TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo

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 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.

Figure 1. TDP2 promotes survival following TOP2-induced DSBs.

A. Clonogenic survival of the indicated DT40 cell line; wild-type, TDP2^−/−/− and TDP2^−/−/− 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 ± s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose when compared to TDP2^−/−/− cells by Two-way ANOVA with Bonferroni post-test 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^flΔ) 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 (+/flΔ) and knock-out (flΔ/flΔ, from now on denoted Tdp2^Δ1–3) mice, using the indicated probe (red line), is shown (bottom right). C. Clonogenic survival of wild-type and Tdp2^Δ1–3 transformed MEFs after 3 h acute exposure to the indicated concentrations of etoposide (left) or the indicated dose of γ-irradiation (right). Average ± s.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).

Figure 2. Deletion of Tdp2 in mouse abolishes 5′-TDP activity and ligation of 5′ phosphotyrosine-blocked ends.

A. Duplex substrate harbouring a 5′phosphotyrosine blunt end (left) was incubated with 9 µg Tdp2^+/+ or Tdp2^Δ1–3 tissue extract from bone marrow (BM), thymus and spleen for 1 h. B. Substrate in “A” was incubated with 1.5 µg of cellular extract from Tdp2^+/+ or Tdp2^Δ1–3 primary MEFs for the indicated time. C. Duplex substrate harbouring a 5′phosphotyrosine self-complementary overhang end (left) was incubated with 10 µg cellular extract from Tdp2^+/+ or Tdp2^Δ1–3 transformed MEFs for 2 h in the presence or absence of 50 mM EDTA. D. Self-ligation of 5′ phosphate (P) and 5′ phosphotyrosine (Y–P) overhang substrates as depicted in “C” incubated for 1.5 h with 3.3 µg cellular extract from Tdp2^+/+ or Tdp2^Δ1–3 transformed MEFs in the presence of T4 DNA ligase. In all cases migration of the 5′ phosphotyrosine substrate (Y–P), 5′ phosphate (P) and ligation (lig) products are indicated. E. Circularization efficiency of a linear plasmid with 5′ phosphotyrosine (Y–P) and 5′ phosphate (P) catalysed by Tdp2^Δ1–3 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 µg of initial substrate DNA (average ± s.e.m. of three independent experiments) is shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated is shown.

Figure 3. TDP2 promotes repair of TOP2-induced DSBs by NHEJ.

A. Clonogenic survival of wild-type, TDP2^−/−/−, KU70^−/− and TDP2^−/−/− KU70^−/− DT40 cells following continuous treatment with the indicated concentrations of etoposide. Average ± s.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. γH2AX foci induction after 30 min 20 µM etoposide treatment and repair at different times following drug removal in confluency arrested Tdp2^+/+ and Tdp2^Δ1–3 primary MEFs. Representative images of the 3 h repair time point including DAPI counterstain (right) and average ± s.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 µM etoposide treatment. Other details as in “C”. E. Confluency arrested primary MEFs exposed to 2Gy γ-irradiation. Other details as in “C”.

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^Δ1–3 transformed MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for representative images. Histogram bars represent the average ± 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^Δ1–3 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 ± s.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”.

Figure 5. The absence of TDP2 increases etoposide induced homologous recombination.

A. Total number of foci per RAD51 foci-containing cell in Tdp2^+/+ and Tdp2^Δ1–3 primary MEFs following 30 min 10 µM etoposide treatment and 2 h recovery (left). Replicating cells were excluded from the analysis. A representative image is shown (right). Average ± s.e.m. from 3 independent experiments and statistical significance by T test is indicated. B. Sister chromatid exchanges (SCEs) scored in Tdp2^+/+ and Tdp2^Δ1–3 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 ± s.e.m. and statistical significance by Mann-Whitney test is also indicated.

Figure 6. The absence of TDP2 causes etoposide sensitivity in vivo.

A. 8-week old wild-type and Tdp2^Δ1–3 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 ± s.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^Δ1–3 animals 6 days after etoposide treatment. C. Macroscopic (left) and histological (right) representative image of spleen and thymus from wild-type and Tdp2^Δ1–3 animals 6 days after treatment. Average weight of these organs ± s.e.m. and statistical significance by Two-way ANOVA with Bonferroni post-test is shown (centre). D. FACS analysis of B-cells in bone marrow (top and bottom-left) and T-cells in thymus (bottom right) in wild-type and Tdp2^Δ1–3 animals 6 days after treatment. See insets to compare etoposide treated samples when required. Average percentage of the indicated cell type among the total number of cells in the corresponding tissue ± s.e.m. of at least 3 animals and statistical significance by Two-way ANOVA with Bonferroni post-test is shown.

Figure 7. The absence of TDP2 increases etoposide-induced genome instability in vivo.

Percentage of micronucleated polychromatic erythrocytes (MN-PCE) among the total number of polychromatic erythrocytes (PCE), examples of which are shown (right), in bone marrow smears of wild-type and Tdp2^Δ1–3 mice 24 h after intraperitoneal injection of 1 mg/kg etoposide or vehicle (10% DMSO). Average ± s.e.m. of 4 (DMSO) and 6 (VP16) animals and statistical significance by paired T test is shown.

Figure 8. Model for the repair of TOP2-induced DSBs.

(a) TDP2-mediated cleavage of the 5′ phosphodiester link between TOP2 peptide and DNA results in 3′ hydroxyl (OH) 5′ phosphste (P) cohesive ends that are easily ligatable by NHEJ resulting in error-free repair. (b) Alternatively, nucleolytic attack on the DNA backbone can also remove the protein adduct from the DSB but genetic information is lost from the ends. Accurate repair of this break would therefore need HR to copy the missing information from the sister chromatid, while NHEJ would result in error-prone repair.