scholarly journals A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae

2016 ◽  
Vol 2 (4) ◽  
pp. e1501914 ◽  
Author(s):  
Marie-Eve Val ◽  
Martial Marbouty ◽  
Francisco de Lemos Martins ◽  
Sean P. Kennedy ◽  
Harry Kemble ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Vibrio Cholerae ◽  
Control Mechanism ◽  
Bacterial Species ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Checkpoint Control ◽  
Initiation Mechanism ◽  
Functional Interactions ◽  
Circular Chromosomes

Bacteria with multiple chromosomes represent up to 10% of all bacterial species. Unlike eukaryotes, these bacteria use chromosome-specific initiators for their replication. In all cases investigated, the machineries for secondary chromosome replication initiation are of plasmid origin. One of the important differences between plasmids and chromosomes is that the latter replicate during a defined period of the cell cycle, ensuring a single round of replication per cell. Vibrio cholerae carries two circular chromosomes, Chr1 and Chr2, which are replicated in a well-orchestrated manner with the cell cycle and coordinated in such a way that replication termination occurs at the same time. However, the mechanism coordinating this synchrony remains speculative. We investigated this mechanism and revealed that initiation of Chr2 replication is triggered by the replication of a 150-bp locus positioned on Chr1, called crtS. This crtS replication–mediated Chr2 replication initiation mechanism explains how the two chromosomes communicate to coordinate their replication. Our study reveals a new checkpoint control mechanism in bacteria, and highlights possible functional interactions mediated by contacts between two chromosomes, an unprecedented observation in bacteria.

scholarly journals Random versus Cell Cycle-Regulated Replication Initiation in Bacteria: Insights from Studying Vibrio cholerae Chromosome 2

2016 ◽  
Vol 81 (1) ◽  
Author(s):  
Revathy Ramachandran ◽  
Jyoti Jha ◽  
Johan Paulsson ◽  
Dhruba Chattoraj
Keyword(s):  
Cell Cycle ◽  
Vibrio Cholerae ◽  
Fixed Time ◽  
Replication Initiation ◽  
Chromosome 1 ◽  
Control Mechanisms ◽  
Chromosome 2 ◽  
Content Type ◽  
Bacterial Chromosomes ◽  
The Difference

SUMMARY Bacterial chromosomes initiate replication at a fixed time in the cell cycle, whereas there is generally no particular time for plasmid replication initiation or chromosomal replication initiation from integrated plasmids. In bacteria with divided genomes, the replication system of one of the chromosomes typically resembles that of bacteria with undivided genomes, whereas the remaining chromosomes have plasmid-like replication systems. For example, in Vibrio cholerae, a bacterium with two chromosomes (chromosome 1 [Chr1] and Chr2), the Chr1 system resembles that of the Escherichia coli chromosome, and the Chr2 system resembles that of iteron-based plasmids. However, Chr2 still initiates replication at a fixed time in the cell cycle and thus offers an opportunity to understand the molecular basis for the difference between random and cell cycle-regulated modes of replication. Here we review studies of replication control in Chr2 and compare it to those of plasmids and chromosomes. We argue that although the Chr2 control mechanisms in many ways are reminiscent of those of plasmids, they also appear to combine more regulatory features than are found on a typical plasmid, including some that are more typical of chromosomes. One of the regulatory mechanisms is especially novel, the coordinated timing of replication initiation of Chr1 and Chr2, providing the first example of communication between chromosomes for replication initiation.

scholarly journals Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Godefroid Charbon ◽  
Belén Mendoza-Chamizo ◽  
Christopher Campion ◽  
Xiaobo Li ◽  
Peter Ruhdal Jensen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Conditions ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Atp Depletion ◽  
Initiator Protein ◽  
Cycle Arrest ◽  
A Cell

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

scholarly journals Two different cell-cycle processes determine the timing of cell division in Escherichia coli

2021 ◽  
Author(s):  
Alexandra Colin ◽  
Gabriele Micali ◽  
Louis Faure ◽  
Marco Cosentino Lagomarsino ◽  
Sven van Teeffelen
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Division ◽  
Single Cells ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Cell Width ◽  
Division Process ◽  
Balanced Contributions ◽  
Independent Process

AbstractCells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting 21 for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

scholarly journals Functions of Fission Yeast Orp2 in DNA Replication and Checkpoint Control

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 599-607
Author(s):  
Joan Kiely ◽  
S B Haase ◽  
Paul Russell ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Cycle Arrest

Abstract orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

scholarly journals Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA

2021 ◽  
Vol 12 ◽  
Author(s):  
Julia E. Grimwade ◽  
Alan C. Leonard
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Chromosome Replication ◽  
Integration Host Factor ◽  
Replication Initiation ◽  
Critical Event ◽  
E Coli ◽  
Chromosome Duplication ◽  
Recognition Sites ◽  
Transcriptional Modulators

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

scholarly journals High Osmolarity Modulates Bacterial Cell Size through Reducing Initiation Volume inEscherichia coli

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Xiongfeng Dai ◽  
Manlu Zhu
Keyword(s):  
Cell Cycle ◽  
Water Content ◽  
Cell Size ◽  
Bacterial Cell ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Hyperosmotic Stress ◽  
Content Type ◽  
High Osmolarity

ABSTRACTBacterial cell size is closely associated with biomass growth and cell cycle progression, including chromosome replication and cell division. It is generally proposed thatEscherichia colicells tightly control the timing of chromosome replication through maintaining a constant cell volume per origin upon initiating chromosome replication (constant initiation volume) under various growth conditions. Here, we quantitatively characterize the cell size and cell cycle ofEscherichia colicells growing exponentially under hyperosmotic stress, which is a common environmental stressor that profoundly affects the bacterial water content. The bacterial cell size is reduced by hyperosmotic stress, even though the C and D periods are remarkably prolonged, indicating a significantly reduced initiation volume. The reduced initiation volume originates from the higher concentration of DnaA initiator protein caused by water loss at high osmolarity. Our study shows suggests a fundamental role of water content in regulating bacterial cell size and has also revealed a new role of the DnaA protein in regulating the chromosome replication elongation beyond regulating the replication initiation process.IMPORTANCEBacterial cell size depends on growth rate, cell cycle progression, and the cell volume per origin upon initiating chromosome replication (initiation volume). Here, we perform the first systematic and quantitative study of the effect of hyperosmotic stress on theE. colicell size and cell cycle. We find that hyperosmotic stress significantly reduces the initiation volume. The reduced initiation volume is attributed to the increased DnaA concentration caused by water loss at high osmolarity, indicating a fundamental role of water content in cell size and cell cycle regulation.

scholarly journals Two different cell-cycle processes determine the timing of cell division in Escherichia coli

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexandra Colin ◽  
Gabriele Micali ◽  
Louis Faure ◽  
Marco Cosentino Lagomarsino ◽  
Sven van Teeffelen
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Division ◽  
Single Cells ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Cell Width ◽  
Division Process ◽  
Balanced Contributions ◽  
Independent Process

Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

scholarly journals Effects of the Chromosome Partitioning Protein Spo0J (ParB) on oriC Positioning and Replication Initiation in Bacillus subtilis

2003 ◽  
Vol 185 (4) ◽  
pp. 1326-1337 ◽  
Author(s):  
Philina S. Lee ◽  
Daniel Chi-Hong Lin ◽  
Shigeki Moriya ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Significant Proportion ◽  
Subcellular Location ◽  
Replication Initiation ◽  
Null Mutant ◽  
Protein Ratio ◽  
Chromosome Partitioning

ABSTRACT Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

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