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Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes

Nature Chemical Biology volume 12, pages 567573 (2016) | Download Citation

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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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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.

Author information

Affiliations

  1. Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.

    • Simon Wisnovsky
    •  & Shana O Kelley
  2. Department of Chemistry, Faculty of Arts and Science, University of Toronto, Toronto, Ontario, Canada.

    • Sae Rin Jean
    •  & Shana O Kelley
  3. Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

    • Sanduni Liyanage
    •  & Aaron Schimmer
  4. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

    • Shana O Kelley

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Shana O Kelley.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–19.

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DOI

https://doi.org/10.1038/nchembio.2102

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