Figure 2

Tdp1 interacts with DNA repair proteins in the mitochondria and maintains mtDNA integrity. (a) Immunoblot of FLAG-HA-tagged Tdp1 immunoprecipitates from mitochondrial extracts of human fibroblasts showing co-precipitation with Ligase III. Human fibroblasts were treated with 1 µM H₂O₂ for 1 hour prior to purification of the mitochondria. The purified mitochondria were digested with proteinase K to remove proteins co-purifying with but external to the mitochondria. Immunoprecipitation (IP) was performed with anti-FLAG. Immunoprecipitated Tdp1 was detected using an anti-HA antibody. (b) mtDNA lesions (left) and mtDNA Taq1 site mutations (right) before and following culture of MEFs with 1 µM H₂O₂ for 4 weeks. (c) Oxygen consumption rate in mitochondria of wt and Tdp1^−/− MEFs after 4 weeks of 1 µM H₂O₂ treatment. The Rho-zero cell line was used as a negative control. (d) ATP production in MEFs subjected to 4 weeks of 1 µM H₂O₂ treatment. (e) Left panel: normalized mitochondria-encoded cytochrome c oxidase II (Cox) and nuclear-encoded S6 ribosomal (S6) RNA levels as measured by qRT-PCR in wt and Rho-zero MEFs. Cox RNA levels were normalized to S6 RNA levels. Cox and S6 were used to determine the presence of transcribed mitochondrial and nuclear DNA in wt and Rho-zero MEFs. Right panel: picogreen staining of MEFs untreated (wt) or treated (Rho-zero) with ethidium bromide. Note the absence of cytoplasmic staining in Rho-zero MEFs indicating the absence of mtDNA. (f) Immunofluorescent staining of Tdp1 in Rho-zero MEFs with or without 1 µM H₂O₂ treatment for 1 hour. (g) Immunoblot of FLAG-HA-tagged Tdp1 immunoprecipitates from Rho-zero MEF mitochondria showing co-precipitation of Ligase III. Human fibroblasts were treated with 1 µM H₂O₂ for 1 hour prior to IP with anti-FLAG. Immunoprecipitated Tdp1 was detected using an anti-HA antibody. (h) Comparison of recombinant Tdp1 activity to Tdp1 activity in mitochondrial lysates of wt, Tdp1^−/−, and Rho-zero MEFs after treatment with 1 µM H₂O₂ for 1 hour. Tdp1 activity was measured in three independent experiments; activity was detected by fluorescence following Tdp1 cleaving a quencher off a synthesized oligonucleotide substrate. The first bar shows fluorescence for purified recombinant Tdp1 without the oligonucleotide substrate; the second bar shows fluorescence for the oligonucleotide substrate without Tdp1 or mitochondrial extract; the third bar shows fluorescence for the oligonucleotide substrate with recombinant Tdp1; the fourth bar shows fluorescence for the oligonucleotide substrate with mitochondrial extracts derived from wt MEFs; the fifth bar shows fluorescence for the oligonucleotide substrate with mitochondrial extracts derived from Tdp1^−/− MEFs; the sixth bar shows fluorescence for the oligonucleotide substrate with mitochondrial extracts derived from Rho-zero MEFs. The oligonucleotide substrate and the purified recombinant N-terminally His-tagged Tdp1 used in this experiment were previously described²⁸. *P < 0.05, **P < 0.01; ***P < 0.001. Error bars represent one standard deviation for three independent experiments. Immunoblots are displayed in cropped format. Scale bar = 10 µm.