2002). By preventing closely spaced origin firing events, the eclipse phenomenon defines a minimal allowed distance between codirectional replication forks in the E. coli chromosome.…”

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confidence: 99%

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of 8 (cells divide more slowly); the "functional" CRC limit of 22 (cells divide extremely slowly); and the "tolerance" CRC limit of 64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. KEYWORDS overinitiation; hydroxyurea; seqA; rep; recA E UKARYOTIC and prokaryotic chromosomes differ in many important aspects (Kuzminov 2014), and one key difference lies in the spatio-temporal organization of chromosomal replication. In contrast to eukaryotes, which perform multibubble replication (Masai et al. 2010), most bacteria replicate their singular chromosome in the unibubble format by initiating bidirectional replication from a designated replication origin (oriC) (Sernova and Gelfand 2008;Leonard and Méchali 2013) and finishing replication within a broad termination zone (ter) (Mirkin and Mirkin 2007;Duggin et al. 2008). Eukaryotes always perform a single replication round in their chromosomes (Masai et al. 2010;Diffley 2011), keeping the ratio of maximally replicated to unreplicated DNA in the same chromosome (the "replication complexity index") strictly at 2 ( Figure 1A). Due to the defined origin and terminus of prokaryotic chromosomes, chromosomal replication complexity in bacteria can be simply expressed as the ori/ter ratio ( Figure 1B). Even though unique cell cycles in some bacteria, such as Caulobacter, also maintain a strict CRC = 2 (Collier 2012), bacterial cells are generally recognized for their ability to support multiple concurrent replication rounds within the same chromosome (Morigen et al. 2009).In reality, under typical growth conditions the ori/ter ratio in exponentially growing bacterial cells is still 2 (Bird et al. 1972;Bipatnath et al. 1998;Wang et al. 2007;Murray and Errington 2008;Rotman et al. 2009;Stokke et al. 2011), showing that bacterial cells, like eukaryotes, also prefer to deal with a single replication round in their chromosomes. However, due to the peculiarity of the prokaryotic chromosome cycle (Kuzminov 2013), whe...

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“…SeqA's binding is stronger when such sites are hemi-methylated by DNA adenine methylase (Dam), a situation that occurs transiently after these sequences are replicated. The binding of SeqA ''sequesters'' the origin (hence its name) and prevents it from accessing Dam and becoming fully methylated for up to one-third of the cell cycle (Campbell and Kleckner 1990;Lu et al 1994;von Freiesleben et al 2000). This sequestration establishes an ''eclipse'' period, a time at which binding of DnaA and reinitiation is actively prevented.…”

Section: R Eplication Initiation In Bacteria Is Controlled Bymentioning

confidence: 99%

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

Molt

Sutera

Moore³

et al. 2009

Genetics

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The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgETTn5-EZ seqAD, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAD 96-120 , ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAD 96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.

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The eclipse period of Escherichia coli

Cited by 83 publications

References 57 publications

SeqA Protein Stimulates the Relaxing and Decatenating Activities of Topoisomerase IV

SeqA Protein Stimulates the Relaxing and Decatenating Activities of Topoisomerase IV

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

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