Eukaryotic topoisomerase II (Topo II) catalyzes DNA relaxation and decatenation in an ATP-dependent reaction. Topo II is a homodimer with three protein-protein interfaces, the N-gate, DNA-gate, and C-gate. Relaxation and decatenation follow a strand-passage mechanism in which one DNA duplex is bound at the DNA-gate in the middle of the enzyme and cleaved, a second DNA duplex is captured by ATP-induced closing of the N-gate through dimerization if the ATPase domains, the DNA-gate opens to enable passage of the second duplex through the gap, then closes for religation of the cleaved duplex, and the C-gate opens to release the second duplex. ATP hydrolysis and re-opening of the N-gate complete the catalytic cycle.

Despite their key role in the topoisomerase reaction, gate opening and closing have not been observed directly. Here we use single-molecule FRET and Saccharomyces cerevisiae Topo II labeled with donor and acceptor fluorophores on opposite sides of the N-gate, the DNA-gate or the C-gate, to investigate the conformational changes of these gates in response to nucleotide, DNA, and the topoisomerase poison etoposide. We show that the N-gate is open in the absence of nucleotides, and closes in response to nucleotide binding. The DNA- and C-gates are in a closed state in the absence of nucleotide and DNA and in the nucleotide-bound state. In the presence of DNA, DNA and nucleotide, or etoposide, conformations with widened or open DNA or C-gates are observed. Consistent with biochemical data, our results point to a tight coupling between the events at different gates and between the conformational changes of these gates.