Study of the cellular response, signaling, and repair pathways to confront DNA double-strand breaks in telophase.

Cell survival depends on the genome integrity throughout the cell cycle and includes the faithful segregation of chromosomes in anaphase. DNA is continuously compromised by both exogenous and endogenous sources, such as ionizing radiation or reactive oxygen species (ROS) from the internal metabolism, respectively. In the last instance, insults to the integrity of the DNA can happen. DNA double-strand breaks (DSBs) are among the most harmful lesions that cells have to face. DNA breakage can result in severe mutations and promote genomic instability, leading to cancer, senescence, or cell death. However, cells have developed different repair pathways to confront DSBs. DSB repair mechanisms can be classified into non-homologous end joining (NHEJ) and homologous recombination (HR). The NHEJ pathway is triggered during the G1 phase, which is characterized by the absence of a sister chromatid to act as an intact DNA template for repair and the low CDK/cyclin activity. On the other hand, HR is used from the S phase onwards, when CDK/cyclin levels rise, and chromosomes comprise two sister chromatids. NHEJ entails an error-prone mechanism since broken DNA ends are barely processed and directly re-joined, causing short insertions and deletions at the flanking sites of the DSB. On the contrary, HR is an error-free repair pathway because the broken DNA is restored by copying the nucleotide information from the homolog intact template, usually the intact sister chromatid. However, how cells deal with DSBs at late anaphase/telophase is still unknown. These latest stages of the cell cycle are paradoxical scenarios. First, CDK/cyclin levels are still high, and this would promote HR-mediated repair. Nonetheless, sister chromatids have been previously segregated in anaphase, supporting a more important role for NHEJ since the intact template is not close and well-aligned to be invaded. In this work, the budding yeast Saccharomyces cerevisiae has been employed as a model to determine the cellular response and the repair pathways triggered to face DSBs in telophase. Cdc15-2 conditional mutants have been used to generate stable telophase blocks where to generate single and multiple DSBs. Fluorescence microscopy analysis has uncovered: i) the approximation of the segregated DNA material, ii) the acceleration of chromosome movement, iii) the structural and dynamics changes produced in the microtubules apparatus, and iv) the generation of coalescence events between segregated sister chromatid loci. Also, cells delay the telophase-to-G1 transition in a Rad9-dependent manner, and the partial dephosphorylation of the Cin8 kinesin motor protein is relevant to promote the reversion of segregation. The molecular monitorization of a single DSB repair also showed that cells favor HR over NHEJ in a Rad9-, Mre11-, and Yku70-independent but cohesin-dependent manner. Additionally, an experimental approximation on the HeLa cells response has also been performed. Cells delayed cytokinesis after being confronted with phleomycin-mediated DSBs at late anaphase/telophase stages. Contrary to what happened in yeast, HeLa cells did not modify the microtubular morphology. Instead, they responded to DSBs by phosphorylating the histone variant gH2A.X. Strikingly, 53BP1, RIF1, and RPA2 foci appeared simultaneously. Although previous works have described that 53BP1 and RIF1 promote NHEJ and counteract resection, telophase-damaged cells showed a pattern where both NHEJ and HR pathways seem to cooperate

Genome-scale genetic interactions and cell imaging confirm cytokinesis as deleterious to transient topoisomerase II deficiency in <i>Saccharomyces cerevisiae</i>

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

Yeast cells can partially revert chromosome segregation to repair late DNA double-strand breaks through homologous recombination

DNA repair in late mitosis sets paradoxical scenarios. Cyclin-dependent kinase (CDK) activity is high, which favors homologous recombination (HR), despite a sister chromatid is not physically close to recombine with. We have found that DNA double-strand breaks partially revert chromosome segregation to find an intact template and repair through HR

A Yeast Mitotic Tale for the Nucleus and the Vacuoles to Embrace

The morphology of the nucleus is roughly spherical in most eukaryotic cells. However, this organelle shape needs to change as the cell travels through narrow intercellular spaces during cell migration and during cell division in organisms that undergo closed mitosis, i.e., without dismantling the nuclear envelope, such as yeast. In addition, the nuclear morphology is often modified under stress and in pathological conditions, being a hallmark of cancer and senescent cells. Thus, understanding nuclear morphological dynamics is of uttermost importance, as pathways and proteins involved in nuclear shaping can be targeted in anticancer, antiaging, and antifungal therapies. Here, we review how and why the nuclear shape changes during mitotic blocks in yeast, introducing novel data that associate these changes with both the nucleolus and the vacuole. Altogether, these findings suggest a close relationship between the nucleolar domain of the nucleus and the autophagic organelle, which we also discuss here. Encouragingly, recent evidence in tumor cell lines has linked aberrant nuclear morphology to defects in lysosomal function

Topoisomerase II deficiency leads to a postreplicative structural shift in all <i>Saccharomyces cerevisiae</i> chromosomes

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 G(2)/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis

Lawsone, Juglone, and β‑Lapachone Derivatives with Enhanced Mitochondrial-Based Toxicity

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 <i>Saccharomyces cerevisiae</i> 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