Publications by authors named "Stephanie A Michaels"

2 Publications

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Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2 short isoform.

EMBO Rep 2018 03 9;19(3). Epub 2018 Feb 9.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2) expressed from an alternative transcription start site. TDP2 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 () and knockout () DT40 cells shows that cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing cells with human restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in cells. Using CRISPR-engineered human cells expressing only the TDP2 isoform, we show that TDP2 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 isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.
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http://dx.doi.org/10.15252/embr.201642139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836098PMC
March 2018