Proteomic Analysis of Human Topoisomerases Reveals Their Distinct and Diverse Cellular Functions

Topoisomerases are essential for resolving topological stress in DNA during key cellular processes. In human cells, six topoisomerases perform specialized yet overlapping functions to manage these challenges. To investigate their distinct and shared roles, as well as their involvement in DNA damage repair, we conducted a comprehensive analysis of the human topoisomerase-associated protein landscape. Using tandem affinity purification coupled with mass spectrometry, we mapped the protein-protein interaction networks of five human topoisomerases under both normal and stressed conditions. Our analysis identified several key interactions that may regulate topoisomerase function. Notably, TOP1 interacts with PUM3, which undergoes a similar relocalization from nucleoli to nucleoplasm following treatment with a TOP1 poison. In addition, we uncovered novel interactions of TOP3A with NSMCE4A, YTHDC2, and NDUFAF7, as well as a previously uncharacterized interaction between TOP3B and the mitochondrial membrane protein TDRKH (TDRD2). We further examined dynamic changes in these interactomes in response to TOP1 and TOP2 poisons and replication stress, distinguishing between interactions in chromatin and soluble fractions. These findings provide new insights into the regulation and functional coordination of human topoisomerases, offering potential biomarkers or therapeutic targets for topoisomerase inhibitors in cancer treatment.