MYC modulates TOP2A diffusion to promote substrate detection and activity [CAD-seq]

Topoisomerases are essential enzymes that alleviate DNA supercoiling by cleaving and resealing DNA strands. Regulation of their abundance and activity is crucial for maintaining genomic stability and supporting nuclear functions. Previously, we showed that the oncoprotein MYC recruits and stimulates topoisomerases to remove DNA entanglements generated by oncogenic transcription. Understanding this mechanism may suggest methods to inhibit MYC-driven topoisomerase activation, thereby selectively targeting tumor-specific transcription. Here, we demonstrate that the essential topoisomerase TOP2A in human cells exists in a dynamic equilibrium between sequestration in the nucleolus, substrate searching in transcription hubs, and active engagement on chromatin. This equilibrium is highly responsive to changes in DNA topology, allowing cells to regulate TOP2A levels in the nucleoplasm. Using single molecule tracking, we show that MYC accelerates TOP2A diffusion in cells. In vitro, we demonstrate that MYC decreases the size of TOP2A condensates, likely by limiting non-specific self interactions. By increasing TOP2A diffusion, MYC promotes substrate binding and increases TOP2A engagement on the chromatin genome-wide, revealing the mechanism underlying MYC stimulation of TOP2A activity.