Role of ATM in the repair of blocked DNA double-strand breaks

La Ataxia-telangiectasia (A-T) es un síndrome causado por mutaciones de pérdida de función en la quinasa ATM (Ataxia Telangiectasia mutated). A-T representa el aradigma de los síndromes caracterizados por predisposición a cáncer y neurodegeneración asociados a deficiencias en la respuesta a daño en el ADN. La función de dicha proteína como regulador principal en la señalización celular en respuesta a roturas de doble cadena en el ADN se ha definido en detalle, aunque aún se desconoce el papel que desempeña en su reparación. Mientras que la deficiencia de ATM causa una alta sensibilidad a agentes que inducen roturas de doble cadena, no se han detectado grandes defectos en la reparación de estas lesiones. En esta Tesis se desarrollan nuevos métodos moleculares y genéticos para modificar la estructura de los extremos de las roturas de doble cadena en el ADN. Mediante estas técnicas se demuestra que ATM es esencial para la correcta reparación de roturas de doble cadena, pero exclusivamente cuando los extremos de la rotura están bloqueados. De esta manera identificamos la función de ATM en la reparación de roturas de doble cadena y sugerimos que los extremos bloqueados en el ADN pueden ser la causa de la Ataxia-telangiectasia.Premio Extraordinario de Doctorado U

Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.Spanish Government SAF2010-21017Spanish Government BES-2011-0473

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

TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In Vivo

Anticancer topoisomerase >poisons> exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.Gobierno Español SAF2010-21017, BFU2010-11042-EEuropean Union PERG07- 2010-268466Queen Elisabeth Medical Foundation GSKE 111

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

The CIP2A–TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers

Pathways for the repair of 5'blocked double-strand breaks

Póster presentado al 22nd IUBMB & 37th FEBS Congress: From Single Molecules to Systems Biology, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.Peer Reviewe

The role of ATM in double-strand break rejoining is specific to blocked DNA ends

Póster presentado en EMBO Conference: The DNA damage response in cell physiology and disease, celebrada en Cape Sounio (Grecia), del 7 al 11 de octubre de 2013Peer Reviewe