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

ARTICLE Received 20 Aug 2013 | Accepted 30 Jan 2014 | Published 27 Feb 2014 DOI: 10.1038/ncomms4347 OPEN ATM specifically mediates repair of double-strand breaks with blocked DNA ends Alejandro Álvarez-Quilón1, Almudena Serrano-Benı́tez1, Jenna Ariel Lieberman1, Cristina Quintero1, Daniel Sánchez-Gutiérrez2, Luis M. Escudero2 & Felipe Cortés-Ledesma1 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. 1 Centro Andaluz de Biologı́a Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla (Departamento de Genética), Sevilla 41092, Spain. 2 Instituto Biomedicina Sevilla (IBiS), Hospital Virgen del Rocı́o-CSIC-Universidad de Sevilla (Departamento de Biologı́a Celular), Sevilla 41013, Spain. Correspondence and requests for materials should be addressed to F.C.-L. (email: ). NATURE COMMUNICATIONS | 5:3347 | DOI: 10.1038/ncomms4347 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE 2 which two topoisomerase subunits are covalently linked to each 50 -terminus of a DSB via a phosphodiester bond between the active-site tyrosine and the 50 -phosphate, is normally very shortlived, because the topoisomerase rapidly religates the DSB once DNA strand passage through the gap has occurred. However, under certain circumstances, such as the presence of nearby DNA lesions, cleavage complexes can be stabilized and interfere with the transcription or replication machinery. If this is the case, trapped TOP2 is degraded, leading to the formation of irreversible DSBs with peptidic blockages at the 50 ends of the DNA. This mechanism underlies the clinical efficacy of a widely used class of antitumour agents that ‘poison’ topoisomerase activity (for example, etoposide)16, thereby prolonging the half-life of the intermediate and increasing the possibility of DSB formation16. Etoposide can therefore be used to generate DSBs homogeneously characterized by covalent peptide blockage of the 50 ends. Tyrosyl DNA phosphodiesterase 2 (TDP2) is the only known enzyme in higher eukaryotes with the physiological capacity to unblock this type of DNA ends, converting them into 50 phosphate/30 hydroxyl ligatable termini17–19. This scenario offers a unique opportunity for the specific induction of clean and blocked DSBs (Fig. 1a). Following etoposide treatment, the majority of the induced DSBs will be efficiently unblocked by TDP2 in wild-type cells. However, when TDP2 is not present, the blockage will be irreversible and will necessarily require alternative nucleolytic processing to allow repair to proceed. This way, clean and blocked DSBs can be specifically induced by etoposide treatment of wild-type and TDP2-deficient cells, respectively. In this study, we exploit this genetic tool to demonstrate that ATM functions specifically in the rejoining of blocked DSBs, in a manner that is independent of the chromatin status of the lesions. Etoposide Tdp2 –/– Tdp2 + 5′ TDP2 5′ 5′ 5′ Unblocking Blocked DNA ends Clean DNA ends Nucleases Processing (DNA cleavage) 1.6 Tdp2 +/+ Tdp2 –/– – + 1.4 Relative units eficiencies in the DNA-damage response (DDR) are the cause of several human genetic syndromes1. Common hallmarks of these disorders include neurodegeneration and/or cancer predisposition, which are a probable consequence of deficient and inaccurate repair of DNA damage. Ataxia telangiectasia (A-T), a rare autosomal recessive syndrome that results from inactivation of the PIKK family Ser/Thr protein kinase ATM (A-T Mutated), is perhaps the paradigm for diseases of this type2,3. It is characterized by a symptomatology that includes progressive cerebellar ataxia, immunodeficiency, radiosensitivity, hypogonadism and increased cancer incidence (mainly leukaemia and lymphoma). Multiple functions have been assigned to ATM and its list of phosphorylation substrates is extensive4. Despite this versatility, its main function, or at least the best understood, is to trigger the initial phosphorylation wave of the DDR to double-strand breaks (DSBs). This fact, together with the exquisite radiosensitivity displayed by ATM-deficient cells and individuals and the symptomatological overlap of A-T with other break repair-defective human syndromes, strongly suggests a link between DSBs and pathology in A-T patients3,5. On the basis of all of this, one could anticipate that ATM would be essential, or at least important, for the repair of DSBs. Strikingly, this is not the case, as ATM loss does not result in obvious defects in the DSB repair rate. It has therefore been proposed that the radiosensitivity and chromosomal instability observed in ATMdeficient cells more likely arises from deficient checkpoint allowing cell cycle progression in the presence of damaged DNA6. However, there is a subset of DSBs, 10–20% depending on the DNAdamaging agent used7,8, which do require ATM. The current understanding is that these DSBs correspond to damage occurring in heterochromatin, where ATM is required to open the chromatin structure, allowing access of the repair machinery9. Interestingly, in addition to this, ATM is involved in specialized DSB-repair mechanisms that are not heterochromatin associated, such as V(D)J, class-switching and meiotic recombination10–12. These processes are related to important aspects of A-T pathogenesis such as immunodeficiency, increased incidence of lymphoma and sterility. Fully understanding the nature of DSBs that specifically require ATM for repair could therefore provide important clues into disease pathogenesis. DSBs can harbour different types of chemical moieties that differ from the canonical 50 phosphate and 30 hydroxyl at the ends13. Cells are therefore endowed with a wide variety of enzymatic activities that can ‘unblock’ DSBs preparing them for repair. However, under certain circumstances, such as the presence of complex or staggered lesions, these activities may be compromised or overwhelmed, resulting in breaks that are ‘blocked,’ in which case the only possibility to allow repair involves the action of nucleases to ‘process’ the ends by cleaving DNA sequence. It is therefore conceivable that clean and blocked DSBs can have different repair (...truncated)