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

TL;DR

This study reveals four conformational states of the Rep helicase during its open-to-closed transition, showing how salt influences intermediate stability and providing insights into its structural dynamics via advanced single-molecule techniques.

Contribution

It uncovers two previously unknown intermediate states in Rep helicase conformational changes and demonstrates salt-dependent stability, advancing understanding of helicase structural dynamics.

Findings

Identified four conformational states, including two new intermediates.

Salt concentration modulates the stability of intermediate states.

Revealed detailed domain movements during the open-closed transition.

Abstract

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule F\"orster 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 to S2 to S3 to S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1 to 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.