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

TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo Fernando Go ´mez-Herreros 1. , Rocı ´o Romero-Granados 2. , Zhihong Zeng 1 , Alejandro A ´lvarez-Quilo ´n 2 , Cristina Quintero 2 , Limei Ju 1 , Lieve Umans 3 , Liesbeth Vermeire 3 , Danny Huylebroeck 3 , Keith W. Caldecott 1 *, Felipe Corte ´s-Ledesma 2 * 1Genome Damage and Stability Centre, University of Sussex, Falmer, United Kingdom, 2Centro Andaluz de Biologı ´a Molecular y Medicina Regenerativa (CABIMER), CSIC– Universidad de Sevilla (Departamento de Gene ´tica), Sevilla, Spain, 3Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, University of Leuven, Leuven, Belgium Abstract 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 59-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. Citation: Go ´mez-Herreros F, Romero-Granados R, Zeng Z, A ´lvarez-Quilo ´n A, Quintero C, et al. (2013) TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo. PLoS Genet 9(3): e1003226. doi:10.1371/journal.pgen.1003226 Editor: Nancy Maizels, University of Washington, United States of America Received July 13, 2012; Accepted November 21, 2012; Published March 7, 2013 Copyright: ß2013 Go ´mez-Herreros et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in FC-L laboratory is funded with grants from the Spanish Government (SAF2010-21017 and BFU2010-11042-E) and the European Union (PERG07- 2010-268466) and with the following fellowships from the Spanish Government: Ramo ´n y Cajal (RYC-2009-03928) for FC-L, JAE-Doc (2010-011) for RR-G, and FPI (BES-2011-047351) for A ´A-Q. Work in the KWC laboratory is funded by MRC grants to KWC (G0901606, G0600776, MR/J006750/1) and supported FG-H, ZZ, and LJ. Generation of the Tdp2 floxed and conditional mice in the DH lab was supported by the EC FP6 Integrated Project EndoTrack, the Interuniversity Attraction Pole IUAP-P6/20 and P7/07 project, and the Queen Elisabeth Medical Foundation (GSKE 1113) and type 3 large-infrastructure support InfraMouse (Hercules Foundation ZW09-03, to DH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (KWC); [email protected] (FC-L) .These authors contributed equally to this work. Introduction The double-stranded helical structure of DNA creates topological problems in all processes that involve opening of the double helix and accessing the genetic information [1,2]. In particular, the transcription and duplication of DNA and its condensation into chromosomes generates knots and tangles that need to be resolved to avoid interference with diverse cellular processes and to ensure faithful chromosome segregation during mitosis. DNA topoisomerases are enzymes that introduce transient breaks in DNA to solve these topological problems. Type II topoisomerases, such as topoisomerase II in eukaryotes (TOP2) are essential homodimeric enzymes that relax, unknot and decatenate DNA molecules by catalyzing the passage of duplex DNA through a transient DNA double strand break (DSB) created by the enzyme [3]. Two isoforms of TOP2, aand b, exist in higher eukaryotes, with primary roles in replication and chromosome segregation and in transcription, respectively. A key intermediate of TOP2 activity is the cleavage complex, in which each of two topoisomerase subunits is covalently linked to the 59- terminus of an enzyme-generated DSB via a phosphodiester bond between the active-site tyrosine and the 59-phosphate. The cleavage complex is normally a very short-lived intermediate, because the topoisomerase rapidly re-ligates the DSB once DNA strand passage through the DSB has occurred. However, under certain circumstances, such as the presence of nearby DNA lesions, cleavage complexes can be stabilized resulting in an increased likelihood of collision with RNA or DNA polymerases [4]. Such collisions can convert cleavage complexes into potentially clastogenic or lethal DSBs that require cellular DNA repair pathways for their removal. PLOS Genetics | www.plosgenetics.org 1 March 2013 | Volume 9 | Issue 3 | e1003226 Cleavage complexes are the target of a widely used class of antitumor agents that ‘poison’ topoisomerase activity, thereby prolonging the half-life of the intermediate and increasing the possibility of DSB formation [4,5]. Thus, these drugs kill tumor cells by inducing high levels of TOP2-associated DSBs. Consequently, TOP2 poisons are commonly used antineoplastic drugs in the treatment of a broad range of tumor types including malignant lymphomas, sarcomas, leukemias, and lung, ovarian, breast and testicular cancers [5]. However, similar to other chemotherapeutic agents, TOP2-targeting drugs are only partially selective for tumour cells, resulting in unwanted toxicity in normal tissues and in therapy-associated chromosome translocations and secondary leukemias [6–14]. Moreover, some breakpoints in such translocations have actually been correlated with preferential sites of cleavage by TOP2 [13–17]. A characteristic feature of TOP2-induced DNA breaks is covalent attachment of the enzyme to 59ends of the DNA, which must be removed by cellular end-processing enzymes if DSB repair is to occur [18]. Until recently, the only known mechanism for removal TOP2 peptide from DNA 59-termini in mammalian cells involved excision of the DNA fragment linked to the peptide using nucleases such as the MRN complex, CtIP or Artemis [19– 21]. Recently, however, we identified a human 59-tyrosyl DNA phosphodiesterase (59-TDP) that can cleave 59-phosphotyrosyl bonds and thereby release TOP2 from DSB termini without the need to also remove DNA sequence [22]. Consequently, this enzyme, which was previously known as signalling protein and transcription cofactor TTRAP/EAPII [23,24], is now denoted tyrosyl DNA phopshodiesterase-2 (TDP2; Human Gene Nomeclature Organisation). Notably, consistent with its enzyme activity, TDP2 is required for cellular resistance to the anti-cancer TOP2 poison etoposide, but is not required for cellular resistance to ionizing radiation or methylmethane sulphonate [22,25]; agents that induce DNA damage independently of TOP2 activity. Following DNA end processing, DSBs can be repaired either by homologous recombination (HR) or by non-homologous end joining (NHEJ) [26]. However, these pathways utilize fundamentally different mechanisms for rejoining DSBs and consequently differ in their accuracy. In particular, HR utilizes undamaged sister chromatids to replace any nucleotides removed from DNA termini during DNA end processing and consequently is normally ‘error-free’. However, this process is available only during S phase or G2, when sister chromatids are available. In contrast, NHEJ is a ‘cut-and-splice’ process in which DSB termini are ligated together following DNA end processing without accurate replacement of missing nucleotides, and thus is potentially ‘error-prone’. Here, we employ avian and murine experimental models to show that TDP2/Tdp2 deletion results in hypersensitivity to a structurally diverse range of anti-cancer TOP2 poisons. Moreover, we present genetic, biochemical and cellular evidence for TDP2 functioning in a mechanism of NHEJ that protects genome integrity in response to TOP2-induced damage. Finally, we show that this TDP2 dependent pathway also operates in vivo, as, upon exposure to TOP2 poisons, it is required for normal adult mouse lymphopoiesis, intestinal mucosa homeostasis and the maintenance of genome stability in the bone marrow. Collectively, our results suggest that TDP2 defines an error-free mechanism of NHEJ in mammals, which is specialized in the repair of TOP2- induced DSBs and reduces both tissue toxicity and genome instability in response to this particular type of DNA damage. These findings suggest the possibility of TDP2 being a significant etiological factor in the clinical tolerance and response to widely used TOP2 poisons. Results TDP2 is required for cellular resistance to clinical TOP2 poisons and is the major 59-TDP activity in the mouse The discovery of TDP2 as the first 59-TDP activity raised the possibility of it being an important factor in the clinical response to TOP2 poisons [22,25]. Indeed, TDP2 deleted avian DT40 cells are hypersensitive to etoposide [22,25]. To address this question further, we examined the sensitivity of TDP2 2/2/2 cells to two additional, structurally diverse, TOP2 poisons. These drugs, denoted doxorubicin and amsacrine (m-AMSA), are employed widely during cancer therapy but in contrast to etoposide, ‘poison’ TOP2 by intercalating into DNA [5]. Nevertheless, similarly to etoposide, TDP2 2/2/2 cells displayed significant hypersensitivity to both doxorubicin and m-AMSA (Figure 1A). Moreover, a functional TDP2 phosphodiesterase domain was required for cellular resistance to this type of drug, because expression of wildtype human TDP2 (hTDP2) rescued the sensitivity of TDP2 2/2/2 DT40 cells to m-AMSA, whereas hTDP2 D262A harbouring an inactivating mutation in the catalytic active site [5] did not (Figure 1A). These results show that TDP2 is required for cellular resistance to a range clinically relevant and structurally diverse TOP2 poisons, and support our contention that this requirement reflects the 59-TDP activity of this enzyme. To determine the impact of TDP2 on TOP2-induced DNA damage in mammals, and thus its possible relevance to anti-cancer therapy, we adopted a mouse model in which the first three exons of Tdp2, plus the 59-UTR, were deleted by Cre-mediated excision (Figure 1B; see Materials and Methods). Mice homozygous for the deleted allele (Tdp2 flD , from here-on denoted Tdp2 D1–3 ) are viable, and so far we have not detected any abnormal pathology (unpublished observations). However, transformed Tdp2 D1–3 mouse embryonic fibloblasts (MEFs) were hypersensitive to etoposide (Figure 1C, left, and Figure S1), but were not hypersensitive to DNA damage induced independently of TOP2 by c-irradiation (Figure 1C, right). Author Summary DNA double-strand breaks (DSBs) are dangerous because they can lead to cellular death and tissue degeneration if not repaired, or to genome rearrangements, which are a common hallmark of cancer, if repaired incorrectly. Although required for all chromosomal transitions in cells, transient DNA cleavage by topoisomerase II (TOP2) is a potential endogenous source of DSBs, which are characteristic in that TOP2 remains covalently bound to the DNA termini. In addition, numerous chemotherapeutic regimes rely on compounds that ‘‘poison’’ TOP2 activity, stimulating the formation of DSBs that target tumour cells. However, these compounds also affect healthy tissue and confer undesirable side effects, including the stimulation of genome rearrangements that can trigger secondary malignancies (mainly acute leukemia). Identifying the factors that participate in the repair of TOP2- induced DSBs and fully understanding their mechanism of action are therefore important for the design of chemotherapeutic regimes that are more effective and safer. Here we demonstrate that TDP2, a recently identified protein that can liberate DSB termini from blocked TOP2, functions as part of established cellular DSB repair processes and is required to safeguard genome integrity upon treatment with TOP2 poisons, both in cells and in mice. These results can therefore have important implications in cancer treatment. TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 2 March 2013 | Volume 9 | Issue 3 | e1003226 Protein extracts from spleen, thymus, and bone marrow from wild type mice possess robust 59-TDP activity, but, importantly, this activity was absent in analogous protein extracts from Tdp2 D1–3 mice, confirming successful inactivation of the enzyme (Figure 2A). Cell extracts prepared from primary Tdp2 D1–3 MEFs also lacked detectable 59-TDP activity (Figure 2B). This was true Figure 1. TDP2 promotes survival following TOP2-induced DSBs. A. Clonogenic survival of the indicated DT40 cell line; wild-type, TDP2 2/2/2 and TDP2 2/2/2 complemented with human TDP2 (hTDP2) and catalytic-dead human TDP2 (hTDP2 262A) or empty vector (Empty V); following continuous treatment with the indicated concentrations of doxorubicin (left) or mAMSA (right). Average 6s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose when compared to TDP2 2/2/2 cells by Two-way ANOVA with Bonferroni posttest is shown. B. Scheme showing the strategy for targeted deletion of the first three exons of Tdp2 in mouse. The wild-type (Tdp2 + ), conditional (Tdp2 flEx1–3,neo ) and deleted (Tdp2 flD ) alleles are depicted. The EcoRI-EcoRI fragment of Tdp2 was used in the targeting construct. Southern-blot analysis of PstI-digested DNA from wild-type (+/+), heterozygous (+/flD) and knock-out (flD/flD, from now on denoted Tdp2 D1–3 ) mice, using the indicated probe (red line), is shown (bottom right). C. Clonogenic survival of wild-type and Tdp2 D1–3 transformed MEFs after 3 h acute exposure to the indicated concentrations of etoposide (left) or the indicated dose of c-irradiation (right). Average 6s.e.m. of three independent experiments and statistical significance by Two-way ANOVA test with Bonferroni post-test is shown. In all figures (*P#0.05; **P#0.01; ***P#0.005). doi:10.1371/journal.pgen.1003226.g001 TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 3 March 2013 | Volume 9 | Issue 3 | e1003226 not only for blunt-ended DSB substrates, but also for DSB substrates harbouring a 4-bp 59-overhang (Figure 2C), characteristic of TOP2-induced DSBs. Additionally, EDTA-mediated chelation of Mg 2+ , which is essential for TDP2 function, completely eliminates 59-TDP activity in wild type MEF extracts. These observations are significant because the related enzyme TDP1, whose activity is Mg 2+ independent, was recently reported to possess weak activity on this type of substrate [27]. Our data therefore suggest that TDP2 is the primary, if not only, source of 59-TDP activity in MEF extracts (Figure 2C). TDP2 creates ligatable DSBs and functions in NHEJ Based on the mechanism of TOP2 cleavage, we anticipated that TDP2 activity would reconstitute ‘clean’ DSBs (59phosphate and 39hydroxyl termini) with 4-bp overhangs, which would be an ideal substrate for ligation by NHEJ. Interestingly, these ligation events would accurately preserve the DNA sequence, suggesting the possibility of an error-free NHEJ mechanism that specifically acts on TOP2-induced DSBs. To test this hypothesis, we examined whether TDP2 action at DSBs typical of those induced by TOP2 creates termini that can be ligated by T4 DNA ligase. Indeed, inclusion of T4 DNA ligase in reactions containing wild type MEF extract resulted in the additional appearance of a product of 46-nt, indicative of the completion of DSB repair by DNA ligation. However, this product was not observed if reactions contained cell extract from Tdp2 D1–3 MEFs, confirming that DNA ligation was dependent on TDP2 activity (Figure 2D). Interestingly, the length of the product is consistent with a ligation event in which DNA sequence is preserved. To analyse ligation events directly catalysed by cell extracts, we generated linear plasmids harbouring 59 phosphate or 59phosphotyrosine ends by PCR amplification with the corresponding modified primers. The incubation of these substrates with NHEJ-competent nuclear extracts [28] results in plasmid circularization events that can be scored as colonies following bacterial transformation. As can be seen in Figure 2E, nuclear extracts from Tdp2 D1–3 MEFs efficiently circularized linear plasmids with 59phosphate ends but not linear plasmids harbouring 59-phosphotyrosine. This difference was lost upon addition of recombinant TDP2 to the reaction, confirming the TDP2–dependent nature of the repair reaction. Collectively, our data suggest that TDP2 activity facilitates NHEJ of 59tyrosine- blocked ends by generating DSBs with ligatable termini, consistent with our hypothesis that this enzyme can support error-free NHEJ of TOP2-induced DNA damage. To genetically test whether TDP2 functions indeed during NHEJ, we generated TDP2 2/2/2 DT40 cells harboring a targeted deletion of Ku70, a core component of the NHEJ pathway (Figure S2). Whilst both TDP2 2/2/2 and KU70 2/2 cells were hypersensitive to etoposide, cells in which both genes were deleted (TDP2 2/2/2 /KU70 2/2 ) were no more hypersensitive than cells in which Ku70 alone was deleted (Figure 3A). In contrast to this epistatic relationship with a core NHEJ factor, transient knockdown of TDP2 further enhances etoposide sensitivity of HR defective (BRCA2 mutated) human fibroblasts (Figure 3B). Based on these genetic relationships, we conclude that TDP2 functions in a NHEJ-mediated and HR-independent pathway for the repair of TOP2-induced DSBs. To further assign a role for TDP2 in the NHEJ pathway for DSB repair, we measured DSB repair rates in primary Tdp2 D1–3 MEFs by immunodetection of cH2AX, a phosphorylated derivative of histone H2AX that arises at sites of chromosomal DSBs [29]. We measured DSB repair in specific phases of the cell cycle, because whilst NHEJ is operative throughout, HR-mediated DSB repair is operative only in S/ G2 [30]. Notably, DSB repair rates were markedly reduced in Figure 2. Deletion of Tdp2 in mouse abolishes 59-TDP activity and ligation of 59phosphotyrosine-blocked ends. A. Duplex substrate harbouring a 59phosphotyrosine blunt end (left) was incubated with 9 mgTdp2 +/+ or Tdp2 D1–3 tissue extract from bone marrow (BM), thymus and spleen for 1 h. B. Substrate in ‘‘A’’ was incubated with 1.5 mg of cellular extract from Tdp2 +/+ or Tdp2 D1–3 primary MEFs for the indicated time. C. Duplex substrate harbouring a 59phosphotyrosine self-complementary overhang end (left) was incubated with 10 mg cellular extract from Tdp2 +/+ or Tdp2 D1–3 transformed MEFs for 2 h in the presence or absence of 50 mM EDTA. D. Self-ligation of 59phosphate (P) and 59phosphotyrosine (Y–P) overhang substrates as depicted in ‘‘C’’ incubated for 1.5 h with 3.3 mg cellular extract from Tdp2 +/+ or Tdp2 D1–3 transformed MEFs in the presence of T4 DNA ligase. In all cases migration of the 59phosphotyrosine substrate (Y–P), 59phosphate (P) and ligation (lig) products are indicated. E. Circularization efficiency of a linear plasmid with 59phosphotyrosine (Y–P) and 59phosphate (P) catalysed by Tdp2 D1–3 transformed MEFs extracts in the presence and absence of recombinant human TDP2 (hTDP2). Reaction products were transformed into E. coli and the number of transformants obtained per mg of initial substrate DNA (average 6s.e.m. of three independent experiments) is shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated is shown. doi:10.1371/journal.pgen.1003226.g002 TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 4 March 2013 | Volume 9 | Issue 3 | e1003226 Tdp2 D1–3 MEFs following etoposide treatment, both in G0/G1 (Figure 3C) and G2 (Figure 3D), consistent with TDP2 functioning, as NHEJ, independently of cell cycle. These results were not specific to murine cells, since similar results were observed in TDP2-depleted human A549 cells (Figure S4). In contrast to treatment with etoposide, the rate of DSB repair was normal in Tdp2 D1–3 MEFs following cirradiation, consistent with a role for TDP2 specifically at TOP2- induced DSBs (Figure 3E). Collectively, these data demonstrate that TDP2 is required in mammalian cells for rapid repair of TOP2- induced DSBs by NHEJ, and for cellular resistance to these lesions. Figure 3. TDP2 promotes repair of TOP2-induced DSBs by NHEJ. A. Clonogenic survival of wild-type, TDP2 2/2/2 ,KU70 2/2 and TDP2 2/2/2 KU70 2/2 DT40 cells following continuous treatment with the indicated concentrations of etoposide. Average 6s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose by Two-way ANOVA with Bonferroni post-test is shown. B. Clonogenic survival of wild-type and BRCA2-mutant human transformed fibroblasts with (Tdp2si) and without (control) TDP2 depletion following 3 h acute exposure to the indicated concentrations of etoposide. Western blot analysis of TDP2 levels in wild type and BRCA2-mutant cell extracts after 48 h of transfection is indicated (inset). Other details as in ‘‘A’’. C. cH2AX foci induction after 30 min 20 mM etoposide treatment and repair at different times following drug removal in confluency arrested Tdp2 +/+ and Tdp2 D1–3 primary MEFs. Representative images of the 3 h repair time point including DAPI counterstain (right) and average 6s.e.m. of at least three independent experiments (left) are shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated. D. G2 primary MEFs (see Matherials and Methods) following 30 min 10 mM etoposide treatment. Other details as in ‘‘C’’. E. Confluency arrested primary MEFs exposed to 2Gy c-irradiation. Other details as in ‘‘C’’. doi:10.1371/journal.pgen.1003226.g003 TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 5 March 2013 | Volume 9 | Issue 3 | e1003226 TDP2 promotes genome stability following TOP2- induced DNA damage We hypothesized that this TDP2-mediated error-free NHEJ mechanism would be important to maintain genome integrity upon exposure to TOP2 poisons. To address this possibility, we quantified the frequency of micronuclei (MN), nucleoplasmic bridges (NB), and chromosomal aberrations following etoposide treatment. These events constitute well-established indicators of genome instability caused by misrepair of DSBs in which acentric, dicentric and aberrant chromosomes or chromosome fragments can be formed. As expected, etoposide increased the number of micronuclei and nucleoplasmic bridges in both transformed Tdp2 +/+ and Tdp2 D1–3 MEFs, but this increase was significantly higher (up to three-fold) in Tdp2 D1–3 cells (Figure 4A). Primary Tdp2 D1–3 MEFs at low passage (P3–4) similarly displayed elevated levels of micronuclei and nucleoplasmic bridges following etoposide treatment, compared to wild type primary MEFs (Figure 4B), although in the case of nucleoplasmic bridges the low number of cells displaying these structures prevented the difference from reaching statistical significance. An additional indicator of genome instability is elevated frequencies of chromosome aberrations. Consequently, we quantified the frequency of chromosome breaks and exchanges in metaphase spreads of transformed Tdp2 +/+ and Tdp2 D1–3 MEFs. In agreement with the increased cell cycle arrest of TDP2 2/2/2 DT40 cells in G2 following etoposide treatment [25], we noted an etoposide-dependent reduction in metaphase cells that was particularly severe in Tdp2 D1–3 MEFs (unpublished observations). However, of those metaphases observed and scored, both chromosome exchanges and breaks were significantly higher (2 to 5-fold) in Tdp2 D1–3 MEFs than in Tdp2 +/+ MEFs (Figure 4C). A similar increase in these events in Tdp2 D1–3 MEFs, compared to wild type cells, was observed if low-passage primary MEFs were employed, ruling out the possibility that the elevated genome instability in Tdp2 D1–3 MEFs was an artefact of cellular transformation (Figure 4D). In the latter case, etoposide treatment almost ablated the appearance of mitotic cells in populations of both wild type and Tdp2 D1–3 MEFs, necessitating the use of caffeine to prevent G2 arrest. Taken together these results demonstrate that loss of TDP2 results in increased genome instability following TOP2-induced DNA strand breakage. Loss of TDP2 results in elevated homologous recombination The above results demonstrate increased genome instability in Tdp2 D1–3 MEFs, consistent with a role for TDP2 in error-free NHEJ-mediated repair of TOP2-induced DSBs. In this scenario, we considered the possibility that loss of TDP2 might also result in channelling of DSB repair towards HR. To address this question, we analyzed the formation of RAD51 foci, a well-established indicator of repair by HR. Following treatment with etoposide, the average number of Rad51 foci per cell was ,3-fold higher in Tdp2 D1–3 than in wild-type MEFs (Figure 5A), in agreement with an increase in the use of HR to repair TOP2-induced DSBs when TDP2 is not present. Furthermore, we compared the frequency of etoposide-induced sister chromatid exchanges (SCEs), a molecular hallmark of HR [31], in wild type and Tdp2 D1–3 MEFs (Figure 5B). Notably, SCE levels increased substantially in transformed MEFs following acute etoposide exposure, being significantly higher in Tdp2 D1–3 cells at two etoposide concentrations tested (1 and 2.5 mM). These data confirm that, upon etoposide treatment, the frequency of HR is elevated in Tdp2 D1–3 MEFs, consistent with TDP2 functioning in NHEJ. Elevated hypersensitivity to TOP2-induced DNA damage in Tdp2 D1–3 mice To address the relevance of TDP2-mediated repair of TOP2- induced DSBs in vivo, we compared the impact of etoposide on adult (8 wk) wild type and Tdp2 D1–3 mice. A single intraperitonal injection of etoposide (75 mg/kg) caused a decrease in body weight in the initial 4 days post-treatment both in wild type and Tdp2 D1–3 animals (Figure 6A). However, whereas Tdp2 +/+ mice exhibited relatively mild and transient weight loss, Tdp2 D1–3 littermates lost weight progressively and were sacrificed at day 6 to prevent suffering. No differences in body weight were observed between mock-treated (with DMSO) wild type and Tdp2 D1–3 mice. Histopathological analysis of Tdp2 D1–3 mice sacrificed 6 days after etoposide treatment revealed marked villous atrophy in the small intestinal mucosa as the likely cause of the drastic weight loss (Figure 6B). This was not observed in either wild-type and/or DMSO treated animals (data not shown), suggesting a protective role for TDP2 against adverse effects of etoposide in vivo. TOP2-induced DNA damage results in increased lymphoid toxicity in Tdp2 D1–3 mice In addition to severe intestinal damage, etoposide administration resulted in elevated splenic and thymic atrophy in Tdp2 D1–3 mice, compared to wild type mice (Figure 6C), consistent with the known hypersensitivity of these organs to this drug [32]. Histological analysis of these tissues revealed a marked reduction in the cellular content in Tdp2 D1–3 animals (Figure 6C, right, note the low density of dark-stained nuclei). In light of these results, we analysed B-cell and T-cell maturation in wild type and Tdp2 D1–3 mice (Figure 6D and Figure S5). In the case of B-cell precursors in bone marrow, treatment with etoposide resulted in a decrease of 30–50% in the fraction of cells that were CD43 + B220 + progenitors (Pro-B cells) and a decrease of .95% in the fraction of cells that were CD43 2 B220 low (Pre-B cells) or CD43 2 B220 high (immature B cells) precursors. In all cases the reduction in B-cell precursors was greater in Tdp2 D1–3 mice, but the differences were not statistically significant at the administered dose. In contrast, in the case of T-cell maturation, whereas etoposide treatment reduced the fraction of CD4 + CD8 + immature T cells by 30– 40% in wild type mice, these cells were almost completely eliminated in Tdp2 D1–3 mice (Figure 6A, bottom right). No effect was observed in CD11b/Mac-1 + myeloid cells in the bone marrow (Figure S6). Taken together, these results suggest that loss of TDP2 increases cellular attrition in the lymphoid system, particularly in the T-cell lineage, in response to TOP2-induced DNA damage. Tdp2 D1–3 mice display increased TOP2-induced genome instability in bone marrow A major side-effect of cancer therapy employing TOP2 poisons is secondary hematological malignancy, and in particular acute leukemia, resulting most likely from error prone/erroneous repair of TOP2-induced DSBs and chromosome translocations [4,7]. Given our findings that TDP2 limits genome rearrangements induced by etoposide in cells, we examined whether TDP2 also promotes genome stability in bone marrow in vivo. We quantified the fraction of micronucleated polychromatic erythrocytes (PCEs) in bone marrow smears from Tdp2 D1–3 and Tdp2 +/+ mice 24 hour after intraperitoneal injection of etoposide (1 mg/kg). The rodent erythrocyte micronucleus test is a standard procedure to detect cytogenetic damage in toxicological studies and is based on the detection of micronuclei in erythrocyte precursors (Hayashi et al 1994). As expected, etoposide increased the fraction of PCEs that were micronucleated in both wild type and Tdp2 D1–3 animals TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 6 March 2013 | Volume 9 | Issue 3 | e1003226 Figure 4. The absence of TDP2 increases etoposide-induced genome instability in mammalian cells. A. Micronuclei (MN, left) and nucleoplasmic bridges (NB, right) in binucleated (following cytochalasin B-mediated cell cycle arrest) Tdp2 +/+ and Tdp2 D1–3 transformed MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for representative images. Histogram bars represent the average 6 s.e.m. of n$600 cells coming from three independent experiments. Statistical significance by Mann-Whitney test. B. Primary MEFs in the absence of cytochalasin B treatment (n$1500). Other details as in ‘‘A’’. C. Break-type (left) and exchange-type (right) chromosomal aberrations in transformed Tdp2 +/+ and Tdp2 D1–3 MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for a representative image. Plots show the number of breaks/exchanges per 100 chromosomes from individual metaphase spreads (n = 100) obtained in at least two independent experiments. Average 6s.e.m. and statistical significance by Mann-Whitney test is also indicated. D. Metaphase spreads from primary MEFs (n = 50). Caffeine was added 4 h after etoposide treatment. Other details as in ‘‘C’’. doi:10.1371/journal.pgen.1003226.g004 TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 7 March 2013 | Volume 9 | Issue 3 | e1003226 (Figure 7). However, this increase was ,2-fold higher in Tdp2 D1–3 mice than in wild type mice, suggesting that TDP2 protects heamatopoietic cells from genome instability induced by anticancer TOP2 poisons. Discussion TDP2 is the major 59-tyrosyl DNA phosphodiesterase in mammals In the current study we observe that Tdp2 deletion ablates detectable 59-TDP activity in different mouse tissues and MEFs, consistent with our previous observations in DT40 cells [25]. It is worth noting that other roles have been assigned to this protein, in other cellular processes such as signal transduction and transcriptional regulation [33]. So far, however, we have been unable to detect any spontaneous phenotype caused by TDP2 loss, either at cellular level or in vivo, while dramatic effects are observed upon etoposide treatment. This suggests that the most important function of TDP2, following Top2 induced DNA damage at least, is related to the 59TDP activity of this enzyme. Additionally, our data suggest that alternative, TDP2–independent, mechanisms of DSB repair are sufficient to cope with the endogenous level of TOP2 damage arising during normal mouse development and life. A role for human TDP1 in repairing TOP2-induced DSBs was recently suggested by a weak 59-TDP activity of human recombinant protein on DSBs possessing 4-bp 59-overhangs, and on a mild sensitivity of TDP1 2/2 DT40 cells to etoposide [27]. This is also consistent with the increased resistance to etoposide reported in cells highly overexpressing TDP1 [34], and with the reported 59-TDP activity of Tdp1 in Saccharomyces cerevisiae [35]. However, while our standard activity assays employs DSBs with blunt-ended 59-phosphotyrosyl termini, in the current study we similarly failed to detect residual 59-TDP activity in Tdp2 D1–3 MEF extracts on DSB substrates with 4-bp 59-overhangs (Figure 2C). In addition, in our hands, TDP1 2/2 DT40 cells are not hypersensitive to etoposide, and deletion of TDP1 in TDP2 2/2/2 DT40 cells does not increase sensitivity to etoposide above that observed by TDP2 deletion alone [36]. Consequently, we conclude that TDP2 is the major if not only 59-TDP activity in mammals (as in DT40 chicken cells), at physiologically relevant enzyme concentrations at least. TDP2 is required for survival and efficient repair upon induction of TOP2-mediated DSBs in mammals We have shown that Tdp2-deleted mouse cells are hypersensitive to TOP2-induced DNA damage, but not to ionizing radiation, in agreement with previous results with TDP2 2/2/2 DT40 cells [25]. Moreover, we demonstrate that this hypersensitivity correlates with a defect in the repair of etoposide-induced DSBs, as measured by immunostaining for sites of cH2AX, which suggests that TDP2-mediated repair promotes tolerance to TOP2-induced DNA damage in mammalian cells. Remarkably, we observed that TDP2 is required for resistance to TOP2-induced DNA damage not only at the cellular level, but also at the whole-organism level. Indeed, etoposide administration in Tdp2 D1–3 mice resulted in both increased mortality due to intestinal damage and in elevated Figure 5. The absence of TDP2 increases etoposide induced homologous recombination. A. Total number of foci per RAD51 focicontaining cell in Tdp2 +/+ and Tdp2 D1–3 primary MEFs following 30 min 10 mM etoposide treatment and 2 h recovery (left). Replicating cells were excluded from the analysis. A representative image is shown (right). Average 6s.e.m. from 3 independent experiments and statistical significance by T test is indicated. B. Sister chromatid exchanges (SCEs) scored in Tdp2 +/+ and Tdp2 D1–3 transformed MEFs after 30 min acute treatment with the indicated concentration of etoposide. Plots show the number of SCEs per chromosome from individual metaphase spreads (n$50) obtained in at least two independent experiments. Average 6s.e.m. and statistical significance by Mann-Whitney test is also indicated. doi:10.1371/journal.pgen.1003226.g005 TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 8 March 2013 | Volume 9 | Issue 3 | e1003226 Figure 6. The absence of TDP2 causes etoposide sensitivity in vivo .A. 8-week old wild-type and Tdp2 D1–3 littermates were intraperitoneally injected with a single 75 mg/kg dose of etoposide or vehicle (DMSO) and body weight was recorded in the following 6 days. Average 6s.e.m. of the percentage of initial body weight from at least 8 mice and statistical significance by One-way ANOVA with Bonferroni post-test is shown. B. Representative image of hematoxylin-eosin stained jejunum slices from wild-type and Tdp2 D1–3 animals 6 days after etoposide treatment. C. TDP2 and Genome Stability PLOS Genetics | www.plosgenetics.org 9 March 2013 | Volume 9 | Issue 3 | e1003226