The segregation of the Escherichia coli origin and terminus of replication

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.     

Dynamics of Escherichia coli chromosome segregation during multifork replication

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.     

Induced chromosomal exchange directs the segregation of recombinant chromatids in mitosis of Drosophila.

In meiosis, the segregation of chromosomes at the reductional division is accomplished by first linking homologs together. Genetic exchange generates the bivalents that direct regular chromosome segregation. We show that genetic exchange in mitosis also generates bivalents and that these bivalents direct mitotic chromosome segregation. After FLP-mediated homologous recombination in G2 of the cell cycle, recombinant chromatids consistently segregate away from each other (x segregation). This pattern of segregation also applies to exchange between heterologs. Most, or all, cases of non-x segregation are the result of exchange in G1. Cytological evidence is presented that confirms the existence of the bivalents that direct this pattern of segregation. Our results implicate sister chromatid cohesion in maintenance of the bivalent. The pattern of chromatid segregation can be altered by providing an additional FRT at a more proximal site on one chromosome. We propose that sister chromatid exchange occurs at the more proximal site, allowing the recombinant chromatids to segregate together. This also allowed the recovery of reciprocal translocations following FLP-mediated heterologous recombination. The observation that exchange can generate a bivalent in mitotic divisions provides support for a simple evolutionary relationship between mitosis and meiosis.     

Recombination and chromosome segregation

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.     

The chromosome cycle of prokaryotes

In both eukaryotes and prokaryotes, chromosomal DNA undergoes replication, condensation–decondensation and segregation, sequentially, in some fixed order. Other conditions, like sister‐chromatid cohesion (SCC), may span several chromosomal events. One set of these chromosomal transactions within a single cell cycle constitutes the ‘chromosome cycle’. For many years it was generally assumed that the prokaryotic chromosome cycle follows major phases of the eukaryotic one: –replication–condensation–segregation–(cell division)–decondensation–, with SCC of unspecified length. Eventually it became evident that, in contrast to the strictly consecutive chromosome cycle of eukaryotes, all stages of the prokaryotic chromosome cycle run concurrently. Thus, prokaryotes practice ‘progressive’ chromosome segregation separated from replication by a brief SCC, and all three transactions move along the chromosome at the same fast rate. In other words, in addition to replication forks, there are ‘segregation forks’ in prokaryotic chromosomes. Moreover, the bulk of prokaryotic DNA outside the replication–segregation transition stays compacted. I consider possible origins of this concurrent replication–segregation and outline the ‘nucleoid administration’ system that organizes the dynamic part of the prokaryotic chromosome cycle.     

Escherichia coli SeqA Structures Relocalize Abruptly upon Termination of Origin Sequestration during Multifork DNA Replication

The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.     

Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division

Following initiation of chromosomal replication in Escherichia coli, newly initiated origins (oriCs) are prevented from further initiations by a mechanism termed sequestration. During the sequestration period (which lasts about one-third of a cell cycle), the origins remain hemimethylated. The SeqA protein binds hemimethylated oriC in vitro. In vivo, the absence of SeqA causes overinitiation and strongly reduces the duration of hemimethylation. The pattern of immunostained SeqA complexes in vivo suggests that SeqA has a role in organizing hemimethylated DNA at the replication forks. We have examined the effects of overexpressing SeqA under different cellular conditions. Our data demonstrate that excess SeqA significantly increases the time oriC is hemimethylated following initiation of replication. In some cells, sequestration continued for more than one generation and resulted in inhibition of primary initiation. SeqA overproduction also interfered with the segregation of sister nucleoids and caused a delay in cell division. These results suggest that SeqA's function in regulation of replication initiation is linked to chromosome segregation and possibly cell division.     

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.     

What determines whether chromosomes segregate reductionally or equationally in meiosis?

Normal meiosis consists of a single round of DNA replication followed by two nuclear divisions. In the 1st division the chromosomes segregate reductionally whereas in the 2nd division they segregate equationally (as they do in mitosis). In certain yeast mutants, a single-division meiosis takes place, in which some chromosomes segregate reductionally while others divide equationally. This autonomous segregation behaviour of individual chromosomes on a common spindle is determined by the centromeres they carry. The relationship between reductional segregation of a pair of chromosomes and their earlier recombinational history is also discussed.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC /Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 μm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.     

The helix clock: a potential biomechanical cell cycle timer

A model based upon helical geometry that provides cylindrically shaped cells with a means to measure their length during growth and to time cell cycle events is presented. The helix clock arises from the change in pitch angle that accompanies the parallel packing of strands on a cylinder surface. A single strand inserted into the cylinder surface nearly perpendicular to the long axis of the cylinder starts the clock running. As additional strands are inserted parallel to those in place, the pitch angle of all strands must reorient. A limit is reached when all strands lie parallel to the long axis of the cylinder. By sensing either the pitch angle or a physical ramification thereof, cells can measure their length during growth and time events of the cell cycle. The helix clock model is discussed in relationship to the bacterial cell cycle. The idea that bacterial cells use one helix hand for cylinder elongation, the other for septation is presented. The negative twist so generated apparently drives folding in the helical bacterial macrofiber system of Bacillus subtilis.     

Establishment of sister chromatid cohesion at the S. cerevisiae replication fork

Two identical sister copies of eukaryotic chromosomes are synthesized during S phase. To facilitate their recognition as pairs for segregation in mitosis, sister chromatids are held together from their synthesis onward by the chromosomal cohesin complex. Replication fork progression is thought to be coupled to establishment of sister chromatid cohesion, facilitating identification of replication products, but evidence for this has remained circumstantial. Here we show that three proteins required for sister chromatid cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along chromosomes with, replication forks. The ring-shaped cohesin complex is loaded onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. Cohesion establishment during DNA replication follows without further cohesin recruitment and without need for cohesin to re-engage an ATP hydrolysis motif that is critical for its initial DNA binding. This provides evidence for cohesion establishment in the context of replication forks and imposes constraints on the mechanism involved.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

An analysis of the factory model for chromosome replication and segregation in bacteria

Recent advances in microscopy have given us important clues as to the nature of chromosome segregation in bacteria. Most current observations favour the view that the process is co-replicational: DNA replication forks are anchored at the cell centre, and the newly replicated DNA is moved towards the cell poles. This scheme can account for orderly segregation even at high growth rates where multiple replication cycles overlap. We argue that there are five distinct activities directly involved in co-replicational segregation dynamics. These we refer to as Push, Direct, Condense, Hold and Clear. We attempt to assign one of these roles to each protein implicated in chromosome segregation. The proposed process is very different from mitosis in eukaryotic cells and perhaps more closely resembles the formation of separate sister chromatids during DNA replication.     

Regulation of Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli B/r

Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.     

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.