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DNA replication introduces double-stranded DNA entanglements, which pose a challenge to cell division and faithful segregation of the genome. During mitosis, Topoisomerase 2A (TOP2A), binds and resolves DNA entanglements by producing a double strand break and then passing the other strand through the break. TOP2A is an essential protein and an important drug target, hence has been extensively studied, but until now the resolution process has not been directly visualised. In this work, I develop an assay for visualising TOP2A activity, mimicking forces that could be applied in a mitotic context, by employing optical tweezers to manually entangle two pieces of DNA(1). I demonstrate that TOP2A DNA resolution is inhibited at high forces, with sharp transition at the half-force of 28 pN. My experiments indicate TOP2A readily binds DNA, however I find resolution is most efficient when TOP2A associates directly at the site of a DNA entanglement. During early mitosis the action of TOP2A chromosome resolution is countered by cohesin and I demonstrate that cohesin readily associated with entangled DNA, and inhibits TOP2A resolution, implicating TOP2A in regulating chromatid cohesion. Collectively, this approach provides novel insights into the important therapeutic target, TOP2A(2).

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(1) Meijering, A.E.C., Bakx, J.A.M., Man, T., Heller, I., Wuite, G.J.L., and Peterman, E.J.G. (2022). Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System. In Methods in Molecular Biology, pp. 75–100. 10.1007/978-1-0716-2229-2_5.

(2) Cutts, E.E., Saravanan, S., Girvan, P., Ambrose, B., Fisher, G.L.M., Rueda, D.S. and Aragon, L. (2025). Substrate accessibility regulation of human TopIIa decatenation by cohesin. Nature Communications, https://doi.org/10.1038/s41467-025-62505-3