Given the existing extensive in vitro characterization of mCI, we decided to further develop it as a probe for detecting RecA* in cells. To better understand the kinetics, cooperativity and affinity of mCI for RecA-ssDNA filaments, we first pursued an in vitro investigation of the interaction between mCI and RecA filaments. With the eventual goal of using mCI to detect RecA* filaments in live cell experiments, we made fusion constructs with fluorescent proteins tagged to the N-terminus of mCI via a 14-amino acid linker. To perform time-lapse imaging, we tagged mCI with the yellow fluorescent protein YPet, and to perform live-cell photoactivatable light microscopy (PALM), we tagged mCI with the photoactivatable red fluorescent protein PAmCherry. Untagged mCI and the two fluorescently labeled constructs, PAmCherry-mCI and YPet-mCI were purified and characterized for RecA-ssDNA binding as described below (See Materials and methods and Supplementary data for details, Figure 2—figure supplement 1A).

We first set out to interrogate the stability of mCI binding to RecA*. To that end, binding of the mCI constructs to ssDNA-RecA filaments was first assayed by surface plasmon resonance (SPR). We immobilized a 5’ biotinylated (dT) 71 ssDNA substrate on the surface of a streptavidin-functionalized SPR chip (Figure 2A) and assembled RecA-ssDNA filaments by injecting 1 μM RecA in buffer supplemented with ATP. This was followed by injection of buffer without RecA, but supplemented with ATPγS to minimize disassembly of the RecA filament on the ssDNA immobilized on the chip surface (Figure 2—figure supplement 1B). Next, the experiment was repeated but now introducing to pre-formed RecA* filaments solutions that not only contain stabilizing ATPγS, but also either untagged or fluorescently tagged mCI proteins. Scaled sensorgrams (Figure 2B) that are corrected for any disassembly of the ssDNA-RecA-ATPγS filament report on kinetics of interactions of mCI (or variant) with the highly stable RecA* filament (see also Figure 2—figure supplement 1C). These sensorgrams reveal that mCI associates with the RecA filament in a biphasic manner. Dissociation of mCI from the RecA filament was slow, with a dissociation halftime (t 1/2 ) of 850 s. In comparison, the fluorescently tagged constructs dissociated faster, but still slowly enough for use as a probe for the detection of RecA*. We measured a t 1/2 = 260 s and 280 s for YPet-mCI and PAmCherry-mCI respectively. Under conditions where the interactions of mCI with ssDNA-RecA filaments could be readily probed, we also attempted to measure interactions of mCI with RecA filaments assembled on 60-mer dsDNA. In this case, we did not detect formation of dsDNA-RecA filaments even in the presence of ATPγS, and hence could not quantify interactions with mCI or tagged variant. It is conceivable that the mCI probe can potentially interact with dsDNA-RecA filaments if they adopt a conformation similar to that of RecA*.

Figure 2 with 2 supplements with 2 supplements see all Download asset Open asset mCI stabilizes ssDNA-RecA filaments in vitro. (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 K D 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. https://doi.org/10.7554/eLife.42761.005

EM studies of RecA-ssDNA filaments have revealed that the pitch of the filament depends on the co-factor bound to it (Egelman and Stasiak, 1986; Egelman and Stasiak, 1988). Notably, ATPγS promotes the formation of the extended filament, whereas, the ADP bound filament exhibits the compressed state. We therefore set out to answer the question: what is the influence of mCI binding on the conformational state of RecA* filaments formed in the presence of ATP? To that end we adopted a single-molecule Förster Resonance Energy Transfer (smFRET) assay that has been previously used to demonstrate the nucleotide dependent conformational states of the RecA* filament (Park et al., 2010). We used a previously described DNA substrate consisting of a biotinylated 18-mer double-stranded region preceded by a 5’-(dT) 40 overhang (‘bio-ds18-(dT) 40 ’, See SI for details, Figure 2C) (Park et al., 2010). This substrate simulates the partly single-stranded and partly double-stranded nature of the DNA substrate that is thought to be generated in the context of replisomes encountering lesions in vivo. The ssDNA region is labelled with a Cy3 donor probe on one end and a Cy5 acceptor probe on the other so that the degree of extension of the ssDNA can be measured by FRET. The DNA substrate was immobilized on a streptavidin-coated surface in a flow cell and the Cy5 FRET signal was measured upon excitation of the Cy3 dye with a 532 nm laser (see SI for details). Consistent with previous FRET investigations of this DNA substrate (Park et al., 2010), the DNA substrate alone exhibited a FRET distribution with a mean value of 0.43 ± 0.07 (mean ± standard deviation of a single Gaussian fit to the data) reflecting the ability of the ssDNA overhang to entropically collapse and sample a large number of conformational states (Figure 2C, G and I; see ‘DNA’ trace). In the presence of ATP and RecA, the resulting FRET distribution exhibited a peak with a mean FRET value of 0.3 ± 0.1, consistent with the formation of a highly dynamic RecA filament undergoing simultaneous assembly and disassembly (Figure 2D, G and I ‘ATP’ trace). Upon incubating the DNA substrate with RecA in the presence of ATPγS, we observed a shift in the FRET distribution to an even lower value of 0.20 ± 0.07, reflecting the formation of a rigid, fully extended ssDNA-RecA filament (Figure 2E, G and I ‘ATPγS’). Since ATPγS traps the RecA filament in an ‘active’ conformation that is capable of LexA repressor autocatalytic cleavage, we interpreted the 0.2 FRET state as corresponding to the active state (Craig and Roberts, 1981). Incubation of RecA with ADP revealed a broad FRET distribution similar to that obtained in the presence of ATP, reflecting unstable RecA filaments assembled on the ssDNA overhang (See Figure 2—figure supplement 2A).

Next, we studied the FRET displayed by the ssDNA-RecA-ATP filament while titrating in purified mCI (Figure 2H and Figure 2—figure supplement 2B) to gain insight into the influence of mCI binding on the stability of ssDNA-RecA-ATP filaments (Figure 2F). In the presence of mCI the FRET substrate exhibited a bi-modal behavior: either molecules exhibited the 0.43 FRET state or the 0.2 FRET state. Upon increasing mCI concentration, the FRET distribution gradually shifted from a mean of 0.43 to 0.20 in response to higher concentrations of mCI (Figure 2H). By fitting the distributions to a sum of two Gaussian fits reflecting the ‘bound’ state (0.20 FRET) and ‘unbound’ state (0.43 FRET), we were able to obtain the bound fraction at every concentration of mCI tested (Figure 2H and J). Fitting these data to the Hill equation yielded an equilibrium dissociation constant of 36 ± 10 nM with a Hill coefficient of 2.4 ± 0.2 (Figure 2J; error bars represent fitting errors). The increase in the population of molecules in the lowest FRET state in response to an increase in mCI concentration demonstrates that mCI stabilizes the RecA filament in the active conformation.

Examination of the FRET traces revealed that in the presence of mCI, the DNA substrate exhibits stochastic transitions from the RecA-bound to the unbound state (e.g. Figure 2I for [mCI] = 10 nM). The frequency of these transitions to the unbound state decreased in the presence of high concentration of mCI (Figure 2I, see also Figure 2—figure supplement 2B). FRET traces of DNA substrates in the presence of RecA and saturating concentrations of mCI (3 μM) exhibited stable, long-lived binding events at a FRET value of 0.20 over the time scale of acquisition (Figure 2I). To obtain off rates from the data, we applied a threshold of 0.3 (Figure 2—figure supplement 2C) to segment the trajectories such that segments with FRET values less than 0.3 were considered to reflect the ‘bound’ state, and those above 0.3 were considered to be the ‘unbound’ state. The cumulative residence time distributions for the binding events (low FRET values) in the FRET trajectories were best fit by a sum of two exponentials decaying according to a fast off rate k off,1 = 0.23 ± 0.06 s−1 and a slow off rate k off,2 = 0.044 ± 0.002 s−1 (Figure 2K). These off-rates were largely independent of the concentration of mCI (Figure 2K). However, strikingly, the fraction of the population decaying following the slower off rate increased from 35% in the absence of mCI to 91% in the presence of 1 μM mCI (Figure 2L).

Inside cells, RecA* performs three key catalytic functions: LexA cleavage, ATP hydrolysis and strand-exchange in its various roles in SOS induction, filament formation and DNA recombination. First, we investigated whether mCI inhibits these catalytic activities of RecA* in vitro. To that end, we measured the influence of mCI binding on the ATPase activity of RecA*. Incubation of pre-formed RecA* filaments on circular ssDNA M13mp18 substrates with micromolar concentrations of mCI revealed a pronounced inhibition of RecA* ATPase activity (Figure 2—figure supplement 2D). The tagged mCI variants did not significantly inhibit RecA* ATPase activity at concentrations under 500 nM (Figure 2—figure supplement 2D).

Current models based on EM reconstructions suggest that both mCI and LexA interact in the groove of the RecA* filament (Galkin et al., 2009; Ndjonka and Bell, 2006; Yu and Egelman, 1993). We next set out to investigate whether mCI could compete with, and inhibit RecA*-catalyzed cleavage of LexA. To that end, we conducted LexA cleavage assays and separated the cleavage products on a SDS-PAGE (Figure 2—figure supplement 2E). Quantification of the percentage of uncleaved LexA as a function of time revealed that even high concentrations of mCI did not inhibit LexA cleavage. However, μM concentrations of mCI induced a delay in the kinetics of LexA cleavage.

RecA* occupies a central role in homologous recombination (HR) where it executes the homology search and strand-exchange required for HR. We therefore investigated whether the strand-exchange activity of RecA* was influenced by mCI. We found that μM concentrations (2–10 μM) of mCI potently inhibited strand exchange (Figure 2—figure supplement 2F). Importantly, tagged mCI constructs did not significantly inhibit strand-exchange activity at concentrations below 500 nM (Figure 2—figure supplement 2F).

Taken together, these in vitro investigations provide insights into the consequences of mCI binding on the activity of RecA*. We found that mCI stabilizes the RecA* filament in the ‘active’ conformation that is capable of LexA cleavage. At high concentrations (5–10 μM), mCI can inhibit ATP hydrolysis and strand-exchange by RecA*, and delay LexA cleavage. This is consistent with mCI binding to the RecA nucleoprotein filament groove as anticipated. Importantly, at low concentrations (10–100 nM) similar to those we eventually employed as a standard in vivo (as described below), these key activities of RecA* are not significantly affected by the presence of mCI or tagged variant. These findings emphasize the suitability of the use of mCI derived probes for visualizing RecA* function.