Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Sudhir Varma,Salim Khiati,Ilaria Dalla Rosa,Stephanie A Michaels,Keli Agama,David V Tulumello,Simone A Baechler,Sae Rin Jean,Lisa M Miller Jenkins,Valentina M Factor,Shana O Kelley,Junko Murai,Shar‐yin N Huang,Yves Pommier

doi:10.15252/embr.201642139

Sudhir Varma, Salim Khiati + Show 12 more

Open Access

https://doi.org/10.15252/embr.201642139

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Journal: EMBO reports	Publication Date: Feb 9, 2018
Citations: 18	License type: cc-by

Affiliation: National Cancer Institute, University of Toronto

Abstract

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Highlights

Type IIA topoisomerases (TOP2a and TOP2b) are essential for decatenating DNA and removing DNA torsional stress generated by transcription, replication, and chromosomal segregation [1,2]
To determine whether the two bands reflect different tyrosyl-DNA phosphodiesterase 2 (TDP2) isoforms, we knocked down TDP2 using a cocktail of 4 siRNA targeting the portion of transcript encoding the catalytic domain of TDP2
Our results show that the levels of all five mitochondrial transcripts in TDP2À/À cells are reduced compared to WT cells, while three out of five transcripts show reduced levels in TDP2S cells (Fig 6A)

Summary

Introduction

Type IIA topoisomerases (TOP2a and TOP2b) are essential for decatenating DNA and removing DNA torsional stress generated by transcription, replication, and chromosomal segregation [1,2]. All these reactions are carried out by the formation of transient DNA double-strand breaks that are covalently linked to the catalytic tyrosine residues of TOP2 homodimers [1,2]. The transient covalent DNA-TOP2 complexes are termed TOP2 cleavage complexes (TOP2cc). Endogenous DNA alterations and anti-cancer drugs, including doxorubicin and etoposide, trap TOP2a and TOP2b cleavage complexes [3,4,5] and extend the normally very short half-lives of TOP2cc [1,2,4,5,6,7]. TDP2 can act on TOP2 trapped on RNA [9] and is known to process replication intermediates for a broad range of viruses (e.g., picornavirus Vpg unlinkase [10] and hepatitis B virus [11])

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