Regulation of blocked-DSB repair by DNA-PKcs and ATM kinases

Dada la amenaza que suponen las roturas del ADN de doble cadena para la integridad del genoma, conocer los mecanismos moleculares implicados de su reparación es extremadamente relevante. En particular, las estructuras de los extremos que presentan las roturas de doble cadena definen su complejidad y son consideradas posibles determinantes de la elección de la ruta por las que van a ser reparadas y del resultado de dicha reparación. Sin embargo, esta cuestión no ha sido suficientemente esclarecida debido a la dificultad de inducir roturas homogéneas con extremos que presenten estructuras definidas. Gracias al reciente desarrollo de un método genético para inducir roturas con extremos homogéneos, hemos diseccionado las rutas requeridas para reparar las roturas de doble cadena inducidas por la Topoisomerasa 2 cuando los extremos se encuentran específicamente limpios o bloqueados en G0/G1. Para ello, hemos caracterizado la implicación de factores identificados en escrutinios genéticos realizados con la técnica CRISPR/Cas9 y otros candidatos relacionados con los factores previamente identificados. De esta manera, hemos identificado que existe una preferencia para reparar las roturas producidas por la Topoisomerasa 2 a través de la actividad de TDP2 en vez del procesamiento llevado a cabo por las nucleasas, que sólo son necesarias cuando los extremos están bloqueados de una forma irreversible. Esta jerarquía contribuye a asegurar la estabilidad del genoma y no se mantiene en ausencia de DNA-PKcs. También demostramos que la función de ATM en la reparación de las roturas bloqueadas está principalmente relacionada con la ruta nucleolítica, aunque también podría estar implicada en la protección de los extremos frente a un excesivo procesamiento. Además, demostramos que esta jerarquía que prioriza la actividad de TDP2 impide la transformación a células malignas y el desarrollo de cáncer

An End to a Means: How DNA-End Structure Shapes the Double-Strand Break Repair Process

Endogenously-arising DNA double-strand breaks (DSBs) rarely harbor canonical 5'-phosphate, 3'-hydroxyl moieties at the ends, which are, regardless of the pathway used, ultimately required for their repair. Cells are therefore endowed with a wide variety of enzymes that can deal with these chemical and structural variations and guarantee the formation of ligatable termini. An important distinction is whether the ends are directly "unblocked" by specific enzymatic activities without affecting the integrity of the DNA molecule and its sequence, or whether they are "processed" by unspecific nucleases that remove nucleotides from the termini. DNA end structure and configuration, therefore, shape the repair process, its requirements, and, importantly, its final outcome. Thus, the molecular mechanisms that coordinate and integrate the cellular response to blocked DSBs, although still largely unexplored, can be particularly relevant for maintaining genome integrity and avoiding malignant transformation and cancer.España, Junta de Andalucía SAF2017-89619-R, CVI-7948European Research Council (ERC-CoG-2014-647359

Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm−/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm−/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.Junta de Andalucía SAF2010-21017, SAF2013-47343-P, SAF2014-55532-R, SAF2017-89619-R, CVI-7948European Research Council ERC-CoG-2014-64735

ATM specifically mediates repair of double-strand breaks with blocked DNA ends

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders

ATM specifically mediates repair of double-strand breaks with blocked DNA ends

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders

Regulation of blocked-DSB repair by DNA-PKcs and ATM kinases

Trabajo presentado para optar al grado de Doctora en Biología Molecular, Biomedicina e Investigación Clínica en la Universidad de Sevilla-CABIMER, Departamento de Biología del Genoma.--Calificación: Sobresaliente CUM LAUDEDada la amenaza que suponen las roturas del ADN de doble cadena para la integridad del genoma, conocer los mecanismos moleculares implicados de su reparación es extremadamente relevante. En particular, las estructuras de los extremos que presentan las roturas de doble cadena definen su complejidad y son consideradas posibles determinantes de la elección de la ruta por las que van a ser reparadas y del resultado de dicha reparación. Sin embargo, esta cuestión no ha sido suficientemente esclarecida debido a la dificultad de inducir roturas homogéneas con extremos que presenten estructuras definidas. Gracias al reciente desarrollo de un método genético para inducir roturas con extremos homogéneos, hemos diseccionado las rutas requeridas para reparar las roturas de doble cadena inducidas por la Topoisomerasa 2 cuando los extremos se encuentran específicamente limpios o bloqueados en G0/G1. Para ello, hemos caracterizado la implicación de factores identificados en escrutinios genéticos realizados con la técnica CRISPR/Cas9 y otros candidatos relacionados con los factores previamente identificados. De esta manera, hemos identificado que existe una preferencia para reparar las roturas producidas por la Topoisomerasa 2 a través de la actividad de TDP2 en vez del procesamiento llevado a cabo por las nucleasas, que sólo son necesarias cuando los extremos están bloqueados de una forma irreversible. Esta jerarquía contribuye a asegurar la estabilidad del genoma y no se mantiene en ausencia de DNA-PKcs. También demostramos que la función de ATM en la reparación de las roturas bloqueadas está principalmente relacionada con la ruta nucleolítica, aunque también podría estar implicada en la protección de los extremos frente a un excesivo procesamiento. Además, demostramos que esta jerarquía que prioriza la actividad de TDP2 impide la transformación a células malignas y el desarrollo de cáncer

An interplay between PIKKs regulates DNA double-strand break end processing and repair

Trabajo presentado en el Abcam Meeting. PI3K-like protein kinases, celebrado en Milán (Italia), del 3 al 5 de noviembre de 201

ATM specifically mediates repair of double-strand breaks with blocked DNA ends

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disordersWork in F.C.-L. laboratory is funded with grants from the Spanish Government (SAF2010-21017 and BFU2010-11042-E, Ministerio de Ciencia e Innovación), the regional Andalusian Government (CVI-7948) and the European Union (PERG07-2010-268466) and with the following fellowships: Formación Personal Investigador (BES-2011-047351, Ministerio de Ciencia e Innovación) for A.A.-Q., Beca Predoctoral AEFAT (Asociación Española Familia Ataxia Telangiectasia) for A.S.-B., Personal Investigador en Formación (Universidad de Sevilla) for J.A.L. and Ramón y Cajal (RYC-2009-03928, Ministerio de Ciencia e Innovación) for F.C.-L. L.M.E. is supported by the Miguel Servet program, (Instituto Carlos III) and the Spanish Government grant (BFU2011-25734, Ministerio de Ciencia e Innovación)Peer Reviewe

GSE4 peptide suppresses oxidative and telomere deficiencies in Ataxia Telangiectasia patient cells

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.RP laboratory is funded by grant P14-01495 and P17-01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds) and CIBER 576/805_ER16PE06P2016 supported by FEDER funds. GG grant “Ministerio de Economía, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER)” (SAF2015-68073-R). CM-G is granted by the CIBERER. Work in FC-L laboratory is funded with grants from the Spanish Government (SAF2014-55532-R, Ministerio de Economía, Industria y Competitividad), the regional Andalusian Government (CVI-7948), the Fundación Ramón Areces (XVII Convocatoria Ciencias de la Vida y Materia), the European Research Council (ERC-CoG-2014-647359), and with a Predoctoral Fellowship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) to AS-B. BER OGG1 was kindly provided by Professor Thomas Helleday’s Lab, Karolinska Institutet, Sweden

A genetic map of the response to DNA damage in human cells

Versión postprint próximamente disponible en: http://hdl.handle.net/10261/228202The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.AAQ, GSM and AMH are recipients of long-term EMBO fellowships (ALTF 910-2017, 795-2017 under a CC-BY-NC-ND 4.0 International license. not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv preprint doi: https://doi.org/10.1101/845446; this version posted November 18, 2019. The copyright holder for this preprint (which was 27 and 124-2019, respectively), NH was supported by a Human Frontier Science Program long-term Fellowship, SER is supported by a fellowship from AIRC and SA was a Banting post-doctoral fellow. ASB was supported by a PhD Studentship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) and an EMBO short-term fellowship for a visit to the DD laboratory. The ICRF187 screen in FCL laboratory was funded by grants from the Spanish Government (SAF2017- 89619-R, European Regional Development Fund) and the European Research Council (ERC-CoG2014-647359). Work in the RSW laboratory was supported in part by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS, 1Z01ES102765). DD is a Canada Research Chair (Tier I) and the work was supported from grants from the CIHR (FDN143343 to DD; FRN 123518, PJT-156330 to AM) Canadian Cancer Society grant 705644 (to DD) with additional support to DD from the Krembil Foundation.N