Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity
Abstract
1. Introduction
2. Results
2.1. Plasmid DNA Relaxation by CTD TOP2A Mutants
2.2. Plasmid DNA Cleavage by CTD TOP2A Mutants
2.3. Plasmid DNA Binding by CTD TOP2A Mutants
2.4. Kinetoplast DNA Decatenation by CTD TOP2A Mutants
2.5. N-Terminal Clamp Stability of TOP2A CTD Mutants
3. Discussion
4. Materials and Methods
4.1. Enzymes, Substrates, and Reagents
4.2. Topoisomerase IIα Plasmid DNA Relaxation Assay
4.3. Topoisomerase IIα Plasmid DNA Cleavage Assay
4.4. Topoisomerase IIα Binding of Plasmid DNA
4.5. Decatenation of Kinetoplast DNA (kDNA) by Topoisomerase IIα
4.6. Clamp Closing
4.7. Data Visualization
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BSA | Bovine serum albumin |
ChT | Chromatin tether domain |
CTD | C-terminal domain |
decat | Decatenated |
DSB | Double strand break |
EMSA | Electrophoretic mobility shift assay |
IDP | Intrinsically disordered proteins |
IDR | Intrinsically disordered protein regions |
kDNA | Kinetoplast DNA |
TOP2 | Topoisomerase II |
TOP2A | Topoisomerase IIα |
TOP2B | Topoisomerase IIβ |
NLS | Nuclear localization sequence |
NT | No topoisomerase II (without the presence of TOP2A) |
N-terminus | Amino-terminus |
Rel | Relaxed |
SC | Supercoiled |
SSB | Single strand break |
WT | Wild-type |
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Chang, J.W.; O’Brian, A.K.; Thomas, A.J.; Hardin, M.R.; Latham, B.D.; Ngabonziza, D.; Simpson, L.G.; Wade, B.D.; Kühnhenrich, L.; Thompson, N.M.; et al. Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity. Int. J. Mol. Sci. 2025, 26, 3604. https://doi.org/10.3390/ijms26083604
Chang JW, O’Brian AK, Thomas AJ, Hardin MR, Latham BD, Ngabonziza D, Simpson LG, Wade BD, Kühnhenrich L, Thompson NM, et al. Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity. International Journal of Molecular Sciences. 2025; 26(8):3604. https://doi.org/10.3390/ijms26083604
Chicago/Turabian StyleChang, Jeong Won, Addison K. O’Brian, Allison J. Thomas, Mattalyn R. Hardin, Brooke D. Latham, Daniel Ngabonziza, Lily G. Simpson, Benjamin D. Wade, Laura Kühnhenrich, Nina M. Thompson, and et al. 2025. "Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity" International Journal of Molecular Sciences 26, no. 8: 3604. https://doi.org/10.3390/ijms26083604
APA StyleChang, J. W., O’Brian, A. K., Thomas, A. J., Hardin, M. R., Latham, B. D., Ngabonziza, D., Simpson, L. G., Wade, B. D., Kühnhenrich, L., Thompson, N. M., Endsley, C. E., & Deweese, J. E. (2025). Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity. International Journal of Molecular Sciences, 26(8), 3604. https://doi.org/10.3390/ijms26083604