Multiple-attempt dynamics of type IA topoisomerases revealed in an orthogonal single-molecule experiment

Topoisomerases are enzymes involved in maintaining the topological state of cellular DNA. Despite many structural, biophysical, and biochemical studies, their dynamic characteristics remain poorly understood. Recent single molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments cannot determine whether the protein remains DNA bound during the pauses or the relationship between domain movements in the protein and topological changes in the DNA. By combining two orthogonal single molecule techniques, we observed that topoisomerase IA is constantly changing conformation and attempting to modify the topology of DNA, but only succeeds in a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand passage attempts that culminate in a successful relaxation event.