Rep structures comprise metastable intermediates between open and closed states whose transitional kinetics are tuned by the ionic environment

Helicases undergo conformational changes in the presence or absence of the DNA substrate and cofactors, however, the forces driving these structural dynamics are poorly understood. Here, we study single molecules of Rep, an accessory helicase which undergoes conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting, fluorescence correlation spectroscopy, rapid single-molecule Förster resonance energy transfer, Anti-Brownian ELectrokinetic trapping and molecular dynamics simulations to provide unparalleled temporal and spatial resolution of Rep's domain movement. Our rapid measurements reveal hitherto hidden intermediates between open and closed conformations, several of which are strongly dependent on sodium chloride concentration. Our findings suggest that the transition between open and closed conformations is not a simple single arm-swing mechanism but comprises different salt-dependent metastable states. They also support an alternative binding model for accessory helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by the ionic environment prior to locking-in upon DNA binding.