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Human topoisomerase II alpha (TOP2) is the main mitotic decatenase 1-3 resolving intertwines between sister chromatids that form during DNA replication 4. Here, we employ quadruple-trap optical tweezers to generate braids between a pair of l-DNA molecules to study, at the single-molecule level and in real-time, how TOP2 untangles these DNAs. We find that TOP2 rapidly resolves single and multiple braids and is inhibited by chemotherapy agent, etoposide. TOP2 is sensitive to DNA conformation, exhibiting a chiral preference for the removal of braids with right-handed crossings and is inhibited when DNAs are held at forces over 20pN. We show that TOP2 must load at the cross between the two DNAs to resolve braids efficiently. TOP2 pre-loaded onto individual DNAs is unable to resolve newly formed braids in the presence of ATP, suggesting that the geometry of pre-bound enzyme is incompatible with the capture of the second DNA. Finally, we show that cohesin binds to braids, preventing TOP2 from resolving them. Our study unveils novel insights into the regulation of TOP2\'s decatenation, underscoring the importance of substrate accessibility and the role of cohesin in modulating TOP2 activity.