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<p><i>Arabidopsis thaliana</i> encodes two Type IB topoisomerases, TOP1α and TOP1β, which are both essential for relaxing DNA supercoiling and torsional stress. Despite sharing this core function, they diverge significantly in expression, subcellular localization, and specialized roles. While TOP1α is predominantly nuclear and well-studied, proteomic data suggests TOP1β also plays a role in mitochondrial DNA maintenance. Importantly, TOP1β is the primary determinant of plant sensitivity to the chemotherapy drug camptothecin (CPT): loss-of-function chemotherapy drug camptothecin (CPT) mutants are highly CPT-resistant, a phenotype not observed with decreased TOP1α expression. To explore the poorly characterized TOP1β, we used advanced structure prediction tools (AlphaFold2, AlphaFold3, and RoseTTAFold) to generate high-fidelity 3D models of DNA-bound <i>Arabidopsis</i> topoisomerase Iβ. Molecular docking with known human topoisomerase I inhibitors revealed conserved drug-binding residues and comparable predicted binding affinities, despite low overall sequence similarity. We then bacterially expressed and purified homogeneous, full-length, poly-His-tagged TOP1β. Its enzymatic activity was confirmed via plasmid relaxation assays. Intrinsic fluorescence quenching spectroscopy showed that CPT analogs bind the <i>apo</i>- TOP1β enzyme weakly, with dissociation constants of approximately 99 µM for irinotecan and 23 µM for topotecan. Nevertheless, both compounds significantly inhibited enzymatic activity in the relaxation assays. These findings offer novel structural and functional insights into plant topoisomerase Iβ and its potential as a target for chemical modulation.</p>

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Investigating Arabidopsis thaliana (L.) Heynh topoisomerase Iβ: heterologous expression, purification, and interaction with human topoisomerase I poisons

  • 期刊论文

  • 发表日期：

    2026年6月28日

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  • Rokono Nagi,
  • Amenti Ozukum,
  • Rajkrishna Mondal,
  • Chitta Ranjan Deb

摘要

Arabidopsis thaliana encodes two Type IB topoisomerases, TOP1α and TOP1β, which are both essential for relaxing DNA supercoiling and torsional stress. Despite sharing this core function, they diverge significantly in expression, subcellular localization, and specialized roles. While TOP1α is predominantly nuclear and well-studied, proteomic data suggests TOP1β also plays a role in mitochondrial DNA maintenance. Importantly, TOP1β is the primary determinant of plant sensitivity to the chemotherapy drug camptothecin (CPT): loss-of-function chemotherapy drug camptothecin (CPT) mutants are highly CPT-resistant, a phenotype not observed with decreased TOP1α expression. To explore the poorly characterized TOP1β, we used advanced structure prediction tools (AlphaFold2, AlphaFold3, and RoseTTAFold) to generate high-fidelity 3D models of DNA-bound Arabidopsis topoisomerase Iβ. Molecular docking with known human topoisomerase I inhibitors revealed conserved drug-binding residues and comparable predicted binding affinities, despite low overall sequence similarity. We then bacterially expressed and purified homogeneous, full-length, poly-His-tagged TOP1β. Its enzymatic activity was confirmed via plasmid relaxation assays. Intrinsic fluorescence quenching spectroscopy showed that CPT analogs bind the apo- TOP1β enzyme weakly, with dissociation constants of approximately 99 µM for irinotecan and 23 µM for topotecan. Nevertheless, both compounds significantly inhibited enzymatic activity in the relaxation assays. These findings offer novel structural and functional insights into plant topoisomerase Iβ and its potential as a target for chemical modulation.