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1.  Asymmetry of Chromosome Replichores Renders the DNA Translocase Activity of FtsK Essential for Cell Division and Cell Shape Maintenance in Escherichia coli 
PLoS Genetics  2008;4(12):e1000288.
Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.
Author Summary
In most bacteria, chromosomes consist of a single replication unit. Replication initiates at a single ori and terminates in the diametrically opposite zone, ter, defining two replication arms. These are also called replichores to account for base composition and sequence motif polarity biases from ori to ter. This organisation as symmetrical replichores is conserved in bacteria. Here, we have investigated the effect of replichore asymmetry in E. coli. We show that it blocks the cell cycle at a step posterior to chromosome replication. This phenotype is not corrected by restoration of the termination of replication opposite ori, strongly suggesting that it is due to the asymmetry of replichore polarity. FtsK, a DNA-translocase associated with the division septum that processes the terminal junction of replichore polarity, is essential for growth and for the controlled blockage of cell growth in cells with asymmetric replichores. These results reveal a reciprocal control between the processing of the terminal junction of the replichores by FtsK and the progression of the cell cycle.
doi:10.1371/journal.pgen.1000288
PMCID: PMC2585057  PMID: 19057667
2.  Independent Segregation of the Two Arms of the Escherichia coli ori Region Requires neither RNA Synthesis nor MreB Dynamics ▿ § ‡  
Journal of Bacteriology  2010;192(23):6143-6153.
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.
doi:10.1128/JB.00861-10
PMCID: PMC2981198  PMID: 20889756
3.  MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves 
Molecular Microbiology  2007;65(6):1485-1492.
The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.
doi:10.1111/j.1365-2958.2007.05881.x
PMCID: PMC2169520  PMID: 17824928
4.  Independent Positioning and Action of Escherichia coli Replisomes in Live Cells 
Cell  2008;133(1):90-102.
Summary
A prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.
doi:10.1016/j.cell.2008.01.044
PMCID: PMC2288635  PMID: 18394992
DNA; CELLCYCLE; MICROBIO
5.  Effects of quinolones on nucleoid segregation in Escherichia coli. 
Antimicrobial Agents and Chemotherapy  1991;35(12):2645-2648.
The effects of quinolone antibiotics on nucleoid segregation in growing Escherichia coli were examined by using fleroxacin (Ro 23-6240, AM 833) as a prototype compound. At levels that were close to its MIC and induced growth arrest and filamentation, fleroxacin caused large nucleoids to appear in midcell, suggesting inhibition of nucleoid segregation. With increasing fleroxacin concentrations, nucleoids became progressively smaller, suggesting inhibition of DNA replication. Removal of fleroxacin restored normal cell and nucleoid morphology in filaments with large nucleoids but not in filaments with small nucleoids. The results are consistent with inhibition of chromosome decatenation at low quinolone concentrations (bacteriostatic effect) and DNA supercoiling at high concentrations (bactericidal effect).
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PMCID: PMC245448  PMID: 1810201
6.  Chromosome Structuring Limits Genome Plasticity in Escherichia coli 
PLoS Genetics  2007;3(12):e226.
Chromosome organizations of related bacterial genera are well conserved despite a very long divergence period. We have assessed the forces limiting bacterial genome plasticity in Escherichia coli by measuring the respective effect of altering different parameters, including DNA replication, compositional skew of replichores, coordination of gene expression with DNA replication, replication-associated gene dosage, and chromosome organization into macrodomains. Chromosomes were rearranged by large inversions. Changes in the compositional skew of replichores, in the coordination of gene expression with DNA replication or in the replication-associated gene dosage have only a moderate effect on cell physiology because large rearrangements inverting the orientation of several hundred genes inside a replichore are only slightly detrimental. By contrast, changing the balance between the two replication arms has a more drastic effect, and the recombinational rescue of replication forks is required for cell viability when one of the chromosome arms is less than half than the other one. Macrodomain organization also appears to be a major factor restricting chromosome plasticity, and two types of inverted configurations severely affect the cell cycle. First, the disruption of the Ter macrodomain with replication forks merging far from the normal replichore junction provoked chromosome segregation defects. The second major problematic configurations resulted from inversions between Ori and Right macrodomains, which perturb nucleoid distribution and early steps of cytokinesis. Consequences for the control of the bacterial cell cycle and for the evolution of bacterial chromosome configuration are discussed.
Author Summary
Genomic analyses have revealed that bacterial genomes are dynamic entities that evolve through various processes including intrachromosome genetic rearrangements, gene duplication, and gene loss or acquisition by gene transfer. Nevertheless, comparison of bacterial chromosomes from related genera revealed a conservation of genetic organization. Most bacterial genomes are circular molecules, and DNA replication proceeds bidirectionally from a single origin to an opposite region where replication forks meet. The replication process imprints the bacterial chromosome because initiation and termination at defined loci result in strand biases due to the mutational differences occurring during leading and lagging strands synthesis. We analyze the strength of different parameters that may limit genome plasticity. We show that the preferential positioning of essential genes on the leading strand, the proximity of genes involved in transcription and translation to the origin of replication on the leading strand, and the presence of biased motifs along the replichores operate only as long-term positive selection determinants. By contrast, selection operates to maintain replication arms of similar lengths. Finally, we demonstrate that spatial structuring of the chromosome impedes strongly genome plasticity. Genetic evidence supports the presence of two steps in the cell cycle controlled by the spatial organization of the chromosome.
doi:10.1371/journal.pgen.0030226
PMCID: PMC2134941  PMID: 18085828
7.  Chromosome Structuring Limits Genome Plasticity in Escherichia coli 
PLoS Genetics  2007;3(12):e226.
Chromosome organizations of related bacterial genera are well conserved despite a very long divergence period. We have assessed the forces limiting bacterial genome plasticity in Escherichia coli by measuring the respective effect of altering different parameters, including DNA replication, compositional skew of replichores, coordination of gene expression with DNA replication, replication-associated gene dosage, and chromosome organization into macrodomains. Chromosomes were rearranged by large inversions. Changes in the compositional skew of replichores, in the coordination of gene expression with DNA replication or in the replication-associated gene dosage have only a moderate effect on cell physiology because large rearrangements inverting the orientation of several hundred genes inside a replichore are only slightly detrimental. By contrast, changing the balance between the two replication arms has a more drastic effect, and the recombinational rescue of replication forks is required for cell viability when one of the chromosome arms is less than half than the other one. Macrodomain organization also appears to be a major factor restricting chromosome plasticity, and two types of inverted configurations severely affect the cell cycle. First, the disruption of the Ter macrodomain with replication forks merging far from the normal replichore junction provoked chromosome segregation defects. The second major problematic configurations resulted from inversions between Ori and Right macrodomains, which perturb nucleoid distribution and early steps of cytokinesis. Consequences for the control of the bacterial cell cycle and for the evolution of bacterial chromosome configuration are discussed.
Author Summary
Genomic analyses have revealed that bacterial genomes are dynamic entities that evolve through various processes including intrachromosome genetic rearrangements, gene duplication, and gene loss or acquisition by gene transfer. Nevertheless, comparison of bacterial chromosomes from related genera revealed a conservation of genetic organization. Most bacterial genomes are circular molecules, and DNA replication proceeds bidirectionally from a single origin to an opposite region where replication forks meet. The replication process imprints the bacterial chromosome because initiation and termination at defined loci result in strand biases due to the mutational differences occurring during leading and lagging strands synthesis. We analyze the strength of different parameters that may limit genome plasticity. We show that the preferential positioning of essential genes on the leading strand, the proximity of genes involved in transcription and translation to the origin of replication on the leading strand, and the presence of biased motifs along the replichores operate only as long-term positive selection determinants. By contrast, selection operates to maintain replication arms of similar lengths. Finally, we demonstrate that spatial structuring of the chromosome impedes strongly genome plasticity. Genetic evidence supports the presence of two steps in the cell cycle controlled by the spatial organization of the chromosome.
doi:10.1371/journal.pgen.0030226
PMCID: PMC2134941  PMID: 18085828
8.  Chromosome and Replisome Dynamics in E. coli: Loss of Sister Cohesion Triggers Global Chromosome Movement and Mediates Chromosome Segregation 
Cell  2005;121(6):899-911.
Summary
Chromosome and replisome dynamics were examined in synchronized E. coli cells undergoing a eukaryotic-like cell cycle. Sister chromosomes remain tightly colocalized for much of S phase and then separate, in a single coordinate transition. Origin and terminus regions behave differently, as functionally independent domains. During separation, sister loci move far apart and the nucleoid becomes bilobed. Origins and terminus regions also move. We infer that sisters are initially linked and that loss of cohesion triggers global chromosome reorganization. This reorganization creates the 2-fold symmetric, ter-in/ori-out conformation which, for E. coli, comprises sister segregation. Analogies with eukaryotic prometaphase suggest that this could be a primordial segregation mechanism to which microtubule-based processes were later added. We see no long-lived replication “factory”; replication initiation timing does not covary with cell mass, and we identify changes in nucleoid position and state that are tightly linked to cell division. We propose that cell division licenses the next round of replication initiation via these changes.
doi:10.1016/j.cell.2005.04.013
PMCID: PMC2973560  PMID: 15960977
9.  Sequence Learning Under Uncertainty in Children: Self-Reflection vs. Self-Assertion 
We know that stochastic feedback impairs children’s associative stimulus–response (S–R) learning (Crone et al., 2004a; Eppinger et al., 2009), but the impact of stochastic feedback on sequence learning that involves deductive reasoning has not been not tested so far. In the current study, 8- to 11-year-old children (N = 171) learned a sequence of four left and right button presses, LLRR, RRLL, LRLR, RLRL, LRRL, and RLLR, which needed to be deduced from feedback because no directional cues were given. One group of children experienced consistent feedback only (deterministic feedback, 100% correct). In this condition, green feedback on the screen indicated that the children had been right when they were right, and red feedback indicated that the children had been wrong when they were wrong. Another group of children experienced inconsistent feedback (stochastic feedback, 85% correct, 15% false), where in some trials, green feedback on the screen could signal that children were right when in fact they were wrong, and red feedback could indicate that they were wrong when in fact they had been right. Independently of age, children’s sequence learning in the stochastic condition was initially much lower than in the deterministic condition, but increased gradually and improved with practice. Responses toward positive vs. negative feedback varied with age. Children were increasingly able to understand that they could have been wrong when feedback indicated they were right (self-reflection), but they remained unable to understand that they could have been right when feedback indicated they were wrong (self-assertion).
doi:10.3389/fpsyg.2012.00127
PMCID: PMC3342618  PMID: 22563324
sequence learning; learning under uncertainty; stochastic feedback; positive and negative feedback
10.  Positioning of replicated chromosomes in Escherichia coli. 
Journal of Bacteriology  1990;172(1):31-39.
The positioning of replicated chromosomes at one-fourth and three-fourths of the cell length was inhibited when protein synthesis was inhibited by chloramphenicol or rifampin or by starvation for amino acids. Under these conditions, the progress of chromosome replication continued and replicated chromosomes were located close to each other as one nucleoid mass at midcell. Cells which already had two separate daughter chromosomes located at the cell quarters divided into two daughter cells under these conditions. When protein synthesis resumed, daughter chromosomes moved from midcell to the cell quarters, respectively, before any detectable increase in cell length was observed. The chromosome positioning occurred even under inhibition of the initiation of chromosome replication and under inactivation of DNA gyrase. The chromosome positioning presumably requires new synthesis of a particular protein(s) or translation itself.
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PMCID: PMC208397  PMID: 2152907
11.  Cytokinesis in Bacteria 
Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.
doi:10.1128/MMBR.67.1.52-65.2003
PMCID: PMC150516  PMID: 12626683
12.  A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli 
Journal of Bacteriology  2014;196(1):36-49.
Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.
doi:10.1128/JB.01230-12
PMCID: PMC3911132  PMID: 24142249
13.  Regulation of Sister Chromosome Cohesion by the Replication Fork Tracking Protein SeqA 
PLoS Genetics  2013;9(8):e1003673.
Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.
Author Summary
Sister chromosome cohesion in eukaryotes maintains genome stability by mediating chromosome segregation and homologous recombination-dependent DNA repair. Here we have investigated the mechanism of cohesion regulation in E. coli by measuring cohesion timing in a broad set of candidate mutant strains. Using a sensitive DNA replication and segregation assay, we show that cohesion is controlled by the conserved DNA decatenation enzyme Topo IV and the abundant DNA binding protein SeqA. Results suggest that cohesion occurs in E. coli by twisting of replicated duplexes around each other behind the replication fork, and immediate resolution of cohered regions is blocked by SeqA. SeqA binds to a sliding 300–400 kb window of hemimethylated DNA behind the fork, and regions binding more SeqA experience longer cohesion periods. An analogous decatenation inhibition function is carried out by the cohesin complex in eukaryotes, indicating that cells mediate pairing and separation of replicated DNA by a conserved mechanism. In both cases, mismanaged cohesion results in failed or inefficient chromosome segregation.
doi:10.1371/journal.pgen.1003673
PMCID: PMC3749930  PMID: 23990792
14.  Type I elements mediate replication fork pausing at conserved upstream sites in the Tetrahymena thermophila ribosomal DNA minichromosome. 
Molecular and Cellular Biology  1997;17(8):4517-4525.
Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome. During vegetative growth, the rDNA is replicated exclusively from origins in the 5' nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5' NTS is not. Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5' NTS. Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required. A conserved tripartite sequence was identified at each pausing site. Mutations in type I elements diminish pausing at proximal RFPs. Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks. Studies with rDNA transformants revealed a strong directional bias for fork pausing. Strong pausing only occurred in forks moving toward the rRNA-coding region. We propose that fork pausing in the 5' NTS evolved to synchronize replication and transcription of the downstream rRNA genes.
PMCID: PMC232305  PMID: 9234709
15.  Inititation and termination of chromosome replication in Escherichia coli subjected to amino acid starvation. 
Journal of Bacteriology  1980;142(1):236-242.
Initiation and termination of chromosome replication in an Escherichia coli auxotroph subjected to amino acid starvation were examined by following the incorporation of [3H]thymidine into the EcoRI restriction fragments of the chromosome. The pattern of incorporation observed upon restoration of the amino acid showed that starvation blocks the process of initiation prior to deoxyribonucleic acid synthesis within any significant portion of the EcoRI fragment which contains the origin of replication, oriC. In this experiment, no incorporation of [3H]thymidine into EcoRI fragments from the terminus of replication was observed, nor was it found when a dnaC initiation mutant was used to prevent incorporation at the origin which might have obscured labeling of terminus fragments. Thus amino acid starvation does not appear to block replication forks shortly before termination of replication. Attempted synchronization of replication initiation by including a period of thymine starvation subsequent to the amino acid starvation led to simultaneous incorporation of [3H]-thymidine into all EcoRI fragments within the 240-kilobase region that surrounds oriC. It is shown that the thymine starvation step allowed initiation and a variable, but limited, amount of replication to occur.
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PMCID: PMC293937  PMID: 6246063
16.  FtsZ Placement in Nucleoid-Free Bacteria 
PLoS ONE  2014;9(3):e91984.
We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement.
doi:10.1371/journal.pone.0091984
PMCID: PMC3956765  PMID: 24638110
17.  Changes in the Min Oscillation Pattern before and after Cell Birth▿ #  
Journal of Bacteriology  2010;192(16):4134-4142.
The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.
doi:10.1128/JB.00364-10
PMCID: PMC2916429  PMID: 20543068
18.  Host DNA replication forks are not preferred targets for bacteriophage Mu transposition. 
Journal of Bacteriology  1985;163(1):282-290.
Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.
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PMCID: PMC219110  PMID: 3159718
19.  Inhibition and restart of initiation of chromosome replication: effects on exponentially growing Escherichia coli cells. 
Journal of Bacteriology  1995;177(7):1670-1682.
Escherichia coli strains in which initiation of chromosome replication could be specifically blocked while other cellular processes continued uninhibited were constructed. Inhibition of replication resulted in a reduced growth rate and in inhibition of cell division after a time period roughly corresponding to the sum of the lengths of the C and D periods. The division inhibition was not mediated by the SOS regulon. The cells became elongated, and a majority contained a centrally located nucleoid with a fully replicated chromosome. The replication block was reversible, and restart of chromosome replication allowed cell division and rapid growth to resume after a time delay. After the resumption, the septum positions were nonrandomly distributed along the length axis of the cells, and a majority of the divisions resulted in at least one newborn cell of normal size and DNA content. With a transient temperature shift, a single synchronous round of chromosome replication and cell division could be induced in the population, making the constructed system useful for studies of cell cycle-specific events. The coordination between chromosome replication, nucleoid segregation, and cell division in E. coli is discussed.
PMCID: PMC176792  PMID: 7896687
20.  Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication▿  
Journal of Bacteriology  2007;189(23):8660-8666.
Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.
doi:10.1128/JB.01212-07
PMCID: PMC2168957  PMID: 17905986
21.  Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex 
The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.
doi:10.1128/MMBR.69.3.501-526.2005
PMCID: PMC1197808  PMID: 16148308
22.  Behavior of Sister Copies of Mini-F Plasmid after Synchronized Plasmid Replication in Escherichia coli Cells 
Journal of Bacteriology  2002;184(11):3142-3145.
To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.
doi:10.1128/JB.184.11.3142-3145.2002
PMCID: PMC135069  PMID: 12003959
23.  Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements 
Molecular cell  2010;39(4):595-605.
Summary
Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes.
Using genomic and mechanistic studies we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S-phase and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs.
We propose that in eukaryotes replication fork barriers, Rrm3 and Top2 coordinate replication fork progression and fusion at termination regions thus counteracting abnormal genomic transitions.
doi:10.1016/j.molcel.2010.07.024
PMCID: PMC3041477  PMID: 20797631
Replication termination; Top2; Rrm3; Replication pausing
24.  The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication 
Molecular microbiology  2009;74(2):467-479.
Summary
Replication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.
doi:10.1111/j.1365-2958.2009.06877.x
PMCID: PMC2967437  PMID: 19737351
25.  On the Mutational Topology of the Bacterial Genome 
G3: Genes|Genomes|Genetics  2013;3(3):399-407.
By sequencing the genomes of 34 mutation accumulation lines of a mismatch-repair defective strain of Escherichia coli that had undergone a total of 12,750 generations, we identified 1625 spontaneous base-pair substitutions spread across the E. coli genome. These mutations are not distributed at random but, instead, fall into a wave-like spatial pattern that is repeated almost exactly in mirror image in the two separately replicated halves of the bacterial chromosome. The pattern is correlated to genomic features, with mutation densities greatest in regions predicted to have high superhelicity. Superimposed upon this pattern are regional hotspots, some of which are located where replication forks may collide or be blocked. These results suggest that, as they traverse the chromosome, the two replication forks encounter parallel structural features that change the fidelity of DNA replication.
doi:10.1534/g3.112.005355
PMCID: PMC3583449  PMID: 23450823
mutation rate; evolution; replication fidelity; chromosome structure; DNA polymerase errors

Results 1-25 (306946)