Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a nuclear and mitochondrial protein that in nuclei and in vitro repairs blocked 3′ DNA termini such as 3′ phosphotyrosine conjugates resulting from stalling of topoisomerase I-DNA intermediates. Its mutation also causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Because Tdp1 colocalizes with mitochondria following oxidative stress, we hypothesized that Tdp1 repairs mitochondrial DNA (mtDNA) and that mtDNA damage mediates entry of Tdp1 into the mitochondria. To test this, we used S. cerevisiae mutants, cultured mouse and human cells, and a Tdp1 knockout mouse. H2O2- and rotenone-induced cellular and intramitochondrial reactive oxygen species (ROS) activated oxidant-responsive kinases P38 and ERK1, and the translocation of Tdp1 from the nucleus to the mitochondria via the TIM/TOM complex. This translocation occurred independently of mtDNA. Within the mitochondria, Tdp1 interacted with Ligase III and reduced mtDNA mutations. Tdp1-deficient tissues had impaired mitochondrial respiration and decreased viability. These observations suggest that Tdp1 maintains mtDNA integrity and support the hypothesis that mitochondrial dysfunction contributes to the pathology of SCAN1.
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Introduction
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a 3′-DNA phosphodiesterase that participates in DNA repair. It resolves stalled topoisomerase I-DNA complexes by cleavage of the 3′-phosphotyrosine bond. Less characterized functions of Tdp1 include the processing of endogenous 3′-DNA lesions such as alkylated bases, phosphoamides and chain-terminating nucleosides1,2,3,4,5,6,7,8.
Tdp1 colocalizes with mitochondria in human and mouse tissue and in cultured human and mouse cells9,10. We previously showed that mitochondrial colocalization of Tdp1 is enhanced by exposure to hydrogen peroxide, suggesting that Tdp1 enters the mitochondria in response to mitochondrial DNA (mtDNA) stress and mediates mtDNA repair9,10,11.
Compared to nuclear DNA, maintenance of mtDNA has several challenges. First, 95% of mtDNA is gene coding, whereas approximately 1–2% of nuclear DNA is12. Second, compared to nuclear DNA, mtDNA has increased exposure to mutagens because it lacks protective histones and DNA compaction13. Third, mtDNA is in close proximity to the reactive oxygen species (ROS) generated by mitochondrial respiration14. Fourth, compared to the nucleus, mitochondria lack certain DNA repair pathways15.
Mitochondria produce oxidative agents as a by-product of electron transport chain activity. The generation of volatile oxidants during respiration causes polymerase-blocking oxidative lesions in mtDNA such as thymine glycols and 8-oxoguanine16,17. The 8-oxoguanine lesions cause guanine to thymine transversions18 and mitochondrial dysfunction by mutation of mtDNA-encoded proteins, tRNAs, and rRNAs. Mitochondrial lysates from breast cancer cells demonstrate base-excision repair (BER) activity9, a repair mechanism for oxidative lesions. Because Tdp1 participates in nuclear BER and colocalizes with mitochondria, it has been hypothesized that Tdp1 also functions in mitochondrial BER6,9,10.
H2O2-induced ROS activates stress-activated protein kinase (SAPK) signaling for import of proteins into the mitochondria. The activation of P38 MAP kinase mediates translocation of Bax to mitochondria where it oligomerizes with Bak and forms a pore on the mitochondrial outer membrane, allowing the release of cytochrome C and other pro-apoptotic proteins19. H2O2 also mediates crosstalk between mitochondria and neighboring organelles and, by peroxidation of mitochondrial membrane lipids, regulates secondary redox signaling in the cytoplasm20,21.
Herein we show that within S. cerevisiae, mouse and human cells, ROS enhances Tdp1 entry into the mitochondrial matrix and that this is independent of new protein synthesis and mtDNA. We also find that intramitochondrial ROS is a signal for translocation of Tdp1 out of the nucleus and acts through P38 and ERK1. Within mitochondria, Tdp1 interacts with the BER protein Ligase III. Consistent with a role for Tdp1 in mtDNA repair, embryonic fibroblasts and myoblasts derived from Tdp1−/− mice have more mtDNA lesions and impaired mitochondrial respiration compared to those derived from wt tissues.
Materials and Methods
Some methods and materials are defined below, whereas others are described in the Supplementary Methods.
Animal subjects
All mice used in this study were housed, bred, sacrificed and studied in accordance to approved and ethical guidelines of the Institutional Review Board of University of British Columbia (protocol # A15–0017). Tdp1−/− mice used in this study were generated as previously described22.
Cell culture
Human dermal fibroblasts were obtained from DSMZ in Germany. Mouse embryonic fibroblasts were harvested from mouse embryos as previously described22. Both cell lines were cultured in DMEM (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated FBS (Hyclone) and 1% antibiotic-antimycotic (Gibco BRL Life Technologies). All cells were grown at 37 °C with 5% CO2 in a humidified environment.
MTT assay
Cell proliferation and viability were measured by MTT assay as previously described23. Briefly, 5 × 103 cells were plated in each well of a 96-well plate and cultured in phenol red-free media. Following treatment of cultured cells, the culture media was supplemented with 10% MTT and incubated for 3 hours. This was followed by a 30-minute incubation in DMSO. Spectrophotometry was done at 565 nm using the Wallac VICTOR2 Multilabel Plate Reader (Beckman-Coulter).
Immunoblotting
Immunoblotting was performed as described22. Briefly, cells were lysed in SDS-sample buffer and denatured by boiling for 5 minutes. The lysate was fractionated on a 10% or 15% gel by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated overnight at 4 °C with blocking solution (0.2% I-Block (Applied Biosystems) and 0.1% Tween 20 in 1x PBS) and gentle agitation. The membrane was then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. After washing, the membranes were incubated with secondary antibodies for 1 hour at room temperature. The signal was detected with the CDP-StarTM chemiluminescent system (Applied Biosystems). The antibodies used for immunoblotting are listed in Supplementary Table 1.
Quantitative immunoblotting
Quantitative fluorescent immunoblotting was performed as previously described24. Briefly, 20 µg of total protein was separated by SDS-PAGE on a 10% gel and then transferred to a PVDF membrane. Subsequently, the membrane was rocked with a blocking solution (1:1 mixture of LI-COR Odyssey blocking buffer and 1 × phosphate buffered saline) for 1 hour at room temperature and incubated for 1 hour at room temperature with primary antibodies diluted in blocking solution and 0.01% Tween 20 (Supplementary Table 1). After washing four times for 5 minutes in washing solution (1x PBS, 0.01% Tween 20), the membrane was incubated for 1 hour at room temperature with secondary antibodies diluted in blocking solution plus 0.01% Tween 20. After washing four times for 5 minutes in washing solution, a final 5-minute wash was performed with 1 × PBS. A laser intensity of 5 for the appropriate channels was used as default when the membrane was scanned. The IRDye® 800 CW Goat anti-Mouse IgG and IRDye® 800 CW Goat anti-Rabbit IgG antibodies were used at a dilution of 1:10,000.
Immunofluorescence microscopy
Immunofluorescence microscopy was performed as described22. Briefly, approximately 5 × 105 cells were grown on a cover slip. Human or mouse cells were washed with 1 × PBS and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB, 22.46 mM NaH2PO4, 77.54 mM Na2HPO4, pH 7.4) for 15 minutes. Cells were permeablized with 0.5% Triton X-100 in 1 × PBS for 15 minutes, blocked for 1 hour at room temperature with blocking buffer (20% horse serum, 1% casein in 1 × PBS), and then incubated overnight at 4 °C with the primary antibodies (Supplementary Table 1). All images were acquired using the Leica TCS SP5 II confocal microscope.
Immunoprecipitation of Tdp1
Briefly, we used the FLAG® HA Tandem Affinity Purification Kit (Sigma-Aldrich) to immunoprecipitate Tdp1-interacting proteins in the mitochondria of cultured MEFs. Preparation of mitochondrial lysate was performed as described in the Supplementary Methods. Preparation and immobilization of the FLAG and HA-tagged Tdp1 protein, as well as capture and elution of prey protein was performed as per the manufacturer’s protocol. The presence of Tdp1-interacting proteins in the eluate was determined by SDS-PAGE and immunoblotting.
Yeast strains
Yeast strains were cultured as described in their source publications. Single gene mutants were obtained from the yeast deletion collection25. The TDP1-GFP strain was obtained from the Yeast GFP collection26. Temperature-sensitive knockout strains of essential genes TIM23 and MIA40 were obtained from the temperature sensitive mutant repository27.
Transfection of cultured cells with siRNA
Human dermal fibroblasts were transfected with ERK1 siRNA (Thermo Scientific/Dharmacon; L-003592-00-0005), p38 siRNA (Cell Signaling; 6562 S) or Tim23 siRNA (Thermo Scientific/Dharmacon; L-190121-00-0005). Rho-zero MEFs were transfected with Erk1 siRNA (Thermo Scientific/Dharmacon; L-040126-00-0005) or p38 siRNA (Cell Signaling; 6417 S). All siRNAs were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol and incubated in appropriate culture media for 48 hours prior to experimentation. Cells were transfected with non-targeting siRNA as control (Thermo Scientific/Dharmacon; D-001810-01-05).
Oligonucleotide assay for Tdp1 activity
Tdp1 enzymatic activity was determined by cleavage of an artificial substrate as described28. Briefly, the assay was run at room temperature in a 96-well plate with a final volume of 100 µL per well. A final concentration of 50 nM DNA was used (5′-/56-TAMN/AGG ATC TAA AAG ACT T/3BHQ_1/-3′). Kinetic analysis was performed using a Varioskan plate reader (Thermo Fisher Scientific) at Ex557/Em582 for the TAMRA fluorophore. To establish Tdp1 cleavage activity, 20 reads were recorded at a kinetic interval of 45 seconds per read.
Homogeneous caspase assay for apoptotic cells
The activity of caspases 3 and 7, which play key effector roles in mammalian cell apoptosis, was measured using the Apo-ONE Homogeneous Caspase-3/7 kit according to the manufacturer’s protocol (Promega). Briefly, 5 × 103 cells were plated per well of a 96-well plate, cultured for 24 hours and treated as described. Fluorescence of the cleaved caspase 3 and 7 substrate was measured at 520 nm using the Wallac VICTOR2 Multilabel Plate Reader (Beckman-Coulter).
TUNEL assay for apoptotic cells
Briefly, 5 × 103 cells were seeded per well in 6-well plates, and following the described treatments, apoptotic cells were visualized by colorimetric labeling of free 3′OH DNA termini using the Apoptag Plus Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer’s protocol (EMD Millipore). 150 cells were counted per sample group. The number of stained nuclei among 150 cells was determined for three independent replicates to quantify apoptotic cells.
Site-directed mutagenesis of Tdp1
The p.S81A mutation was introduced into pcDNA3.1(−)•TDP1WT by site-directed mutagenesis according to the manufacturer’s protocol using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). Tdp1−/− MEFs were transfected with recombinant WT or mutant Tdp1 using Lipofectamine 3000 (Invitrogen). The sequences of the oligonucleotides used are listed in Supplementary Table 2. The mutant vector was validated by Sanger sequencing (Macrogen).
Statistical analysis
Microsoft Excel was used to compute the group means and SDs for all treatment and control groups. Statistical significance between individual groups was established by the Student t test and a P value of less than 0.05 was judged significant. The Bonferroni correction was applied to all comparisons of replicate means for the duration of the experiments.
Results
H2O2 treatment of cells causes Tdp1 to enter the mitochondria
In a previous study, we found that the colocalization of Tdp1 with mitochondria in MEFs and human dermal fibroblasts is enhanced upon treatment with H2O210. We treated both human dermal fibroblasts and MEFs with varying concentrations of H2O2 and found that a non-lethal dose of 1 µM H2O2 for 24 hours enhances colocalization of Tdp1 and mitochondria (Supplementary Figure 1a and b). To determine whether Tdp1 was associating with mitochondria or entering the mitochondrial matrix, we treated mitochondria purified from H2O2-exposed human dermal fibroblasts with proteinase K in the presence and absence of Triton X-100. Tdp1 was only digested by proteinase K following permeabilization of the mitochondrial membrane with Triton X-100 (Fig. 1a), suggesting that H2O2 treatment causes Tdp1 to enter the mitochondria. The BER protein Ligase III is observed in the mitochondrial matrix, whereas XRCC1 is not (Fig. 1a).
Entry of Tdp1 into the mitochondria is not dependent on de novo protein synthesis
To determine whether Tdp1 entry into the mitochondria required de novo protein synthesis, we treated human dermal fibroblasts with 10 µM cycloheximide, a ribosome translocation inhibitor. Ubiquitylated proteins accumulated in cycloheximide-treated human fibroblasts, indicating blocked protein synthesis (Fig. 1b). As detected by immunoblotting and immunofluorescence, mitochondrial accumulation of Tdp1 increased upon cycloheximide and H2O2 treatment (Fig. 1c and d). Consistent with translocation of Tdp1 from the nucleus to the mitochondria, live-cell imaging of H2O2-treated human dermal fibroblasts showed loss of Tdp1 from the nucleus and increased Tdp1 accumulation within the mitochondria (Fig. 1e).
Elevated intramitochondrial ROS is sufficient to increase mitochondrial Tdp1 accumulation
To determine whether mitochondrial Tdp1 accumulation is correlated with elevated mitochondrial superoxide levels, we stained human dermal fibroblasts with the superoxide indicator mitoSOX. Compared to untreated cells, treatment of cells with H2O2, or with 10 µM rotenone, a mitochondrial complex I inhibitor that increases levels of intramitochondrial oxidants29, resulted in elevated mitochondrial superoxide levels as well as mitochondrial Tdp1 accumulation (Fig. 1f). We postulate that accumulation of Tdp1 within the mitochondria did not occur because rotenone dissipates the proton motive force needed for transfer of proteins into the intermembrane space and occludes the mitochondrial outer membrane import channel30,31.
Tdp1 translocation is not associated with activation of caspases 3 and 7
Although ROS stress can initiate mitochondria-dependent apoptosis32,33, neither rotenone nor H2O2 treatment caused an elevation in cellular caspase-3/7 activity (Fig. 1g, left panel) or nuclear DNA fragmentation (Fig. 1g, right panel). This minimizes the likelihood that Tdp1 translocation is a response to apoptotic mitochondrial signaling.
Mitochondrial Tdp1 interacts with BER constituents and maintains mtDNA integrity
We hypothesized that Tdp1 interacts with mtDNA repair proteins to maintain mtDNA integrity. To test for interaction with DNA repair proteins, we expressed FLAG-HA-tagged Tdp1 in human dermal fibroblasts and immunoprecipitated the tagged Tdp1 from purified mitochondria following treatment of the cells with H2O2. To remove proteins co-purifying with but external to the mitochondria, the purified mitochondria were digested with proteinase K. Following inactivation of the proteinase K with PMSF, the mitochondria were washed and then lysed. The lysates were used for anti-FLAG immunoprecipitation of the FLAG-HA-tagged Tdp1. By immunoblotting, we detected co-precipitation of Ligase III with Tdp1 (Fig. 2a), consistent with Tdp1 being a component of the mitochondrial BER complex34,35.
To determine if Tdp1 deficiency compromised mtDNA integrity, we harvested MEFs from Tdp1−/− mice and assessed the level of polymerase-impeding lesions, random mutations, and deletions within the mtDNA36,37,38. As assessed by interference with Taq polymerase extension, both wt and Tdp1−/− cells had comparable amounts of baseline lesions (Fig. 2b, left panel). After 4 weeks of culture, Tdp1−/− MEFs harbored approximately 30% more mtDNA lesions than wt MEFs (Fig. 2b, left panel), and after 4 weeks of culture in the presence of 1 µM H2O2, Tdp1−/− MEFs had 200% more mtDNA lesions than wt MEFs (Fig. 2b, left panel). To assess accumulation of mtDNA point mutations, we digested the mtDNA with Taq1 and amplified the mtDNA using primers hybridizing to sequences flanking the Taq1 recognition sites. Because mutations of the Taq1 recognition sites block Taq1 digestion, the amount of PCR product is proportional to the abundance of Taq1 site mutations. This showed that Tdp1−/− and wt MEFs harbored similar quantities of mtDNA mutations at baseline. However, after culturing in the presence of 1 µM H2O2 for 4 weeks, Tdp1−/− cells had 40% more mtDNA point mutations than wt cells (Fig. 2b, right panel).
Because dysfunctional mtDNA repair and incomplete resolution of DNA breaks during mtDNA replication are thought to cause the formation of deleted mtDNA products and give rise to mitochondrial dysfunction39,40,41, we checked if the absence of Tdp1 favors the accumulation of mtDNA deletions. Using mtDNA extracted from wt, Tdp1−/−, and PolG null MEFs cultured in the presence or absence of 1 µM H2O2 for 4 weeks, we quantified two common mitochondrial deletion products (D1 and D13) as previously described37. Compared to wt MEFs, the Tdp1−/− MEFs did not have markedly more of these mtDNA deletions under baseline conditions or when treated with H2O2 (data not shown).
Tdp1 deficiency impairs mitochondrial respiration
To test whether the increase in mtDNA lesions and mutations correlated with mitochondrial respiratory dysfunction, we performed a Seahorse assay on mitochondria isolated from wt and Tdp1−/− MEFs. At baseline, the wt and Tdp1−/− MEF mitochondria had no significant difference in oxygen consumption rate, whereas following 4 weeks of treatment with 1 µM H2O2, Tdp1−/− MEF mitochondria consumed 30% less oxygen than wt MEF mitochondria (Fig. 2c). Similarly, despite being comparable at baseline, ATP production from Tdp1−/− MEF mitochondria was 25% less than from wt MEF mitochondria after 4 weeks of treatment with 1 µM H2O2 (Fig. 2d).
Tdp1 translocates into mitochondria devoid of mtDNA
The above data define a role for Tdp1 in maintenance of mtDNA and suggest that mtDNA stress is a possible signal for Tdp1 translocation into the mitochondria. To test this, we generated Rho-zero MEFs, cells devoid of mtDNA by treatment with ethidium bromide, and validated the absence of mtDNA by loss of expression of the mtDNA-encoded complex IV subunit by qRT-PCR and by loss of mtDNA staining with PicoGreen (Fig. 2e). H2O2 treatment of the Rho-zero MEFs caused translocation of Tdp1 to the mitochondria similar to that observed in wt MEFs (Fig. 2f). In the mitochondria of Rho-zero cells, FLAG-HA-tagged Tdp1 formed a complex with Ligase III (Fig. 2g), and the intramitochondrial Tdp1 was enzymatically active as judged by cleavage of a 3′-quencher from a fluorescent oligonucleotide28 (Fig. 2h). Tdp1 translocation into the mitochondria is thus not mediated by mtDNA.
S. cerevisiae screen identifies P38, ERK, the TIM/TOM complex and Mia40 as contributors to H2O2-dependent translocation of Tdp1 into the mitochondria
To determine if intramitochondrial ROS is the origin of the signal for translocation of Tdp1, we used S. cerevisiae to screen for factors participating in the H2O2-dependent translocation of Tdp1 to the mitochondrial matrix. After 5 µM H2O2 treatment, we observed Tdp1 expression in the cytoplasm of yeast by fluorescence microscopy and in the mitochondrial fraction by immunoblotting (Fig. 3a and b). H2O2 exposure is a known activator of Rho5, mitogen-activated protein (MAP) kinase P38, ERK1 and JNK142. P38 and ERK1 are respectively orthologs of yeast HOG1 and FUS3. Knocking out each of HOG1 and FUS3, but not JNK1, reduced Tdp1 translocation into the mitochondria by 70–80% at 1 hour following exposure to 5 µM H2O2 (Fig. 3c). Indicative of HOG1 and FUS3 redundantly regulating Tdp1 translocation, the double-knockout of HOG1 and FUS3 inhibited Tdp1 translocation more than the individual knockouts (95% vs. 70–80%, Fig. 3d). Suggesting that Rho5 mediates this activation of HOG1 and FUS3, knockout of Rho5 alone and double-knockout of RHO5 and HOG1 reduced Tdp1 translocation into the mitochondria by 95% at 1 hour following exposure to 5 µM H2O2 (Fig. 3e).
Knockout of the mitochondrial intermembrane transporter MIA40, a redox-responsive protein43, reduced H2O2-dependent translocation of Tdp1 by 85% (Fig. 3f). Knockout of TIM23, a subunit of the inner membrane translocase of the canonical mitochondrial protein import complex completely abrogated Tdp1 import (Fig. 3f). These results indicate that Tdp1 transport proceeds through the canonical TIM-TOM complexes and is chaperoned through the intermembrane space by Mia40 (Fig. 3g).
siRNA knockdown of P38, ERK1, and TIM23 impedes H2O2-dependent translocation of Tdp1 into the mitochondria of human dermal fibroblasts
Similar to S. cerevisiae, exposure of cultured human dermal fibroblasts to H2O2 activates mitogen-activated protein (MAP) kinases P38, ERK1 and JNK (the respective human orthologs of HOG1, FUS3, JNK1)44. We found that exposure of human dermal fibroblasts to 1 µM H2O2 for 1 hour promoted the maximal phosphorylation of P38 and ERK1 but did not promote the phosphorylation of JNK (Supplementary Figure 1c). Given that Tdp1 translocated into the mitochondria within 6–7 minutes at 1 µM H2O2 (Fig. 1e), we focused on P38 and ERK1 as mediators of H2O2-dependent mitochondrial Tdp1 translocation. Using siRNA, an 80% knockdown of P38 resulted in a 50% reduction in H2O2-mediated mitochondrial Tdp1 translocation, and a 70% knockdown of ERK1 resulted in a 20% reduction in H2O2-dependent mitochondrial Tdp1 translocation (Fig. 4a and Supplementary Figure 1d). Concurrent 80% knockdown of P38 and ERK1 resulted in an 80% reduction in H2O2-dependent mitochondrial Tdp1 translocation (Fig. 4c). Similarly, knockdown of P38 or ERK1 in Rho-zero MEFs also impaired H2O2-dependent transport of Tdp1 into the mitochondria (Fig. 4b).
The decrease in mitochondrial Tdp1 after P38 and ERK1 knockdown is due to retention of nuclear Tdp1 (Supplementary Figure 2a and b). Confirming that intramitochondrial ROS generated by addition of 1 µM rotenone activated the same pathways, 80% knockdown of P38 or 75% knockdown of ERK1 increased the retention of Tdp1 in the nucleus (Supplementary Figure 2c and d).
P38 and ERK1 translocate from the cytoplasm into the nucleus upon phosphorylation and activate nuclear substrates45,46. Within 5 minutes of adding 1 µM H2O2 or 1 µM rotenone to human dermal fibroblasts, we detected increased nuclear P38 and ERK1 (Fig. 4d). This timing is consistent with Tdp1 translocation out of the nucleus and into the mitochondria (Fig. 1e) and suggests that P38 and ERK1 activate nuclear targets facilitating Tdp1 translocation from the nucleus to the mitochondria.
Consistent with the yeast studies, siRNA knockdown of TIM23 in human dermal fibroblasts reduced Tdp1 translocation into the mitochondria and resulted in Tdp1 aggregation in the cytoplasm (Fig. 4d). Mitochondrial import of Tdp1 via the canonical TIM-TOM pathway is thus conserved between human and yeast cells.
Tdp1 does not contain a canonical mitochondria-targeting presequence identifiable by mitochondrial targeting sequence predictors TargetP, MitoProt and MitoFates (data not shown). Additionally, serial truncations from either the N- or C-terminus of Tdp1 did not identify a distinct mitochondrial-targeting signal; rather they identified contributions from multiple regions of the Tdp1 protein to Tdp1 mitochondrial entry (data not shown). In addition, site-directed mutagenesis of Ser81, a phosphorylation site in Tdp1, showed that changing serine 81 to alanine was insufficient to block H2O2-dependent translocation of Tdp1 into the mitochondria (Fig. 4e)47.
siRNA knockdown of Tdp1, P38, ERK1, and TIM23 increases H2O2-dependent mtDNA mutations in human dermal fibroblasts
Similar to our observation in Tdp1−/− MEFs, siRNA knockdown of Tdp1 in human fibroblasts led to an increase in random mtDNA mutations after treatment with H2O2 (Supplementary Figure 2e). Because P38, ERK1 and TIM23 enable the translocation of Tdp1 into mitochondria, we hypothesized that transient knockdown of any of these proteins increases random mtDNA mutations in human fibroblasts treated with H2O2. Indeed, knocking down P38, ERK1 and TIM23 in the presence of H2O2 led to an increase in random mutations by 45%, 20% and 65%, respectively, over the course of 96 hours. (Supplementary Figure 2e). Further study is required to determine if inhibition of these pathways contributes to the increase in mtDNA mutations by impeding transport of other DNA repair factors besides Tdp1.
Tdp1 −/− mice accumulate mutations in mtDNA and have impaired mitochondrial respiration
Given that long-term culture of Tdp1−/− MEFs in low dose H2O2 is detrimental to cultured cells, we examined the mtDNA integrity and the mitochondrial respiratory function of skeletal muscle from 1-month-old to 6-month-old Tdp1−/− mice. Analysis of polymerase-interrupting lesions in mtDNA showed a 70% to 400% increase in these lesions in 4-, 5- and 6-month-old Tdp1−/− mice (Fig. 5a). Quantification of mutations that inhibited Taq1 digestion showed that myoblast mtDNA from 1-month-old Tdp1−/− mice and wt mice had a comparable mutation burden (Fig. 5a), whereas myoblast mtDNA from 2-, 3-, 4-, 5- and 6-month-old Tdp1−/− mice had mtDNA mutation loads 20%–120% higher than wt mice myoblast mtDNA (Fig. 5b).
Since mutations in mtDNA-encoded genes can alter mitochondrial function, we examined mitochondrial respiration using myoblasts isolated from skeletal muscle. Comparative Seahorse analysis of myoblast cultures from wt and Tdp1−/− mice showed that Tdp1−/− myoblasts isolated from 3-, 4-, 5-, and 6-month-old mice were unable to achieve full respiratory potential; that is, uncoupling of the electron transport chain by FCCP treatment detected a 30–60% reduction in oxygen consumption rate in Tdp1−/− myoblasts compared to wt myoblasts (Fig. 5c).
Discussion
Since the discovery of Tdp1 as the main 3′-DNA phosphodiesterase, the biochemical activity of Tdp1 has been well characterized3,5,6,35,48,49,50,51,52,53. We herein report the first delineation of Tdp1 transport into the mitochondria and its significance for mitochondrial function. Within S. cerevisiae, mouse and human cells, intramitochondrial ROS induce Tdp1 translocation from the nucleus to the mitochondria via P38- and ERK1-dependent mechanisms. This translocation is independent of new protein synthesis and of mtDNA. Our findings also show that Tdp1 interacts with BER protein Ligase III within mitochondria and that cultured cells and tissues deficient for Tdp1 have more mtDNA lesions, accumulate mtDNA mutations, and have impaired mitochondrial respiration.
Given that exogenous rotenone stimulated the transport of Tdp1 out of the nucleus via the P38 and ERK1 signaling cascades, intramitochondrial ROS appears sufficient for Tdp1 translocation from the nucleus to the mitochondria. Unlike H2O2, however, rotenone treatment did not result in the entry of Tdp1 into the mitochondrial matrix. Two factors account for this failure. First, inhibition of complex I by rotenone blocks electron transfer between complex I and co-enzyme Q and prevents the concurrent transfer of hydrogen ions out of the matrix into the intermembrane space; this decreases the proton motive force driving transfer of proteins into the intermembrane space30. Second, rotenone impedes mitochondrial import by occluding the mitochondrial outer membrane import channel31.
Although intramitochondrial ROS likely signals translocation of Tdp1 from the nucleus to the mitochondrial matrix, the mediating MAP kinase P38 and ERK1 reside in the cytoplasm. This implies that either the intramitochondrial ROS or a signal from the intramitochondrial ROS crosses the mitochondrial membranes to activate these signaling cascades. A possible mediator is H2O2, a membrane-permeable signaling molecule produced by NADPH oxidases (NOX), cyclooxygenases and lipooxygenases54. H2O2 might therefore be the messenger activating the extra-mitochondrial pathways of stress-activated protein kinases; it also inhibits phosphatases that attenuate signaling of cell survival pathways, and in the nucleus it activates transcription factors by oxidation of cysteine residues55. Further studies are required to determine if H2O2 is the mitochondrial to cytoplasmic messenger mediating Tdp1 translocation.
The mechanisms by which the P38 and ERK1 signaling cascades trigger Tdp1 translocation from the nucleus to the mitochondrial matrix are unknown. The rapid translocation of P38 and ERK1 from the cytoplasm to the nucleus following generation of intramitochondrial ROS suggests that Tdp1 or a nuclear chaperone of Tdp1 is the target of P38 and ERK1. Although phosphorylation of Tdp1 serine 81 activates repair of topoisomerase-I mediated DNA damage, promotes Tdp1 interaction with Ligase III, and enhances the stability of Tdp1 in vitro34,47, it is not required for translocation of Tdp1. This suggests that, if Tdp1 is a direct target of P38 and ERK1, then other serines or threonines are phosphorylated.
A surprising observation was that mtDNA was unnecessary for translocation of Tdp1 into the mitochondria or its incorporation into the BER complex. These findings are consistent with a prior study showing that components of the BER complex reside in the mitochondria of Rho-zero cells56. We conclude that Tdp1 is recruited by and incorporated into the mitochondrial BER complex under conditions predisposing to DNA damage, i.e., reactive oxygen species, and not by the DNA damage itself. We also hypothesize that the increase in and the coprecipitation of Tdp1 and Ligase III from the mitochondrial matrix of Rho-zero cells after H2O2 treatment indicates that Tdp1 interacts in the mitochondria with Ligase III, a component of the BER complex9 and that, independent of DNA damage, it participates in the formation of the mitochondrial BER complex under conditions predisposing to DNA damage, i.e., reactive oxygen species.
Previous studies have shown that oxidative base lesions induce the formation of stalled topoisomerase I-DNA cleavage complexes within the nucleus57 and that Tdp1 participates in repair of such complexes6. Knowing that there is a mitochondrial topoisomerase I58, we postulate that, similar to the nucleus, oxidative agents in the mitochondrial matrix cause formation of 8-oxoguanine lesions and subsequently of stalled mitochondrial topoisomerase I-DNA cleavage complexes and that Tdp1 participates in the repair of these. Additionally, because the promiscuity of mitochondrial DNA polymerase γ (polG) can lead to the incorporation of chain-terminating nucleoside analogs, which are used as drugs in antiviral and anticancer therapy, mitochondrial Tdp1 might also repair blocked 3′-mtDNA ends caused by the incorporation of these nucleoside analogs3.
Maintenance of mtDNA is crucial because the mitochondrial genome consists almost entirely of open reading frames encoding essential proteins, rRNAs, or tRNAs. Despite the importance of mtDNA repair, little detail is known about mtDNA repair mechanisms or the transport of DNA repair proteins into the mitochondria. Approximately 1,500 nuclear-encoded proteins are transported to the human mitochondria59. Most of these proteins thread through mitochondrial membrane translocases coincident with protein synthesis60. Many proteins destined for the matrix contain matrix-targeting pre-sequences recognized by the outer and inner membrane translocase (TOM/TIM) subunits61, which form a channel to the matrix60. In the intermembrane space, chaperones guide proteins with these signal sequences62. Once in the matrix, the pre-sequence is cleaved by Matrix Processing Peptidase (MPP)63. We did not, however, identify a Tdp1 targeting pre-sequence; therefore Tdp1 classifies as a mitochondrial matrix protein without an identifiable targeting pre-sequence64,65.
In the absence of a targeting pre-sequence, Tdp1 mitochondrial translocation might rely on cryptic mitochondrial targeting residues. The presence of such residues usually results in truncated isoforms of the protein within the mitochondria following post-translational endoprotease processing of the protein into a translocation-active form66,67. This is unlikely for Tdp1 because truncated isoforms of Tdp1 were not detected by immunoblotting of mitochondrial extracts.
The p.H493R mutation in Tdp1 causes Spinocerebellar Ataxia with Axonal Neuropathy 1 (SCAN1), a neurodegenerative disease68. This mutation reduces the enzymatic activity of Tdp1 by 25-fold and causes accumulation of Tdp1-DNA reaction intermediates22,69. Our findings that skeletal muscle isolated from Tdp1−/− mice have an increased mtDNA mutation load and that myoblast mitochondria isolated from Tdp1−/− mice have significantly reduced respiratory capacity suggest that mitochondrial dysfunction contributes to the pathogenesis of SCAN1.
In summary, we show that cytoplasmic or mitochondrial ROS promotes the translocation of Tdp1 from the nucleus to the mitochondria of yeast, mouse and human cells and that translocation occurs independently of protein synthesis and the presence of mtDNA. Transport of nuclear Tdp1 to the mitochondria is mediated by P38 and ERK1 MAP kinase activation, and Tdp1 entry into the mitochondrial matrix is through the TIM/TOM complexes. Within the mitochondria, Tdp1 associates with the BER complex, and consistent with a role for Tdp1 in mtDNA repair, mtDNA from Tdp1−/− mouse tissue has increased mutations and lesions. Functionally, this translates into Tdp1−/− mouse myoblasts having impaired mitochondrial respiration.
These findings define a mitochondrial role for Tdp1, although further work is needed to shed light on mtDNA integrity and mitochondrial function in the neurons of Tdp1−/− mice as precedent for understanding the neuropathology of SCAN1 patients. Additionally, given that several cancers have elevated Tdp1 expression23,70,71 and that evasion of apoptotic cell death increases tumorigenesis72, one might also hypothesize that the mitochondrial localization of Tdp1 favors the survival of cancer cells.
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Acknowledgements
We thank Drs. Marie Morimoto, Supipi Duffy, and Catherine Pallen for their criticism, suggestions and review of this manuscript. This work was supported in part by grants to C.F. Boerkoel from the United Mitochondrial Disease Foundation, the Child & Family Research Institute, the Canadian Institutes of Health (248178), and the Michael Smith Foundation for Health Research (CI-SCH-O1899(07–1). C.J. Lim is supported by grants from CIHR, NSERC and CFI. C.F. Boerkoel is a scholar of the Michael Smith Foundation for Health Research and a Clinical Investigator of the B.C. Children’s Hospital Research Institute.
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H.K.F. co-designed the study, performed all experiments (excepting those stated below) and wrote the manuscript. K.C. performed the co-immunoprecipitation experiments. L.F. performed experiments to study the mitochondrial translocation signal in Tdp1. C.J.L. provided tools and expertise for live-cell imaging. C.F.B. co-designed the study and supervised the research. All authors declare no conflicts of interest. This manuscript has been read and approved by all authors. The necessary ethics committee approval was secured for the study reported.
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Fam, H.K., Choi, K., Fougner, L. et al. Reactive oxygen species stress increases accumulation of tyrosyl-DNA phsosphodiesterase 1 within mitochondria. Sci Rep 8, 4304 (2018). https://doi.org/10.1038/s41598-018-22547-8
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DOI: https://doi.org/10.1038/s41598-018-22547-8
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