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.
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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.
The authors declare no competing financial interests.
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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
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