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. 2025:2928:197-204.
doi: 10.1007/978-1-0716-4550-5_16.

Kinetoplast DNA Decatenation Assay with Eukaryotic Topoisomerase II

Addison K O'Brian  1 , Mattalyn R Hardin  1 , Allison J Thomas  1 , Brooke D Latham  1 , Joseph Deweese  2   3
Affiliations

Kinetoplast DNA Decatenation Assay with Eukaryotic Topoisomerase II

Addison K O'Brian et al. Methods Mol Biol. 2025.

Abstract

The interlinking of DNA during replication and other DNA processes is resolved by type II DNA topoisomerases. In humans, topoisomerase IIα is the main enzyme involved in this process using a transient double-stranded DNA break to unlink sister chromatids during and after replication to allow for mitosis. To measure decatenation, a consistent catenated substrate called kinetoplast DNA (kDNA) is commonly used. kDNA is often purified from the trypanosome Crithidia fasciculata. kDNA represents catenated or interlinked circles of DNA. Early work on topoisomerases identified kDNA as a useful substrate for measuring decatenation by DNA topoisomerase II. This protocol will describe a common method for measuring decatenation of kDNA by eukaryotic type II topoisomerases.

Keywords: Decatenation; Gel electrophoresis; Kinetoplast DNA; Minicircles; Topoisomerase II; kDNA.

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References

    1. Pommier Y, Sun Y, Huang SN, Nitiss JL (2016) Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nat Rev Mol Cell Biol 17(11):703–721. https://doi.org/10.1038/nrm.2016.111 - DOI - PubMed - PMC
    1. Marini JC, Miller KG, Englund PT (1980) Decatenation of kinetoplast DNA by topoisomerases. J Biol Chem 255(11):4976–4979 - DOI - PubMed
    1. Miller KG, Liu LF, Englund PT (1981) A homogeneous type II DNA topoisomerase from HeLa cell nuclei. J Biol Chem 256(17):9334–9339 - DOI - PubMed
    1. Dougherty AC, Hawaz MG, Hoang KG, Trac J, Keck JM, Ayes C, Deweese JE (2021) Exploration of the role of the C-terminal domain of human DNA topoisomerase IIalpha in catalytic activity. ACS Omega 6(40):25892–25903. https://doi.org/10.1021/acsomega.1c02083 - DOI - PubMed - PMC
    1. Gibson EG, Oviatt AA, Cacho M, Neuman KC, Chan PF, Osheroff N (2019) Bimodal actions of a naphthyridone/aminopiperidine-based antibacterial that targets gyrase and topoisomerase IV. Biochemistry 58(44):4447–4455. https://doi.org/10.1021/acs.biochem.9b00805 - DOI - PubMed

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