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The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness.

The transactive response DNA-binding protein (TARDBP/TDP-43) influences the processing of diverse transcripts, including that of histone deacetylase 6 (HDAC6). Here, we assessed TDP-43 activity in terms of regulating CD4+ T-cell permissivity to HIV-1 infection. We observed that overexpression of wt-TDP-43 increased both mRNA and protein levels of HDAC6, resulting in impaired HIV-1 infection independently of the viral envelope glycoprotein complex (Env) tropism. Consistently, using an HIV-1 Env-mediated cell-to-cell fusion model, the overexpression of TDP-43 levels negatively affected viral Env fusion capacity. Silencing of endogenous TDP-43 significantly decreased HDAC6 levels and increased the fusogenic and infection activities of the HIV-1 Env. Using pseudovirus bearing primary viral Envs from HIV-1 individuals, overexpression of wt-TDP-43 strongly reduced the infection activity of Envs from viremic non-progressors (VNP) and rapid progressors (RP) patients down to the levels of the inefficient HIV-1 Envs observed in long-term non-progressor elite controllers (LTNP-EC). On the contrary, silencing endogenous TDP-43 significantly favored the infectivity of primary Envs from VNP and RP individuals, and notably increased the infection of those from LTNP-EC. Taken together, our results indicate that TDP-43 shapes cell permissivity to HIV-1 infection, affecting viral Env fusion and infection capacities by altering the HDAC6 levels and associated tubulin-deacetylase anti-HIV-1 activity. This work is supported by the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” RD12/0017/0002, RD12/0017/0028, RD12/0017/0034, RD16/0025/0011, RDCIII16/0002/0005 and RD16/0025/0041 as part of the Plan Nacional R + D+I and co-funded by the Spanish “Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER)”. J.B. is a researcher from “Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol” supported by the Health Department of the Catalonian Government/Generalitat de Catalunya and ISCIII grant numbers PI17/01318 and PI20/00093 (to J.B.). Work in CC Lab was supported by grants SAF (2010-17226) and (2016-77894-R) from MINECO (Spain), FIS (PI 13/02269, ISCIII) and PI20/00093. Work in CF Lab was supported by the Cabildo Insular de Tenerife (grants CGIEU0000219140 and “Apuestas científicas del ITER para colaborar en la lucha contra la COVID-19”); the agreement with the Instituto Tecnológico y de Energías Renovables (ITER) to strengthen scientific and technological education, training research, development and innovation in Genomics, Personalized Medicine and Biotechnology (grant number OA17/008). A.V.-F.’s Lab is supported by the European Regional Development Fund (ERDF), RTI2018-093747-B-100 (“Ministerio de Ciencia e Innovación”, Spain), “Ministerio de Ciencia, Innovación y Universidades” (Spain), ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund), UNLL10-3E-783 (ERDF and “Fundación CajaCanarias”) and “SEGAI-ULL”. S.P-Y is funded by “Fundación Doctor Manuel Morales” (La Palma, Spain) and “Contrato Predoctoral Ministerio-ULL Formación de Doctores” (2019 Program) (“Ministerio de Ciencia, Innovación y Universidades”, Spain). R.C.-R. is funded by RD16/0025/0011 and ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund). J.G.-L. is funded by the “Juan de la Cierva de Incorporación” Spanish Program (IJC2019-038902-I) (“Ayudas Juan de la Cierva de incorporación; Agencia Estatal de Investigación. Ministerio de Ciencia e Innovación”). Sí

The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

Condensin plays a central role in the organisation of chromosomes by compacting chromatin into loops during mitosis. Condensin achieves this through a loop extrusion mechanism that remains poorly understood. To identify the molecular steps of yeast condensin during loop formation, we used optical tweezers with fluorescence detection. We find that single yeast condensin complexes use ATP to extrude DNA through distinct 15 nm steps, thus advancing ~45 base pairs (bp) per step. Under increasing load, the condensin step size remains constant while step-dwell times increase, and stalls at forces >1 pN. We also show that nucleosome arrays hinder processive condensin extrusion and demonstrate that the histone chaperone FACT is required for compaction of nucleosomal arrays by condensin. Importantly, FACT-assisted compaction on nucleosomes also occurs through distinct 15 nm steps. Finally, we show that FACT is required for correct condensin localisation in vivo. Our results establish that loop extrusion by yeast condensin involves a 45 bp stroke that requires FACT for condensin function on chromatin.

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role in determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids are maintained during anaphase in the absence of cohesin. Here we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, TEV-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wildtype cells and show that they play a role in the organisation of centromeric regions. Our data demonstrate that in yeast cells, cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.One sentence summaryCohesin complexes on yeast chromosomes provide organisation to segregating chromatids.

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.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT (‘facilitates chromatin transcription’) in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding., The work in the L.A. laboratory was supported by a Wellcome Trust Senior Investigator award to L.A. (100955, ‘Functional dissection of mitotic chromatin’) and the London Institute of Medical Research (LMS), which receives its core funding from the UK Medical Research Council. This research was further supported by funding from The European Research Council (R.K.), Agence Nationale pour la Recherche (R.K.) and the the Spanish Ministerio de Economía, Industria y Competitividad (BFU2017-89622-P; F.A.).

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.

Homologous recombination (HR) is a preferred mechanism to deal with DNA replication impairments. However, HR synapsis gives rise to joint molecules (JMs) between the nascent sister chromatids, challenging chromosome segregation in anaphase. Joint molecules are resolved by the actions of several structure-selective endonucleases (SSEs), helicases and topoisomerases. Previously, we showed that yeast double mutants for the Mus81-Mms4 and Yen1 SSEs lead to anaphase bridges (ABs) after replication stress. Here, we have studied the role of the Mph1 helicase in preventing these anaphase aberrations. Mph1, the yeast ortholog of Fanconi anaemia protein M (FANCM), is involved in the removal of the D-loop, the first JM to arise in canonical HR. Surprisingly, the absence of Mph1 alone did not increase ABs, rather, it blocked cells in G2. Interestingly, in the search for genetic interactions with functionally related helicases and translocases, we found additive effects on the G2 block and post-G2 aberrations between mph1&Delta, and knockout mutants for Srs2, Rad54 and Rad5. Based on these interactions, we suggest that Mph1 acts coordinately with these helicases in the non-canonical HR-driven fork regression mechanism to bypass stalled replication forks.

Cycling events in nature start and end to restart again and again. In the cell cycle, whose purpose is to become two where there was only one, cyclin-dependent kinases (CDKs) are the beginning and, therefore, phosphatases must play a role in the ending. Since CDKs are drivers of the cell cycle and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only master cell cycle phosphatase in the model yeast Saccharomyces cerevisiae, Cdc14. Transient inactivation is expected to better mimic the pharmacological action of drugs. Interestingly, we have found that yeast cells tolerate badly a relatively brief inactivation of Cdc14 when cells are already committed into anaphase, the first cell cycle stage where this phosphatase plays important roles. First, we noticed that the segregation of distal regions in the chromosome arm that carries the ribosomal DNA array was irreversibly impaired, leading to an anaphase bridge (AB). Next, we found that this AB could eventually be severed by cytokinesis and led to two different types of genetically compromised daughter cells. All these previous studies were done in haploid cells. We have now recently expanded this analysis to diploid cells and used the advantage of making hybrid diploids to study chromosome rearrangements and changes in the ploidy of the surviving progeny. We have found that the consequences for the genome integrity were far more dramatic than originally envisioned.

β-Lapachone (β-lap) is a promising antitumour drug currently undergoing clinical trials. Although it is known that β-lap generates reactive oxygen species (ROS), its actual mechanism of action is still controversial. Especially important is to determine whether concomitant DNA or microtubule damage is the key target of its antitumour properties and whether DNA damage is mediated by topoisomerases as previously suggested. Here, we have searched for determinants of β-lap cytotoxicity in the model organism Saccharomyces cerevisiae through a mechanism-driven approach whereby several pathways of the DNA and microtubule integrity responses, as well as the anti-oxidant response, were downregulated and the outcome of β-lap treatment examined. We also included in the analysis several β-lap derivatives expected to modify drug bioavailability and activity. We found that neither topoisomerase II nor microtubules contributed to yeast sensitivity to β-lap and its equitoxic derivative 3-bromo-β-lapachone. Instead, we found that oxidative and related environmental stresses were primarily responsible for toxicity. Accordingly, Yap1, the central transcription factor in the antioxidant response in yeast, together with several components involved in stress tolerance (i.e., Snf1 and Hog1) and chromatin remodelling (i.e., the SWR1 and RSC complexes), played major roles in protection against β-lapachone. Critically, we show that dioxygen enhanced toxicity and that ROS scavengers protected cells from it. Furthermore, we show that both quinones resulted in cell death in a manner which cytologically resembled apoptosis/necrosis. We thus conclude that β-lap is toxic to yeast through massive ROS production that either directly kills the cells or else triggers programmed cell death.

Downregulation of separase, condensin, Smc5/6, topoisomerase II and Cdc14 in Saccharomyces cerevisiae yields anaphase bridges formed by unresolved sister chromatids (SCBs). Here we report that the overlapping actions of the structure-selective endonucleases (SSEs) Mus81-Mms4/EME1 and Yen1/GEN1, but not Slx1-Slx4, are also essential to prevent the formation of spontaneous SCBs that depend on the homologous recombination pathway. We further show that the frequency of SCBs is boosted after mild replication stress and that they contain joint molecules enriched in non-canonical forms of the Holliday junction (HJ), including nicked-HJ (nHJ). We show that SCBs are mostly reversible upon activation of either Mus81-Mms4 or Yen1 in late anaphase, which is concomitant with the disappearance of non-canonical HJs and restoration of viable progeny. On the basis of these findings, we propose a model where unresolved recombination intermediates are a source of mitotic SCBs, and Mus81-Mms4 and Yen1 play a central role in their resolution in vivo.

The only canonical Holliday junction (HJ) resolvase identified in eukaryotes thus far is Yen1/GEN1. Nevertheless, Yen1/GEN1 appears to have a minor role in HJ resolution, and, instead, other structure-specific endonucleases (SSE) that recognize branched DNA play the leading roles, Mus81-Mms4/EME1 being the most important in budding yeast. Interestingly, cells tightly regulate the activity of each HJ resolvase during the yeast cell cycle. Thus, Mus81-Mms4 is activated in G2/M, while Yen1 gets activated shortly afterwards. Nevertheless, cytological studies have shown that Yen1 is sequestered out of the nucleus when cyclin-dependent kinase activity is high, i.e., all of the cell cycle but G1. We here show that the mitotic master phosphatase Cdc14 targets Yen1 to the nucleus in early anaphase through the FEAR network. We will further show that this FEAR-mediated Cdc14-driven event is sufficient to back-up Mus81-Mms4 in removing branched DNA structures, which are especially found in the long chromosome arms upon replication stress. Finally, we found that MEN-driven Cdc14 re-activation in late anaphase is essential to keep Yen1 in the nucleus until the next G1. Our results highlight the essential role that early-activated Cdc14, i.e., through the FEAR network, has in removing all kind of non-proteinaceous linkages that preclude faithful sister chromatid segregation in anaphase. In addition, our results support the general idea of Yen1 acting as a last resource endonuclease to deal with any remaining HJ that might compromise genetic stability during chromosome segregation. © 2014 Landes Bioscience., This work was supported by Instituto de Salud Carlos III (PS09/00106 and PS12/00280), Fundación Canaria de Investigación y Salud (08/42 to J.G.L.) and Spanish Ministry of Education (FPU fellowship AP2009-2511 to J.G.L.). All these programs were co-financed with the European Commission's ERDF structural funds.

β-Lapachone (β-lap) is a promising antitumoral agent. DNA base oxidation and alkylation are among the expected damages by β-lap. Herein, we have explored the role that the homologous recombination pathway (HR), a critical DNA repair process in Saccharomyces cerevisiae, has in the cytotoxic profile of β-lap. We have further compared β-lap to the closely related compound menadione and the well-known alkylating agent methyl methanesulfonate (MMS). Surprisingly, we found that β-lap does not trigger HR, as seen for (i) the mutant sensitivity profiles, (ii) concentration-dependent arrest profiles, (iii) absence of nuclear DNA repair factories, and (iv) frequency of recombination between direct repeats.

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction., Author Summary When cells divide they must segregate copies of their chromosomes to each of their daughters. A particular harmful situation arises when those copies are glued to each other (i.e., nondisjunction) at the moment of division. Previously, it has been possible to genetically favour this scenario, yet it has been difficult to limit the extent of nondisjunction to a single chromosome. We have developed and studied a yeast model where we control nondisjunction of one of its sixteen chromosomes. We show that dividing cells manage to complete nuclear and cell fission and therefore break that chromosome. We further show that new daughter cells then trigger a DNA damage response, yet only after they initiate a new round of replication. Remarkably, an uncommon repair strategy seems to be used to deal with this damage, which involves part of the homologous recombination machinery (i.e., RPA complex and Rad52) but lacks its primary sensor Mre11. Importantly though, both daughter cells arrest their cell cycle in G2 to prevent further damage from occurring. After a while, the cell that still carries an entire copy of the chromosome often survives, leading to aberrant forms of the chromosome in the progeny.