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 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.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Chromosome segregation

The ability to visualise specific genes and proteins within bacterial cells is revolutionising knowledge of chromosome segregation. The essential elements appear to be the driving force behind DNA replication, which occurs at fixed cellular positions, the condensation of newly replicated DNA by a chromosome condensation machine located at the cell 1/4 and 3/4 positions, and molecular machines that act at midcell to allow chromosome separation after replication and movement of the sister chromosomes away from the division septum prior to cell division. This review attempts to provide a perspective on current views of the bacterial chromosome segregation mechanism and how it relates to other cellular processes.     

Re-replication of a Centromere Induces Chromosomal Instability and Aneuploidy

The faithful inheritance of chromosomes during cell division requires their precise replication and segregation. Numerous mechanisms ensure that each of these fundamental cell cycle events is performed with a high degree of fidelity. The fidelity of chromosomal replication is maintained in part by re-replication controls that ensure there are no more than two copies of every genomic segment to distribute to the two daughter cells. This control is enforced by inhibiting replication initiation proteins from reinitiating replication origins within a single cell cycle. Here we show in Saccharomyces cerevisiae that re-replication control is important for the fidelity of chromosome segregation. In particular, we demonstrate that transient re-replication of centromeric DNA due to disruption of re-replication control greatly induces aneuploidy of the re-replicated chromosome. Some of this aneuploidy arises from missegregation of both sister chromatids to one daughter cell. Aneuploidy can also arise from the generation of an extra sister chromatid via homologous recombination, suggesting that centromeric re-replication can trigger breakage and repair events that expand chromosome number without causing chromosomal rearrangements. Thus, we have identified a potential new non-mitotic source of aneuploidy that can arise from a defect in re-replication control. Given the emerging connections between the deregulation of replication initiation proteins and oncogenesis, this finding may be relevant to the aneuploidy that is prevalent in cancer.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

Bacterial DNA segregation: its motors and positional control

A model for DNA segregation in bacteria is proposed which involves not merely growth of the cell membrane and wall, as previously assumed, but also the active movement of one of the two chromosome sister origins by a DNA helicase enzyme and of the chromosome termini and the bulk of the chromosomes by supercoiling tension exerted by DNA gyrase. This provides a unified mechanism for DNA chromosome movement in prosthecate budding bacteria as well as for bacteria that undergo binary fission. The positional control of DNA segregation and the plane of cell division depend, I suggest, on four things: (1) the attachment of the daughter chromosome termini to the cell wall in a position adjacent to the new cell poles at about the time of septation, (2) the displacement of the parental chromosome terminus from this attachment site by the mobile origin, which attaches itself instead to the wall at that point, (3) the movement of the chromosome terminus to a new location in between the daughter origins by the tension of supercoiling, and (4) the determination of the location of the future septum at the position occupied by the chromosome terminus at the time of septal initiation; septum-initiation proteins are postulated to achieve this by binding directly or indirectly to the chromosome terminus. This mechanism automatically ensures ordered DNA segregation in rapidly growing bacteria with more than two sister origins of replication.     

Partitioning of the Escherichia coli chromosome: superhelicity and condensation

Segregation in Escherichia coli, the process of separating the replicated chromosomes into daughter progeny cells, seems to start long before the duplication of the genome reaches completion. Soon after initiation in mid-cell region, the daughter oriCs rapidly move apart to fixed positions inside the cell (quarter length positions from each pole) and are anchored there by yet unknown mechanism(s). As replication proceeds, the rest of the chromosome is sequentially unwound and then refolded. At termination, the two sister chromosomes are unlinked by decatenation and separated by supercoiling and/or condensation. Muk and Seq proteins are involved in different stages of this replication-cum-partition process and thus can be categorized as important partition proteins along with topoisomerases. E. coli strains, lacking mukB or seqA functions, are defective in segregation and cell division. The nucleoids in these mutant strains exhibit altered condensation and superhelicity as can be demonstrated by sedimentation analysis and by fluorescence microscopy. As the supercoiling of an extrachromosomal element (a plasmid DNA) was also influenced by the mukB and seqA mutations we concluded that the MukB and SeqA proteins are possibly involved in maintaining the general supercoiling activity in the cell. The segregation of E. coli chromosome might therefore be predominantly driven by factors that operate by affecting the superhelicity and condensation of the nucleoid (MukB, SeqA, topoisomerases and additional unknown proteins). A picture thus emerges in which replication and partition are no longer compartmentalized into separable stages with clear gaps (S and M phases in eukaryotes) but are parallel processes that proceed concomitantly through a cell cycle continuum.     

Spo13 maintains centromeric cohesion and kinetochore coorientation during meiosis I

BACKGROUND: The meiotic cell cycle, the cell division cycle that leads to the generation of gametes, is unique in that a single DNA replication phase is followed by two chromosome segregation phases. During meiosis I, homologous chromosomes are segregated, and during meiosis II, as in mitosis, sister chromatids are partitioned. For homolog segregation to occur during meiosis I, physical linkages called chiasmata need to form between homologs, sister chromatid cohesion has to be lost in a stepwise manner, and sister kinetochores must attach to microtubules emanating from the same spindle pole (coorientation). RESULTS: Here we show that the meiosis-specific factor Spo13 functions in two key aspects of meiotic chromosome segregation. In cells lacking SPO13, cohesin, which is the protein complex that holds sister chromatids together, is not protected from removal around kinetochores during meiosis I but is instead lost along the entire length of the chromosomes. We furthermore find that Spo13 promotes sister kinetochore coorientation by maintaining the monopolin complex at kinetochores. In the absence of SPO13, Mam1 and Lrs4 disassociate from kinetochores prematurely during pro-metaphase I and metaphase I, resulting in a partial defect in sister kinetochore coorientation in spo13 Delta cells. CONCLUSIONS: Our results indicate that Spo13 has the ability to regulate both the stepwise loss of sister chromatid cohesion and kinetochore coorientation, two essential features of meiotic chromosome segregation.     

Segregation of the replication terminus of the two Vibrio cholerae chromosomes

Genome duplication and segregation normally are completed before cell division in all organisms. The temporal relation of duplication and segregation, however, can vary in bacteria. Chromosomal regions can segregate towards opposite poles as they are replicated or can stay cohered for a considerable period before segregation. The bacterium Vibrio cholerae has two differently sized circular chromosomes, chromosome I (chrI) and chrII, of about 3 and 1 Mbp, respectively. The two chromosomes initiate replication synchronously, and the shorter chrII is expected to complete replication earlier than the longer chrI. A question arises as to whether the segregation of chrII also is completed before that of chrI. We fluorescently labeled the terminus regions of chrI and chrII and followed their movements during the bacterial cell cycle. The chrI terminus behaved similarly to that of the Escherichia coli chromosome in that it segregated at the very end of the cell division cycle: cells showed a single fluorescent focus even when the division septum was nearly complete. In contrast, the single focus representing the chrII terminus could divide at the midcell position well before cell septation was conspicuous. There were also cells where the single focus for chrII lingered at midcell until the end of a division cycle, like the terminus of chrI. The single focus in these cells overlapped with the terminus focus for chrI in all cases. It appears that there could be coordination between the two chromosomes through the replication and/or segregation of the terminus region to ensure their segregation to daughter cells.     

Chromosome strand segregation during sporulation in Bacillus subtilis

After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.     

The Chromosomal Association of the Smc5/6 Complex Depends on Cohesion and Predicts the Level of Sister Chromatid Entanglement

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.     

Replication-related activities establish cohesion between sister chromatids

Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase. The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaroytic cells. Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle. Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage. Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol σ (formerly Trf4p/Pol κ), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment. Here, we review the molecular events in the chromosome cycle with respect to cohesion. Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans.     

Condensin restructures chromosomes in preparation for meiotic divisions

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.     

Diversity and redundancy in bacterial chromosome segregation mechanisms

Bacterial cells are much smaller and have a much simpler overall structure and organization than eukaryotes. Several prominent differences in cell organization are relevant to the mechanisms of chromosome segregation, particularly the lack of an overt chromosome condensation/decondensation cycle and the lack of a microtubule-based spindle. Although bacterial chromosomes have a rather dispersed appearance, they nevertheless have an underlying high level of spatial organization. During the DNA replication cycle, early replicated (oriC) regions are localized towards the cell poles, whereas the late replicated terminus (terC) region is medially located. This spatial organization is thought to be driven by an active segregation mechanism that separates the sister chromosomes continuously as replication proceeds. Comparisons of various well-characterized bacteria suggest that the mechanisms of chromosome segregation are likely to be diverse, and that in many bacteria, multiple overlapping mechanisms may contribute to efficient segregation. One system in which the molecular mechanisms of chromosome segregation are beginning to be elucidated is that of sporulating cells of Bacillus subtilis. The key components of this system have been identified, and their functions are understood, in outline. Although this system appears to be specialized, most of the functions are conserved widely throughout the bacteria.     

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.     

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

Dynein participates in chromosome segregation in fission yeast

Background information. In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi‐orientation). At the metaphase—anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule‐dependent forces. Results. We have studied the role of the minus‐end‐directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle‐assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2‐dependent spindle‐assembly checkpoint. Conclusions. These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi‐orientation during early mitosis.