The transitional kinetics between open and closed Rep structures can be tuned by salt via two intermediate states.

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of the superfamily 1A DNA helicase Rep, which undergoes conformational transitions during bacterial DNA replication, repair, and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Förster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping, and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Rep's domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Rep's open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A, and 2B along the S1→S2→S3→S4 transitional pathway comprising an initial truncated swing of 2B, which then rolls across the 1B surface, followed by combined rotations of 1B, 2A, and 2B. High forward and reverse rates for S1→S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking in upon DNA binding.