Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport

A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine, Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers, In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct, This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays, However, these experiments do not directly distinguish between a ''one-gate'' and a ''two-gate'' mechanism for the enzyme.