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

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

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267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation
(Oxford University Press, 2016) Dopazo, Joaquín; Amadoz, Alicia; Bleda, Marta; Garcia Alonso, Luz; Daub, Josephine T.; Antiñolo Gil, Guillermo; Bhattacharya, Shomi S.; Universidad de Sevilla. Departamento de Cirugía; Ministerio de Economía y Competitividad (MINECO). España
Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms.
A CDK-regulated chromatin segregase promoting chromosome replication
(Springer Nature, 2021) Chacin, Erika; Bansal, Priyanka; Reusswig, Karl-Uwe; Díaz Santín, Luis M.; Ortega Moreno, Pedro; Vizjak, Petra; Gómez González, Belén; Müller-Planitz, Felix; Aguilera López, Andrés; Pfander, Boris; Cheung, Alan C. M.; Kurat, Christoph F.; Universidad de Sevilla. Departamento de Genética
The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme—Yta7—entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.
A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs
(Elsevier B.V., 2019) García Muse, Tatiana; Galindo Díaz, U.; García Rubio, María Luisa; Martin, Julie S.; Polanowska, Jolanta; O'Reilly, N.; Aguilera López, Andrés; Boulton, Simon J.; Universidad de Sevilla. Departamento de Genética
Accurate meiotic chromosome segregation critically depends on the formation of inter-homolog crossovers initiated by double-strand breaks (DSBs). Inaccuracies in this process can drive aneuploidy and developmental defects, but how meiotic cells are protected from unscheduled DNA breaks remains unexplored. Here we define a checkpoint response to persistent meiotic DSBs in C. elegans that phosphorylates the synaptonemal complex (SC) to switch repair partner from the homolog to the sister chromatid. A key target of this response is the core SC component SYP-1, which is phosphorylated in response to ionizing radiation (IR) or unrepaired meiotic DSBs. Failure to phosphorylate (syp-16A) or dephosphorylate (syp-16D) SYP-1 in response to DNA damage results in chromosome non-dysjunction, hyper-sensitivity to IR-induced DSBs, and synthetic lethality with loss of brc-1BRCA1. Since BRC-1 is required for inter-sister repair, these observations reveal that checkpoint-dependent SYP-1 phosphorylation safeguards the germline against persistent meiotic DSBs by channelling repair to the sister chromatid.
A population of hematopoietic stem cells derives from GATA4-expressing progenitors located in the placenta and lateral mesoderm of mice
(Ferrata Storti Foundation, 2017) Cañete, Ana; Carmona, Rita; Ariza, Laura; Sánchez, Mª José; Rojas, Anabel; Muñoz Chápuli, Ramón; Ministerio de Economía. España; Instituto de Salud Carlos III
GATA transcription factors are expressed in the mesoderm and endoderm during development. GATA1-3, but not GATA4, are critically involved in hematopoiesis. An enhancer (G2) of the mouse Gata4 gene directs its expression throughout the lateral mesoderm and the allantois, beginning at embryonic day 7.5, becoming restricted to the septum transversum by embryonic day 10.5, and disappearing by midgestation. We have studied the developmental fate of the G2-Gata4 cell lineage using a G2-Gata4Cre;R26REYFP mouse line. We found a substantial number of YFP+ hematopoietic cells of lymphoid, myeloid and erythroid lineages in embryos. Fetal CD41+ /cKit+ /CD34+ and Lin– /cKit+ /CD31+ YFP+ hematopoietic progenitors were much more abundant in the placenta than in the aorta-gonad-mesonephros area. They were clonogenic in the MethoCult assay and fully reconstituted hematopoiesis in myeloablated mice. YFP+ cells represented about 20% of the hematopoietic system of adult mice. Adult YFP+ hematopoietic stem cells constituted a long-term repopulating, transplantable population. Thus, a lineage of adult hematopoietic stem cells is characterized by the expression of GATA4 in their embryonic progenitors and probably by its extraembryonic (placental) origin, although GATA4 appeared not to be required for hematopoietic stem cell differentiation. Both lineages basically showed similar physiological behavior in normal mice, but clinically relevant properties of this particular hematopoietic stem cell population should be checked in physiopathological conditions.
A positioned +1 nucleosome enhances promoter-proximal pausing
(Oxford University Press, 2015) Jimeno González, Silvia; Ceballos Chávez, María; Reyes , José Carlos; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER); Ministerio de Economía y Competitividad (MINECO). España
Chromatin distribution is not uniform along the human genome. In most genes there is a promoter-associated nucleosome free region (NFR) followed by an array of nucleosomes towards the gene body in which the first (+1) nucleosome is strongly positioned. The function of this characteristic chromatin distribution in transcription is not fully understood. Here we show in vivo that the +1 nucleosome plays a role in modulating RNA polymerase II (RNAPII) promoter-proximal pausing. When a +1 nucleosome is strongly positioned, elongating RNAPII has a tendency to stall at the promoter-proximal region, recruits more negative elongation factor (NELF) and produces less mRNA. The nucleosome-induced pause favors pre-mRNA quality control by promoting the addition of the cap to the nascent RNA. Moreover, the uncapped RNAs produced in the absence of a positioned nucleosome are degraded by the 5′-3′ exonuclease XRN2. Interestingly, reducing the levels of the chromatin remodeler ISWI factor SNF2H decreases +1 nucleosome positioning and increases RNAPII pause release. This work demonstrates a function for +1 nucleosome in regulation of transcription elongation, pre-mRNA processing and gene expression.
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.
A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells
(Springer Nature, 2021) Dagg, Rebecca A.; Zonderland, Gijs; Puig Lombardi, Emilia; Rossetti, Giacomo G.; Groelly, Florian J.; Barroso Ceballos, Sonia Inés; Tacconi, Eliana M. C.; Wright, Benjamin; Lockstone, Helen; Aguilera López, Andrés; Halazonetis, Thanos D.; Tarsounas, Madalena; Universidad de Sevilla. Departamento de Genética
BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.
Actin nucleators safeguard replication forks by limiting nascent strand degradation
(Oxford University Press, 2023) Nieminuszczy, J.; Martin, P. R.; Broderick, R.; Krwawicz, J.; Kanellou, A.; Mocanu, C.; Bousgouni, V.; Smith, C.; Aguilera López, Andrés; Niedzwiedz, Wojciech; Universidad de Sevilla. Departamento de Genética; Cancer Reserch. United Kingdom; National Institutes of Health. United States; European Research Council (ERC)
Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that β-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing β-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing β-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.
Adaptive response of saccharomyces hosts to totiviridae L-A dsRNA viruses Is achieved through intrinsically balanced action of targeted transcription factors
(Multidisciplinary Digital Publishing Institute (MDPI), 2022) Ravoitytė, Bazilė; Lukša, Juliana; Wellinger, Ralf Erik; Serva, Saulius; Servienė, Elena; Universidad de Sevilla. Departamento de Genética; Fondo Social Europeo (FSO)
Totiviridae L-A virus is a widespread yeast dsRNA virus. The persistence of the L-A virus alone appears to be symptomless, but the concomitant presence of a satellite M virus provides a killer trait for the host cell. The presence of L-A dsRNA is common in laboratory, industrial, and wild yeasts, but little is known about the impact of the L-A virus on the host’s gene expression. In this work, based on high-throughput RNA sequencing data analysis, the impact of the L-A virus on whole-genome expression in three different Saccharomyces paradoxus and S. cerevisiae host strains was analyzed. In the presence of the L-A virus, moderate alterations in gene expression were detected, with the least impact on respiration-deficient cells. Remarkably, the transcriptional adaptation of essential genes was limited to genes involved in ribosome biogenesis. Transcriptional responses to L-A maintenance were, nevertheless, similar to those induced upon stress or nutrient availability. Based on these data, we further dissected yeast transcriptional regulators that, in turn, modulate the cellular L-A dsRNA levels. Our findings point to totivirus-driven fine-tuning of the transcriptional landscape in yeasts and uncover signaling pathways employed by dsRNA viruses to establish the stable, yet allegedly profitless, viral infection of fungi.
Adipose-derived mesenchymal stem cells (AdMSC) for the treatment of secondary-progressive multiple sclerosis: A triple blinded, placebo controlled, randomized phase I/II safety and feasibility study
(Public Library of Science, 2018) Fernández, Oscar; Izquierdo Ayuso, Guillermo; Fernández Montesinos, Rafael; Pozo Pérez, David; Leyva, Laura; Universidad de Sevilla. Departamento de Bioquímica Médica y Biología Molecular e Inmunología; Universidad de Sevilla. Departamento de Citología e Histología Normal y Patológica
Background Currently available treatments for secondary progressive multiple sclerosis(SPMS) have limited efficacy and/or safety concerns. Adipose-mesenchymal derived stem cells(AdMSCs) represent a promising option and can be readily obtained using minimally invasive procedures. Go to: Patients and methods In this triple-blind, placebo-controlled study, cell samples were obtained from consenting patients by lipectomy and subsequently expanded. Patients were randomized to a single infusion of placebo, low-dose(1x106cells/kg) or high-dose(4x106cells/kg) autologous AdMSC product and followed for 12 months. Safety was monitored recording adverse events, laboratory parameters, vital signs and spirometry. Expanded disability status score (EDSS), magnetic-resonance-imaging, and other measures of possible treatment effects were also recorded. Go to: Results Thirty-four patients underwent lipectomy for AdMSCs collection, were randomized and thirty were infused (11 placebo, 10 low-dose and 9 high-dose); 4 randomized patients were not infused because of karyotype abnormalities in the cell product. Only one serious adverse event was observed in the treatment arms (urinary infection, considered not related to study treatment). No other safety parameters showed changes. Measures of treatment effect showed an inconclusive trend of efficacy. Conclusion Infusion of autologous AdMSCs is safe and feasible in patients with SPMS. Larger studies and probably treatment at earlier phases would be needed to investigate the potential therapeutic benefit of this technique.
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.
An improved system for estradiol-dependent regulation of gene expression in yeast
(2007) Chávez de Diego, Sebastián; Cebolla Ramírez, Ángel; Arévalo Rodríguez, Miguel; Quintero Carrasco, María José; Maya Miles, Douglas; Universidad de Sevilla. Departamento de Genética; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Analysis of the relationship between coexpression domains and chromatin 3D organization
(Public Library of Science, 2017) Soler Oliva, María Eugenia; Guerrero Martínez, José A.; Bachetti, Valentina; Reyes, José C.; Universidad de Sevilla. Departamento de Genética
Gene order is not random in eukaryotic chromosomes, and co-regulated genes tend to be clustered. The mechanisms that determine co-regulation of large regions of the genome and its connection with chromatin three-dimensional (3D) organization are still unclear however. Here we have adapted a recently described method for identifying chromatin topologically associating domains (TADs) to identify coexpression domains (which we term ªCODsº). Using human normal breast and breast cancer RNA-seq data, we have identified approximately 500 CODs. CODs in the normal and breast cancer genomes share similar characteristics but differ in their gene composition. COD genes have a greater tendency to be coexpressed with genes that reside in other CODs than with non-COD genes. Such inter-COD coexpression is maintained over large chromosomal distances in the normal genome but is partially lost in the cancer genome. Analyzing the relationship between CODs and chromatin 3D organization using Hi-C contact data, we find that CODs do not correspond to TADs. In fact, intra-TAD gene coexpression is the same as random for most chromosomes. However, the contact profile is similar between gene pairs that reside either in the same COD or in coexpressed CODs. These data indicate that co-regulated genes in the genome present similar patterns of contacts irrespective of the frequency of physical chromatin contacts between them.
Application of flow focusing to the break-up of a magnetite suspension jet for the production of paramagnetic microparticles
(2011) Martín Banderas, Lucía; González Prieto, Román; Rodríguez Gil, Alfonso; Fernández Arévalo, María Mercedes; Flores Mosquera, María; Chávez de Diego, Sebastián; Gañán-Calvo, Alfonso M.; Universidad de Sevilla. Departamento de Farmacia y Tecnología Farmacéutica; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Ingeniería Aeroespacial y Mecánica de Fluidos; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Paramagnetic particles offer an extensive improvement in the magnetic separation or purification of a wide variety of protein molecules. Most commercial paramagnetic particles are synthesized by laborious and costly procedures. A straightforward production of paramagnetic microparticles with homogeneous and selectable sizes using flow focusing (FF) technology is described in this work. The development of an initial formulation of a stable iron oxide suspension compatible with the FF requirements is also reported. The obtained particles, below 10 microns in diameter and presenting smooth and reactive surface, were codified with an organic fluorophore and showed excellent properties for covalent attachment of biomolecules such as proteins and its subsequent recognition by flow cytometry. Furthermore, particles with suitable magnetite content resulted as well-suited for commercial magnet separators for these purposes.
Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor
(Wiley-Blackwell, 2012) Peña, Álvaro; Gewartowski, Kamil; Mroczek, Seweryn; Cuéllar, Jorge; Szykowska, Aleksandra; Prokop, Andrzej; Czarnocki-Cieciura, Mariusz; Piwowarski, Jan; Tous Rivera, Cristina; Aguilera López, Andrés; Carrascosa, José L.; Valpuesta, José María; Dziembowski, Andrzej; Universidad de Sevilla. Departamento de Genética
The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a β-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.
ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors
(MDPI, 2022) Pladevall Morera, David; Castejón Griñán, María; Aguilera, Paula; Gaardahl, Karina; Ingham, Andreas; Brosnan Cashman, Jacqueline A.; Meeker, Alan K.; López Contreras, Andrés J.; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); Danish Cancer Society; Danish National Research Foundation
High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)–the current standard of care treatment for GBM patients–causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mutations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi.
Brahma is required for proper expression of the floral repressor FLC in Arabidopsis
(2011) Amasino, Richard M.; Farrona Cortés, Sara; Hurtado Álvarez, Lidia; Florencio Bellido, Francisco Javier; Reyes Rosa, José Carlos; March Díaz, Rosana Rocío; Schmitz, Robert J.; Turck, Franziska; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER); Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
BRCA1 Accelerates CtIP-Mediated DNA-End Resection
(Elsevier, 2014) Cruz García, Andrés; López Saavedra, Ana; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética
DNA-end resection is a highly regulated and critical step in the response and repair of DNA double-strand breaks. In higher eukaryotes, CtIP regulates resection by integrating cellular signals via its posttranslational modifications and protein-protein interactions, including cell-cycle-controlled interaction with BRCA1. The role of BRCA1 in DNA-end resection is not clear. Here, we develop an assay to study DNA resection in higher eukaryotes at high resolution. We demonstrate that the BRCA1-CtIP interaction, albeit not essential for resection, modulates the speed at which this process takes place.
BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis
(Nature Research, 2024-05-20) Salas Lloret, Daniel; García Rodríguez, Néstor; Soto Hidalgo, Emily; González Vinceiro, Lourdes; Espejo Serrano, Carmen; Giebel, Lisanne; Huertas Sánchez, Pablo; González Prieto, Román; Universidad de Sevilla. Departamento de Biología Celular; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Union (UE)
Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.
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.

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