(A) Schematic of SPR experiment probing association and dissociation kinetics of mCI from ssDNA-RecA-ATPγS filaments on the surface of an SPR chip. ssDNA-RecA-ATPγS filaments were assembled on a biotinylated (dT)71 ssDNA molecule. (B) mCI (blue), YPet-mCI (yellow) or PAmCherry-mCI (red) were then flowed into the flow cell at time t = 0 for 400 s to monitor the association phase. Dissociation of mCI from ssDNA-RecA-ATPγS filaments was observed by leaving out mCI (or variant) from the injection buffer. Sensorgram reveals biphasic association of mCI (or variant, 1 μM) to RecA* filaments, followed by a slow dissociation from the ssDNA-RecA-ATPγS filament. Sensorgrams presented here are corrected for slow disassembly of the RecA-ATPγS filament, and data are scaled to the binding curve of YPet-mCI for purposes of comparison (see also Figure 2—figure supplement 1C for unscaled data). (C) Schematic of single-molecule FRET assay used to probe the influence of mCI binding on the conformational state of the ssDNA-RecA-ATP filament assembled on a ssDNA (dT)40 overhang. Biotinylated substrate DNA (bio-ds18-(dT)40 containing donor and acceptor fluorophores) was immobilized on a functionalized coverslip via a streptavidin-biotin interaction. (D) RecA binds the ssDNA overhang dynamically to form a ssDNA-RecA filament. (E) In the presence of ATPγS, RecA forms a stable filament. (F) Incubation with mCI leads to a RecA filament decorated with mCI. (G) FRET distributions observed from the substrate alone (n = 101 molecules), with RecA-ATP (1 μM RecA, 1 mM ATP, n = 179 molecules) and RecA-ATPγS (1 mM ATPγS, n = 87 molecules) from at least three independent experiments. (H) Titration of mCI shifts the RecA-ATP distribution to that of the active filament. (I) Example FRET traces of DNA substrate alone or when bound to RecA in the presence of ATPγS, or when bound to RecA in the presence of ATP and mCI (0, 10, 100, 300, 1000 and 3000 nM mCI; n = 179, 139, 77, 70, 172, 68 molecules respectively from at least three independent experiments). Dashed lines represent ‘bound’ (FRET = 0.2 dark green) and ‘unbound’ (FRET = 0.4 light green) states. (J) Fitting of the Hill equation to the percentage of bound fraction as a function [mCI] reveals a KD of 36 ± 10 nM and a cooperativity of 2.4 ± 0.2. Errors represent fitting errors to the entire data set. (K) Off-rates measured from binding of mCI to ssDNA-RecA-ATP filaments (L) Percentage amplitude of the detected rate-constants as a function of [mCI] reveals enrichment of the population decaying according to the slow off-rate as a function of [mCI] (between 40–50 molecules were analyzed at each concentration; Error bars represent fitting errors). See also Figure 2—figure supplements 1 and 2.