Biology subjects

Ayra-Plasencia, J.

Publications and source records attributed to Ayra-Plasencia, J..

5 recordsLinked to original sources

bioRxiv2023

Msc1 is a nuclear envelope protein that reinforces DNA repair in late mitosis.

Precise double-strand break (DSB) repair is paramount for genome stability. Homologous recombination (HR) repairs DSBs when cyclin-dependent kinase (CDK) activity is high, which correlates with the availability of the sister chromatid as a template. However, anaphase and telophase are paradoxical scenarios since high CDK favors HR despite sister chromatids being no longer aligned. To identify factors specifically involved in DSB repair in late mitosis, we have undertaken comparative proteomics in Saccharomyces cerevisiae and found that Msc1, a poorly characterized nuclear envelope protein, is significantly enriched upon both random and guided DSBs. We further show that {Delta}msc1 is more sensitive to DSBs in late mitosis, and has a delayed repair of DBSs, as indicated by increased Rad53 hyperphosphorylation, fewer Rad52 repair factories, and slower HR completion. We discuss how Msc1 may favor the formation of Rad52 factories and the timely completion of HR before cytokinesis.

molecular biology↗

bioRxiv2021

Nuclear envelope reshaping around the vacuole determines the morphology of the ribosomal DNA

The ribosomal DNA array (rDNA) 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 morphologies that arise as spatial adaptations to changes in the nuclear morphology triggered during the 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 any nuclear component yet colocalizes with the vacuole. We finally show that the formation and maintenance of the rDNA loop and the SUL require TORC1 and membrane synthesis. We propose that the rDNA-associated nuclear envelope (NE) reshapes into a loop to accommodate the vacuole, with the nucleus becoming bilobed.

cell biology↗

bioRxiv2020

Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae 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 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.

molecular biology↗

bioRxiv2018

DNA damage in telophase leads to coalescence between segregated sister chromatid loci.

The generation of DNA double strand breaks (DSBs) pose a high risk for the maintenance of the genome. Cells repair DSBs through two major mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is usually preferred when a sister chromatid is available, thus cells have coupled the activity of the cycling dependent kinase (CDK) to the selection of HR (Symington et al. 2014). Paradoxically, there is a window in the cell cycle where CDK is high despite a sister chromatid is not physically available for HR: late anaphase/telophase. We have here studied in budding yeast the response to DSBs generated in telophase by means of the radiomimetic drug phleomycin. We first show that phleomycin treatment activates the DNA damage response and leads to a delay in the telophase-to-G1 transition. Outstandingly, we also found a partial reversion of sister chromatid segregation, which includes approximation of spindle pole bodies (SPBs) and sister centromeres, de novo formation of anaphase bridges, trafficking of DNA back and forth through the cytokinetic plane and events of coalescence between segregated sister telomeres. We importantly show that phleomycin promotes a massive change in the structure and dynamic of mitotic microtubules (MTs), which coincides with dephosphorylation and re-localization of kinesin-5 Cin8. We propose that anaphase is not entirely irreversible and that there could still be a window to repair DSBs using the sister chromatid after segregation.

cell biology↗

bioRxiv2017

Genome-scale genetic interactions position the Mitotic Exit Network as a major antagonist of transient Topoisomerase II deficiency.

Topoisomerase II (Top2) is the essential protein that resolves DNA catenations. When the 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, tumours become resistant to these drugs by downregulating Top2. Here, we have compared two isogenic yeast 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. We found that top2-5 transits through anaphase faster than top2-4. In order to define the biological importance of this difference, we performed genome-scale Synthetic Gene Array (SGA) analyses during chronic sublethal Top2 downregulation and acute, yet transient, Top2 inactivation. We find that downregulation of cell cycle progression, especially the Mitotic Exit Network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5, and this correlated with destabilization of anaphase bridges by execution of MEN. We suggest that mitotic exit may be a therapeutic target to hypersensitize cancer cells carrying downregulating mutations in TOP2.

genetics↗