Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2^S short isoform

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2^S) expressed from an alternative transcription start site. TDP2^S 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 TDP2^S isoform, we show that TDP2^S 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 TDP2^S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.