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Sister chromatids are tethered together by the cohesin complex from the time they are made until their separation at anaphase. The ability of cohesin to tether sister chromatids together depends on acetylation of its Smc3 subunit by members of the Eco1 family of cohesin acetyltransferases. Vertebrates express two orthologs of Eco1, called Esco1 and Esco2, both of which are capable of modifying Smc3, but their relative contributions to sister chromatid cohesion are unknown. We therefore set out to determine the precise contributions of Esco1 and Esco2 to cohesion in vertebrate cells. Herewe show that cohesion establishment is critically dependent upon Esco2. Although most Smc3 acetylation is Esco1 dependent, inactivation of the ESCO1 gene has little effect on mitotic cohesion. The unique ability of Esco2 to promote cohesion is mediated by sequences in the N terminus of the protein. We propose that Esco1-dependent modification of Smc3 regulates almost exclusively the noncohesive activities of cohesin, such as DNA repair, transcriptional control, chromosome loop formation, and/or stabilization. Collectively, our data indicate that Esco1 and Esco2 contribute to distinct and separable activities of cohesin in vertebrate cells.

Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2′-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis. It remains impossible using conventional Hi-C to differentiate interactions between and along sister chromatids. SisterC relies on selective destruction of nascent DNA and in combination with Hi-C offers a means to study intra- and inter-sister interactions independently.

The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S‐phase can persist into mitosis, where they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this ‘unfinished S‐phase business’ can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis. Furthermore, we discuss the potential ways in which cells might not only tolerate this inevitable aspect of chromosome biology, but also exploit it to assist in the maintenance of genome stability. Hickson and colleagues review emerging concepts on how replication problems are frequently processed only in subsequent cell‐cycle stages, and postulate that cells may even exploit this to ensure genome stability maintenance during cell division.

In most organisms, meiotic chromosome segregation is dependent on crossovers (COs), which enable pairs of homologous chromosomes to segregate to opposite poles at meiosis I. In mammals, the majority of meiotic chromosome segregation errors result from a lack of COs between homologs. Observations in Homo sapiens and Drosophila melanogaster have revealed a second class of exceptional events in which a CO occurred near the centromere of the missegregated chromosome. We show that in wild-type strains of Saccharomyces cerevisiae, most spore inviability is due to precocious separation of sister chromatids (PSSC) and that PSSC is often associated with centromere-proximal crossing over. COs, as opposed to nonreciprocal recombination events (NCOs), are preferentially associated with missegregation. Strains mutant for the RecQ homolog, SGS1, display reduced spore viability and increased crossing over. Much of the spore inviability in sgs1 results from PSSC, and these events are often associated with centromere-proximal COs, just as in wild type. When crossing over in sgs1 is reduced by the introduction of a nonnull allele of SPO11, spore viability is improved, suggesting that the increased PSSC is due to increased crossing over. We present a model for PSSC in which a centromere-proximal CO promotes local loss of sister-chromatid cohesion.

The replicated copies of each chromosome, the sister chromatids, are attached prior to their segregation in mitosis and meiosis. This association or cohesion is critical for each sister chromatid to bind to microtubules from opposite spindle poles and thus segregate away from each other at anaphase of mitosis or meiosis II. The cohesin protein complex is essential for cohesion in both mitosis and meiosis, and cleavage of one of the subunits is sufficient for loss of cohesion at anaphase. The localization of the cohesin complex and other cohesion proteins permits evaluation of the positions of sister-chromatid associations within the chromosome structure, as well as the relationship between cohesion and condensation. Recently, two key riddles in the mechanism of meiotic chromosome segregation have yielded to molecular answers. First, analysis of the cohesin complex in meiosis provides molecular support for the long-standing hypothesis that sister-chromatid cohesion links homologs in meiosis I by stabilizing chiasmata. Second, the isolation of the monopolin protein that controls kinetochore behavior in meiosis I defines a functional basis by which sister kinetochores are directed toward the same pole in meiosis I.

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.

Key Points The polo-like kinases 1–5 (PLK1–5), and aurora kinases, aurora kinase A, aurora kinase B and aurora kinase C have essential roles during cell division, and a large number of inhibitors for these kinases are currently being evaluated as anticancer drugs in Phase I and Phase II clinical trials. Information on the selectivity of these compounds in vivo is limited, but it is likely that off-target effects within the same kinase families will affect efficacy and toxicity profiles. The efficacy of polo-like kinase and aurora kinase inhibitors probably primarily depends on their capacity to severely disrupt normal cell division, implying a limited tumour specificity of these types of drugs. It is unclear whether PLK1 and the aurora kinases have oncogenic or tumour suppressor activity when deregulated. However, the increased cancer incidence in heterozygous mice suggests a role for them as tumour suppressors, probably through the prevention of chromosome instability. PLK1 and the aurora kinases co-regulate multiple processes in the dividing cell, such as entry into mitosis, mitotic spindle formation, sister chromatid resolution, chromosome–spindle connections and cytokinesis. Although some overlap in clinical performance can be expected of PLK1 and aurora kinase inhibitors, these kinases can also antagonize one another. Consequently, the outcome of selective PLK1, aurora kinase A or aurora kinase B inhibition, compared with combined inhibition, will certainly be different. Several treatment approaches have been proposed, which consist of PLK1, aurora kinase A or aurora kinase B inhibition combined with conventional chemotherapeutic agents that might enhance the efficacy and specificity of these drugs. The polo-like kinases and aurora kinases have various roles in mitosis, and inhibitors of these kinases are being tested in clinical trials. Recent data have shown that extensive crosstalk exists between the polo-like kinase and aurora kinase pathways, which might affect the efficacy of these drugs. This Review discusses the biology of these kinases and how drugs that inhibit them might be used in the clinic. Large numbers of inhibitors for polo-like kinases and aurora kinases are currently being evaluated as anticancer drugs. Interest in these drugs is fuelled by the idea that these kinases have unique functions in mitosis. Within the polo-like kinase family, the emphasis for targeted therapies has been on polo-like kinase 1 (PLK1), and in the aurora kinase family drugs have been developed to specifically target aurora kinase A (AURKA; also known as STK6) and/or aurora kinase B (AURKB; also known as STK12). Information on the selectivity of these compounds in vivo is limited, but it is likely that off-target effects within the same kinase families will affect efficacy and toxicity profiles. In addition, it is becoming clear that interplay between polo-like kinases and aurora kinases is much more extensive than initially anticipated, and that both kinase families are important factors in the response to classical chemotherapeutics that damage the genome or the mitotic spindle. In this Review we discuss the implications of these novel insights on the clinical applicability of polo-like kinase and aurora kinase inhibitors.

During cell division, mitotic chromosomes assemble and are equally distributed into two new daughter cells. The chromosome organisation of the two chromatids is essential for even distribution of genetic materials. Although the 11-nm fibre or nucleosome structure is well-understood as a fundamental fibrous structure of chromosomes, the reports on organisation of 30-nm basic chromatin fibres have been controversial, with debates on the contribution of 30-nm or thicker fibres to the higher order inner structure of chromosomes. Here, we used focused ion beam/scanning electron microscopy (FIB/SEM) to show that both 11-nm and 30-nm fibres are present in the human metaphase chromosome, although the higher-order periodical structure could not be detected under the conditions employed. We directly dissected the chromosome every 10-nm and observed 224 cross-section SEM images. We demonstrated that the chromosome consisted of chromatin fibres of an average diameter of 16.9-nm. The majority of the chromatin fibres had diameters between 5 and 25-nm, while those with 30-nm were in the minority. The reduced packaging ratio of the chromatin fibres was detected at axial regions of each chromatid. Our results provide a strong basis for further discussions on the chromosome higher-order structure.

Mitosis is a highly dynamic process, aimed at separating identical copies of genomic material into two daughter cells. A failure of the mitotic process generates cells that carry abnormal chromosome numbers. Such cells are predisposed to become tumorigenic upon continuous cell division and thus need to be removed from the population to avoid cancer formation. Cells that fail in mitotic progression indeed activate cell death or cell cycle arrest pathways; however, these mechanisms are not well understood. Growing evidence suggests that the formation of de novo DNA damage during and after mitotic failure is one of the causal factors that initiate those pathways. Here, we analyze several distinct malfunctions during mitosis and cytokinesis that lead to de novo DNA damage generation.