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Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes

An Author Correction to this article was published on 20 August 2018

This article has been updated

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.

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Figure 1: Using mtDNA-damaging agents to identify novel mtDNA repair and replication factors.
Figure 2: RAD23A plays a role in mitochondrial oxidative DNA damage repair.
Figure 3: Mitochondria possess dsDNA break-repair capability involving XRCC4.
Figure 4: DNA polymerase θ localizes to mitochondria and is involved in maintaining mtDNA integrity.

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.

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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.

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Authors

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

Correspondence to Shana O Kelley.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–19. (PDF 14781 kb)

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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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