Artículos (Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER))

URI permanente para esta colecciónhttps://hdl.handle.net/11441/11090

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A Rfa1-MN-based system reveals new factors involved in the rescue of broken replication forks
(Public Library of Science, 2025-04-01) Amiama Roig, Ana; Barrientos Moreno, Marta; Cruz Zambrano, Esther; López Ruiz, Luz M.; González Prieto, Román; Ríos Orelogio, Gabriel; Prado, Félix; Universidad de Sevilla. Departamento de Biología Celular; Ministerio de Ciencia e Innovación (MICIN). España
The integrity of the replication forks is essential for an accurate and timely completion of genome duplication. However, little is known about how cells deal with broken replication forks. We have generated in yeast a system based on a chimera of the largest subunit of the ssDNA binding complex RPA fused to the micrococcal nuclease (Rfa1-MN) to induce double-strand breaks (DSBs) at replication forks and searched for mutants affected in their repair. Our results show that the core homologous recombination (HR) proteins involved in the formation of the ssDNA/Rad51 filament are essential for the repair of DSBs at forks, whereas non-homologous end joining plays no role. Apart from the endonucleases Mus81 and Yen1, the repair process employs fork-associated HR factors, break-induced replication (BIR)-associated factors and replisome components involved in sister chromatid cohesion and fork stability, pointing to replication fork restart by BIR followed by fork restoration. Notably, we also found factors controlling the length of G1, suggesting that a minimal number of active origins facilitates the repair by converging forks. Our study has also revealed a requirement for checkpoint functions, including the synthesis of Dun1-mediated dNTPs. Finally, our screening revealed minimal impact from the loss of chromatin factors, suggesting that the partially disassembled nucleosome structure at the replication fork facilitates the accessibility of the repair machinery. In conclusion, this study provides an overview of the factors and mechanisms that cooperate to repair broken forks.
Radiotherapy Resistance Driven by Asparagine Endopeptidase through ATR Pathway Modulation in Breast Cancer
(BioMed Central, 2025) Morillo Huesca, Macarena; G. López Cepero, Ignacio; Conesa Bakkali, Ryan; Tomé, Mercedes; Watts, Colin; Huertas Sánchez, Pablo; Moreno Bueno, Gema; Durán, Raúl V.; Martínez Fábregas, Jonathan; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Genética; European Union (UE). H2020; Ministerio de Ciencia e Innovación (MICIN). España; Universidad de Sevilla; Instituto de Salud Carlos III
Background: Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear. Methods: High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify protein and mRNA levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings. Results: Here we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients resulted in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients. Conclusion: The newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.
Alpha-catenin-dependent recruitment of the centrosomal protein cap350 to adherens junctions allows epithelial cells to acquire a columnar shape
(Public Library Science, 2015-03-12) Gavilán Dorronzoro, María de la Paz; Arjona, Marina; Zurbano, Ángel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.; Universidad de Sevilla. Departamento de Citología e Histología Normal y Patológica; Junta de Andalucía; Gobierno de España
Abstract Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskele ton. How this complex process is controlled at the molecular level is still largely unknown. Here, wereport that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of AP350with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking downjunction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity wasnotaffected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350mayactasanadaptorbetweenadherensjunctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.
The Integrated Genomic Surveillance System of Andalusia (SIEGA) Provides a One Health Regional Resource Connected with the Clinic
(Nature Portfolio, 2024) Casimiro-Soriguer Camacho, Inés; Pérez Florido, J.; Robles, E. A.; Lara, M.; Aguado, A.; Iglesias, M.A.; Lepe Jiménez, José Antonio; García, F.; Pérez Alegre, M.; Andújar, E.; Pérez de Camino Cantos, Dolores; Dopazo, J.; Universidad de Sevilla. Departamento de Microbiología; Instituto de Salud Carlos III; Junta de Andalucía; European Union (UE). H2020; Ministerio de Ciencia e Innovación (MICIN). España
The One Health approach, recognizing the interconnectedness of human, animal, and environmental health, has gained signifcance amid emerging zoonotic diseases and antibiotic resistance concerns. This paper aims to demonstrate the utility of a collaborative tool, the SIEGA, for monitoring infectious diseases across domains, fostering a comprehensive understanding of disease dynamics and risk factors, highlighting the pivotal role of One Health surveillance systems. Raw whole-genome sequencing is processed through diferent species-specifc open software that additionally reports the presence of genes associated to anti-microbial resistances and virulence. The SIEGA application is a Laboratory Information Management System, that allows customizing reports, detect transmission chains, and promptly alert on alarming genetic similarities. The SIEGA initiative has successfully accumulated a comprehensive collection of more than 1900 bacterial genomes, including Salmonella enterica, Listeria monocytogenes, Campylobacter jejuni, Escherichia coli, Yersinia enterocolitica and Legionella pneumophila, showcasing its potential in monitoring pathogen transmission, resistance patterns, and virulence factors. SIEGA enables customizable reports and prompt detection of transmission chains, highlighting its contribution to enhancing vigilance and response capabilities. Here we show the potential of genomics in One Health surveillance when supported by an appropriate bioinformatic tool. By facilitating precise disease control strategies and antimicrobial resistance management, SIEGA enhances global health security and reduces the burden of infectious diseases. The integration of health data from humans, animals, and the environment, coupled with advanced genomics, underscores the importance of a holistic One Health approach in mitigating health threats.
LRH-1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype
(Wiley, 2024-12-19) Cobo Vuilleumier, Nadia; Rodríguez Fernández, Silvia; López Noriega, Livia; Lorenzo, Petra I.; Franco, Jaime M.; Lachaud, Christian Claude; Dorronsoro, Akaitz; González Prieto, Román; Gauthier, Benoit R.; Instituto de Salud Carlos III; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Junta de Andalucía; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
Background The complex aetiology of type 1 diabetes (T1D), characterised by a detrimental cross-talk between the immune system and insulin-producing beta cells, has hindered the development of effective disease-modifying therapies. The discovery that the pharmacological activation of LRH-1/NR5A2 can reverse hyperglycaemia in mouse models of T1D by attenuating the autoimmune attack coupled to beta cell survival/regeneration prompted us to investigate whether immune tolerisation could be translated to individuals with T1D by LRH-1/NR5A2 activation and improve islet survival. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from individuals with and without T1D and derived into various immune cells, including macrophages and dendritic cells. Cell subpopulations were then treated or not with BL001, a pharmacological agonist of LRH-1/NR5A2, and processed for: (1) Cell surface marker profiling, (2) cytokine secretome profiling, (3) autologous T-cell proliferation, (4) RNAseq and (5) proteomic analysis. BL001-target gene expression levels were confirmed by quantitative PCR. Mitochondrial function was evaluated through the measurement of oxygen consumption rate using a Seahorse XF analyser. Co-cultures of PBMCs and iPSCs-derived islet organoids were performed to assess the impact of BL001 on beta cell viability. Results LRH-1/NR5A2 activation induced a genetic and immunometabolic reprogramming of T1D immune cells, marked by reduced pro-inflammatory markers and cytokine secretion, along with enhanced mitohormesis in pro-inflammatory M1 macrophages and mitochondrial turnover in mature dendritic cells. These changes induced a shift from a pro-inflammatory to an anti-inflammatory/tolerogenic state, resulting in the inhibition of CD4+ and CD8+ T-cell proliferation. BL001 treatment also increased CD4+/CD25+/FoxP3+ regulatory T-cells and Th2 cells within PBMCs while decreasing CD8+ T-cell proliferation. Additionally, BL001 alleviated PBMC-induced apoptosis and maintained insulin expression in human iPSC-derived islet organoids. Conclusion These findings demonstrate the potential of LRH-1/NR5A2 activation to modulate immune responses and support beta cell viability in T1D, suggesting a new therapeutic approach.
Non-Canonical, Extralysosomal Activities of Lysosomal Peptidases in Physiological and Pathological Conditions: New Clinical Opportunities for Cancer Therapy
(Multidisciplinary Digital Publishing Institute (MDPI), 2025-01-07) Conesa Bakkali, Ryan; Morillo Huesca, Macarena; Martínez Fábregas, Jonathan; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Junta de Andalucía; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; Universidad de Sevilla
Lysosomes are subcellular compartments characterised by an acidic pH, containing an ample variety of acid hydrolases involved in the recycling of biopolymers. Among these hydrolases, lysosomal proteases have merely been considered as end-destination proteases responsible for the digestion of waste proteins, trafficked to the lysosomal compartment through autophagy and endocytosis. However, recent reports have started to unravel specific roles for these proteases in the regulation of initially unexpected biological processes, both under physiological and pathological conditions. Furthermore, some lysosomal proteases are no longer restricted to the lysosomal compartment, as more novel non-canonical, extralysosomal targets are being identified. Currently, lysosomal proteases are accepted to play key functions in the extracellular milieu, attached to the plasma membrane and even in the cytosolic and nuclear compartments of the cell. Under physiological conditions, lysosomal proteases, through non-canonical, extralysosomal activities, have been linked to cell differentiation, regulation of gene expression, and cell division. Under pathological conditions, these proteases have been linked to cancer, mostly through their extralysosomal activities in the cytosol and nuclei of cells. In this review, we aim to provide a comprehensive summary of our current knowledge about the extralysosomal, non-canonical functions of lysosomal proteases, both under physiological and pathological conditions, with a particular interest in cancer, that could potentially offer new opportunities for clinical intervention.
Histone post-translational modifications — cause and consequence of genome function
(Springer Nature, 2022-03-25) Millán Zambrano, Gonzalo; Burton, Adam; Bannister, Andrew J.; Schneider, Robert; Universidad de Sevilla. Departamento de Genética; German Research Foundation (DFG); Cancer Research. United Kingdom; Wellcome Trust; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE)
Much has been learned since the early 1960s about histone post-translational modifications (PTMs) and how they affect DNA-templated processes at the molecular level. This understanding has been bolstered in the past decade by the identification of new types of histone PTM, the advent of new genome-wide mapping approaches and methods to deposit or remove PTMs in a locally and temporally controlled manner. Now, with the availability of vast amounts of data across various biological systems, the functional role of PTMs in important processes (such as transcription, recombination, replication, DNA repair and the modulation of genomic architecture) is slowly emerging. This Review explores the contribution of histone PTMs to the regulation of genome function by discussing when these modifications play a causative (or instructive) role in DNA-templated processes and when they are deposited as a consequence of such processes, to reinforce and record the event. Important advances in the field showing that histone PTMs can exert both direct and indirect effects on genome function are also presented.
BRCA2 Prevents R-loop Accumulation and Associates with TREX-2 mRNA Export Factor PCID2
(Springer Nature, 2014) Bhatia, Vaibhav; Barroso Ceballos, Sonia Inés; García Rubio, María Luisa; Tumini, Emanuela; Herrera Moyano, Emilia; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de Andalucía
Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA-RNA hybrids and a displaced single-stranded DNA. Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.
Coordinated Control of Replication and Transcription by a SAPK Protects Genomic Integrity
(Springer Nature, 2013) Duch, Alba; Felipe Abrio, Irene; Barroso Ceballos, Sonia Inés; Yaakov, Gilad; García Rubio, María Luisa; Aguilera López, Andrés; Nadal, Eulàlia de; Posas, Francesc; Universidad de Sevilla. Departamento de Genética; Gobierno de España
Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1 3A) do not delay replication upon stress and show a marked increase in transcription- associated recombination, genomic instability and Rad52 foci. In contrast, mrc1 3A induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.
The molecular chaperone ALYREF promotes R-loop resolution and maintains genome stability
(American Society for Biochemistry and Molecular Biology Inc., 2024-12) Bhandari, Jay; Guillén Mendoza, Cristina; Banks, Kathryn; Eliaz, Lillian; Southwell, Sierra; Eyaa, Darriel; Luna Varo, Rosa María; Aguilera López, Andrés; Xue, Xiaoyu; Universidad de Sevilla. Departamento de Genética; Texas State University; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC)
Unscheduled R-loops usually cause DNA damage and replication stress, and are therefore a major threat to genome stability. Several RNA processing factors, including the conserved THO complex and its associated RNA and DNA-RNA helicase UAP56, prevent R-loop accumulation in cells. Here, we investigate the function of ALYREF, an RNA export adapter associated with UAP56 and the THO complex, in R-loop regulation. We demonstrate that purified ALYREF promotes UAP56-mediated R-loop dissociation in vitro, and this stimulation is dependent on its interaction with UAP56 and R-loops. Importantly, we show that ALYREF binds DNA-RNA hybrids and R-loops. Consistently, ALYREF depletion causes R-loop accumulation and R-loop–mediated genome instability in cells. We propose that ALYREF, apart from its known role in RNA metabolism and export, is a key cellular R-loop coregulator, which binds R-loops and stimulates UAP56-driven resolution of unscheduled R-loops during transcription.
Preparation of a radiobiology beam line at the 18 MeV proton cyclotron facility at CNA
(Elsevier, 2020-06) Baratto Roldán, Anna; Jiménez Ramos, María del Carmen; Jimeno González, Sonia; Huertas Sánchez, Pablo; García López, Francisco Javier; Gallardo Fuentes, María Isabel; Cortés Giraldo, Miguel Antonio; Espino Navas, José Manuel; Universidad de Sevilla. Departamento de Economía Aplicada II; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Física Atómica, Molecular y Nuclear; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Proton therapy has gained interest in recent years due to its excellent clinical outcomes. However, the lack of accurate biological data, especially in the Bragg peak region of clinical beams, makes it difficult to implement biophysically optimized treatment plans in clinical practice. In this context, low energy proton accelerator facilities provide the perfect environment to collect good radiobiological data, as they can produce high LET beams with narrow energy distributions. This study presents the radiobiology beam line that has been designed at the 18 MeV proton cyclotron facility at the National Centre of Accelerators (CNA, Seville, Spain), to perform irradiations of mono-layer cell cultures. To ensure that all the cells receive the same dose with a suitable dose rate, low beam intensities and broad and homogeneous beam profiles are necessary. To do so, at the CNA an unfocused beam has been used, broadened with a 500 μm thick aluminium scattering foil. Homogeneous dose profiles, with deviations lower than 10% have been obtained over a circular surface of 35 mm diameter for an incident average energy of 12.8 MeV. Further, a Monte Carlo simulation of the beam line has been developed with Geant4, and benchmarked towards experimental measurements, with differences generally below 1%. Once validated, the code has been used, together with an ionization chamber, for dosimetry studies, to characterize the beam and monitor the dose. Finally, cultures of Human Bone Osteosarcoma cells (U2OS) have been successfully irradiated at the radiobiology beam line, investigating the effects of radiation in terms of DNA damage induction.
Differential Effect of the Overexpression of Rad2/XPG Family Endonucleases on Genome Integrity in Yeast and Human Cells
(Elsevier, 2017) Jimeno González, Sonia; Herrera Moyano, Emilia; Ortega Moreno, Pedro; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España
Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.
Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation
(Elsevier, 2018-07-01) López-López, Manuel; López-Cornejo, María del Pilar; Martín, Victoria I.; Ostos Marcos, Francisco José; Checa Rodríguez, Cintia; Prados Carvajal, Rosario; Lebrón Romero, José Antonio; Huertas Sánchez, Pablo; Moyá Morán, María Luisa; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Biología Celular; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Universidad de Sevilla; Ministerio de Economía y Competitividad (MINECO). España
The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency
The nucleolus: Coordinating Stress Response and Genomic Stability
(Elsevier, 2024) González Arzola, Katiuska; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
The perception that the nucleoli are merely the organelles where ribosome biogenesis occurs is challenged. Only around 30 % of nucleolar proteins are solely involved in producing ribosomes. Instead, the nucleolus plays a critical role in controlling protein trafficking during stress and, according to its dynamic nature, undergoes continuous protein exchange with nucleoplasm under various cellular stressors. Hence, the concept of nucleolar stress has evolved as cellular insults that disrupt the structure and function of the nucleolus. Considering the emerging role of this organelle in DNA repair and the fact that rDNAs are the most fragile genomic loci, therapies targeting the nucleoli are increasingly being developed. Besides, drugs that target ribosome synthesis and induce nucleolar stress can be used in cancer therapy. In contrast, agents that regulate nucleolar activity may be a potential treatment for neurodegeneration caused by abnormal protein accumulation in the nucleolus. Here, I explore the roles of nucleoli beyond their ribosomal functions, highlighting the factors triggering nucleolar stress and their impact on genomic stability.
Inadequate control of thyroid hormones sensitizes to hepatocarcinogenesis and unhealthy aging
(Impact Journals LLC, 2019-09) López Noriega, Livia; Capilla González, Vivian; Cobo Vuilleumier, Nadia; Martin Vázquez, Eugenia; Lorenzo, Petra I.; Martínez Force, Enrique; Soriano Navarro, Mario; García Fernández, María; Romero Zerbo, Silvana Y.; Díaz Contreras, Irene; Gauthier, Benoit R.; Martín Montalvo, Alejandro; Universidad de Sevilla. Departamento de Biología Celular
An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals.
Human DNA Topoisomerase I Poisoning Causes R Loop-mediated Genome Instability Attenuated by Transcription Factor IIS
(American Association for the Advancement of Science, 2024) Duardo, Renée C.; Marinello, Jessica; Russo, Marco; Morelli, Sara; Pepe, Simona; Guerra, Federico; Gómez González, Belén; Aguilera López, Andrés; Capranico, Giovanni; Universidad de Sevilla. Departamento de Genética; Associazione italiana per la Ricerca sul cancro (AIRC). Italy; Ministry of University and Research. Rome, Italy; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); European Union (UE)
DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.
Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells
(Public Library of Science, 2024-09-10) Gaillard, Hélène; Ciudad, Toni; Aguilera López, Andrés; Wellinger, Ralf Erik; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Junta de Andalucía; Universidad de Sevilla; Ministerio de Economía y Competitividad (MINECO). España
The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.
Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast
(Elsevier, 2023-06) Williams, Jonathan D.; Zhu, Demi; García Rubio, María Luisa; Shaltz, Samantha; Aguilera López, Andrés; Jinks-Robertson, Sue; Universidad de Sevilla. Departamento de Genética; National Institutes of Health. United States; Ministerio de Ciencia e Innovación (MICIN). España
Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.
PLK1 Regulates CtIP and DNA2 Interplay in Long-range DNA end Resection.
(Cold Spring Harbor Laboratory Press, 2023) Ceppi, Ilaria; Cannavo, Elda; Bret, Helene; Camarillo Daza, María Rosa; Vivalda, Francesca; Thakur, Roshan Singh; Romero Franco, Amador; Sartori, Alessandro A.; Huertas Sánchez, Pablo; Guerois, Raphael; Cejka, Petr; Universidad de Sevilla. Departamento de Genética; European Research Council (ERC); Swiss National Science Foundation (SNFS); Ministerio de Ciencia e Innovación (MICIN). España; French government
DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11–RAD50–NBS1 (MRN) to endonucleolytically cleave 5′-terminatedDNAto bypass protein blocks. CtIP also promotes the DNA2 helicase–nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.
EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells
(Oxford University Press, 2024-06-24) García Rodríguez, Néstor; Domínguez García, Iria; Domínguez Pérez, María del Carmen; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Ministerio de Ciencia e Innovación (MICIN). España
DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

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