Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

Many enzymes undergo conformational changes when bound to a substrate, however, structural plasticity in substrate-free enzymes, and potential associated benefits, is poorly understood. Here, we study the structural dynamics of Rep, an accessory helicase which undergoes conformational transitions in performing crucial roles in bacterial DNA replication, repair and recombination. Using time-correlated single-photon counting, fluorescence correlation spectroscopy, rapid single-molecule Förster resonance energy transfer, Anti-Brownian ELectrokinetic trapping and molecular dynamics simulations, we find that free Rep adopts four conformations, revealing that DNA absence confers structural plasticity beyond simple two-state binding. Our fast measurement technologies reveal hitherto hidden transient intermediate transitions between open and closed conformations, several of which are strongly dependent on sodium chloride concentration. Our findings 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.