Abstract
Efficient and accurate replication and repair of mitochondrial DNA is essential for cellular viability, yet only a minimal complement of mitochondrial proteins with relevant activities have been identified. Here, we describe an approach to screen for new pathways involved in the maintenance of mitochondrial DNA (mtDNA) that leverages the activities of DNA-damaging probes exhibiting specific subcellular localization. By conducting a siRNA screen of known nuclear DNA maintenance factors, and monitoring synergistic effects of gene depletion on the activity of mitochondria-specific DNA-damaging agents, we identify a series of proteins not previously recognized to act within mitochondria. These include proteins that function in pathways of oxidative DNA damage repair and dsDNA break repair, along with a novel mitochondrial DNA polymerase, POLθ, that facilitates efficient DNA replication in an environment prone to oxidative stress. POLθ expression levels affect the mutational rate of mitochondrial DNA, but this protein also appears critical for efficient mtDNA replication.
Similar content being viewed by others
Change history
02 April 2018
In the version of this article initially published, Sanduni Liyanage and Aaron Schimmer were not properly acknowledged as co-authors. Both authors have now been included in the current author list, and their contributions are now specified in the author contributions statement. The error has been corrected in the PDF and HTML versions of this article.
References
Friedman, J.R. & Nunnari, J. Mitochondrial form and function. Nature 505, 335–343 (2014).
Falkenberg, M., Larsson, N.G. & Gustafsson, C.M. DNA replication and transcription in mammalian mitochondria. Annu. Rev. Biochem. 76, 679–699 (2007).
Kazak, L., Reyes, A. & Holt, I.J. Minimizing the damage: repair pathways keep mitochondrial DNA intact. Nat. Rev. Mol. Cell Biol. 13, 659–671 (2012).
Pinto, M. & Moraes, C.T. Mechanisms linking mtDNA damage and aging. Free Radic. Biol. Med. 85, 250–258 (2015).
Trifunovic, A. et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423 (2004).
Chatterjee, A., Mambo, E. & Sidransky, D. Mitochondrial DNA mutations in human cancer. Oncogene 25, 4663–4674 (2006).
Ishikawa, K. et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320, 661–664 (2008).
LeDoux, S.P., Druzhyna, N.M., Hollensworth, S.B., Harrison, J.F. & Wilson, G.L. Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults. Neuroscience 145, 1249–1259 (2007).
Rin Jean, S. et al. Molecular vehicles for mitochondrial chemical biology and drug delivery. ACS Chem. Biol. 9, 323–333 (2014).
Horton, K.L., Stewart, K.M., Fonseca, S.B., Guo, Q. & Kelley, S.O. Mitochondria-penetrating peptides. Chem. Biol. 15, 375–382 (2008).
Fonseca, S.B. et al. Rerouting chlorambucil to mitochondria combats drug deactivation and resistance in cancer cells. Chem. Biol. 18, 445–453 (2011).
Chamberlain, G.R., Tulumello, D.V. & Kelley, S.O. Targeted delivery of doxorubicin to mitochondria. ACS Chem. Biol. 8, 1389–1395 (2013).
Wisnovsky, S.P. et al. Targeting mitochondrial DNA with a platinum-based anticancer agent. Chem. Biol. 20, 1323–1328 (2013).
Tann, A.W. et al. Apoptosis induced by persistent single-strand breaks in mitochondrial genome: critical role of EXOG (5′-EXO/endonuclease) in their repair. J. Biol. Chem. 286, 31975–31983 (2011).
Santos, J.H., Hunakova, L., Chen, Y., Bortner, C. & Van Houten, B. Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J. Biol. Chem. 278, 1728–1734 (2003).
Santos, J.H., Meyer, J.N., Mandavilli, B.S. & Van Houten, B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 314, 183–199 (2006).
Carreon, J.R., Mahon, K.P. Jr. & Kelley, S.O. Thiazole orange-peptide conjugates: sensitivity of DNA binding to chemical structure. Org. Lett. 6, 517–519 (2004).
Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson, J.D. & Will, Y. Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97, 539–547 (2007).
Bellance, N. et al. Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity. Int. J. Biochem. Cell Biol. 41, 2566–2577 (2009).
Mourtada, R. et al. Re-directing an alkylating agent to mitochondria alters drug target and cell death mechanism. PLoS ONE 8, e60253 (2013).
Pohjoismäki, J.L.O. et al. Oxidative stress during mitochondrial biogenesis compromises mtDNA integrity in growing hearts and induces a global DNA repair response. Nucleic Acids Res. 40, 6595–6607 (2012).
Mahon, K.P. et al. Deconvolution of the cellular oxidative stress response with organelle-specific peptide conjugates. Chem. Biol. 14, 923–930 (2007).
Ashley, N., Harris, D. & Poulton, J. Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. Exp. Cell Res. 303, 432–446 (2005).
Sheng, Z. et al. 8-Oxoguanine causes neurodegeneration during MUTYH-mediated DNA base excision repair. J. Clin. Invest. 122, 4344–4361 (2012).
Weterings, E. & Chen, D.J. The endless tale of non-homologous end-joining. Cell Res. 18, 114–124 (2008).
Bacman, S.R., Williams, S.L. & Moraes, C.T. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res. 37, 4218–4226 (2009).
Lakshmipathy, U. & Campbell, C. Double strand break rejoining by mammalian mitochondrial extracts. Nucleic Acids Res. 27, 1198–1204 (1999).
Grawunder, U. et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 (1997).
Gao, Y. et al. DNA ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair. Nature 471, 240–244 (2011).
Lange, S.S., Takata, K.-I. & Wood, R.D. DNA polymerases and cancer. Nat. Rev. Cancer 11, 96–110 (2011).
Holt, I.J. & Reyes, A. Human mitochondrial DNA replication. Cold Spring Harb. Perspect. Biol. 4, a012971 (2012).
Fernandez-Vidal, A. et al. A role for DNA polymerase θ in the timing of DNA replication. Nat. Commun. 5, 4285 (2014).
Yousefzadeh, M.J. et al. Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. PLoS Genetics 10, e1004654 (2014).
Yoon, J.H., Roy Choudhury, J., Park, J., Prakash, S. & Prakash, L. A role for DNA polymerase θ in promoting replication through oxidative DNA lesion, thymine glycol, in human cells. J. Biol. Chem. 289, 13177–13185 (2014).
Ceccaldi, R. et al. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518, 258–262 (2015).
Mateos-Gomez, P.A. et al. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).
Rhee, H.W. et al. Proteomic map** of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).
Williams, C.C., Jan, C.H. & Weissman, J.S. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling. Science 346, 748–751 (2014).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Li, H., et al. 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Acknowledgements
We acknowledge the Canadian Institutes of Health Research for their support of this work. We also thank J.H.J. Hoeijmakers (Erasmus University Medical Center) for providing Rad23a+/+ and Rad23a−/− MEFs.
Author information
Authors and Affiliations
Contributions
S.W. performed siRNA knockdown screens, western-blot-based assays, co-immunoprecipitation, JC-1 staining, PCR-based assays, 8-oxoguanine staining, DNA end-joining assays, mtDNA immunoprecipitation, preparation for deep sequencing and mitochondrial respiration assays, and cell toxicity measurements. S.R.J. performed and analyzed the localization imaging studies and cellular superoxide detection. S.W. and S.R.J. synthesized the targeted chemical probes. S.W., S.R.J., and S.O.K. wrote the manuscript. S.L. collected the data shown in Figure 4e, and A.S. is her thesis supervisor.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–19. (PDF 14781 kb)
Rights and permissions
About this article
Cite this article
Wisnovsky, S., Jean, S., Liyanage, S. et al. Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes. Nat Chem Biol 12, 567–573 (2016). https://doi.org/10.1038/nchembio.2102
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.2102
- Springer Nature America, Inc.
This article is cited by
-
Fabrication of self-assembled peptide nanoparticles for in vitro assessment of cell apoptosis pathway and in vivo therapeutic efficacy
Microchimica Acta (2022)
-
Molecular basis of microhomology-mediated end-joining by purified full-length Polθ
Nature Communications (2019)