Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2. Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1. We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G 1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.

Naphthoquinones are among the most active natural products obtained from plants and microorganisms. Naphthoquinones exert their biological activities through pleiotropic mechanisms that include reactivity against cell nucleophiles, generation of reactive oxygen species (ROS), and inhibition of proteins. Here, we report a mechanistic antiproliferative study performed in the yeast Saccharomyces cerevisiae for several derivatives of three important natural naphthoquinones: lawsone, juglone, and β-lapachone. We have found that (i) the free hydroxyl group of lawsone and juglone modulates toxicity; (ii) lawsone and juglone derivatives differ in their mechanisms of action, with ROS generation being more important for the former; and (iii) a subset of derivatives possess the capability to disrupt mitochondrial function, with β-lapachones being the most potent compounds in this respect. In addition, we have cross-compared yeast results with antibacterial and antitumor activities. We discuss the relationship between the mechanistic findings, the antiproliferative activities, and the physicochemical properties of the naphthoquinones.

The ribosomal DNA (rDNA) array of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct presentations that arise as spatial adaptations to changes in the nuclear morphology triggered during mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of nuclear components yet colocalizes with the vacuole. We show that the rDNA-associated nuclear envelope (NE) often reshapes into a ladle to accommodate the vacuole in the SUL, with the nucleus becoming bilobed and doughnut-shaped. Finally, we demonstrate that the formation of the rDNA loop and the SUL require TORC1, membrane synthesis and functional vacuoles, yet is independent of nucleus–vacuole junctions and rDNA-NE tethering.

Background Chronic obstructive pulmonary disease (COPD) has been proposed as a disease of accelerated aging. Several cross-sectional studies have related a shorter telomere length (TL), a marker of biological aging, with COPD outcomes. Whether accelerated telomere shortening over time relates to worse outcomes in COPD patients, is not known. Methods Relative telomere length (T/S) was determined by qPCR in DNA samples from peripheral blood in 263 patients at baseline and up to 10 years post enrolment. Yearly clinical and lung function data of 134 patients with at least two-time measures of T/S over this time were included in the analysis. Results At baseline, T/S inversely correlated with age (r = − 0.236; p < 0.001), but there was no relationship between T/S and clinical and lung function variables (p > 0.05). Over 10 years of observation, there was a median shortening of TL of 183 bp/year for COPD patients. After adjusting for age, gender, active smoking and mean T/S, patients that shortened their telomeres the most over time, had worse gas exchange, more lung hyperinflation and extrapulmonary affection during the follow-up, (PaO2 p < 0.0001; KCO p = 0.042; IC/TLC p < 0.0001; 6MWD p = 0.004 and BODE index p = 0.009). Patients in the lowest tertile of T/S through the follow-up period had an increased risk of death [HR = 5.48, (1.23–24.42) p = 0.026]. Conclusions This prospective study shows an association between accelerated telomere shortening and progressive worsening of pulmonary gas exchange, lung hyperinflation and extrapulmonary affection in COPD patients. Moreover, persistently shorter telomeres over this observation time increase the risk for all-cause mortality.

It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double‐strand breaks (DSBs), likely to find a donor sequence for homology‐directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and even anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses and GFP-tagged DNA damage markers suggest that non-recombinational modifications of late replication intermediates may account for the shift in the PFGE behavior. The fact that this shift does not trigger G2/M checkpoints further supports this statement since checkpoints are active for other replicative stresses in the absence of Top2. We propose that the prolonged absence of Top2 activity leads to a general chromosome structural change. This change might interlock chromatids together with catenations and thus contribute to the formation of anaphase bridges in top2 mutants.