Although the RecB2109CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3′-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the χ-modified wild-type RecBCD enzyme. However, we further show that the RecB2109CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the χ-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites. https://doi.org/10.7554/eLife.08646.001 eLife digest Chromosomes consist of two strands of DNA that are intertwined as a helix. These strands can peal apart to form single-stranded DNA before the DNA is copied and for other processes in cells. Single-stranded DNA can also form if double-stranded DNA is damaged by harmful radiation or chemicals so that only one strand can be copied or when the damaged strand is selectively degraded by enzymes during the course of repair. Proteins called single-stranded binding proteins (or SSBs for short) bind to single-stranded DNA molecules to protect them. A molecule of single-stranded DNA wraps around a group of four SSB proteins (known as a tetramer). The degree to which DNA is wrapped around the SSB tetramer depends on the environmental conditions. For example, in the presence of high levels of salt—which is typical inside cells – single-stranded DNA wraps around all four subunits of the SSB. However, at lower salt levels, the DNA only wraps around some of the units in the SSB tetramer. A process called recombination can repair breaks in DNA. During this process, a broken DNA molecule that contains single-stranded DNA can pair with a matching (or complementary) strand from an intact double-stranded DNA molecule that carries an identical genetic sequence. A protein called RecO helps to anneal two complementary DNA strands together with the help of the RecR protein. However, for RecR and RecO to achieve this task, they need to work together with the resident SSB proteins that occupy single-stranded DNA. How they find matching sequences when SSB proteins are in the way is not clear. Bell et al. used techniques called TIRF microscopy and single-molecule force spectroscopy to directly observe how SSB from the bacterium E. coli binds to and coats individual molecules of single-stranded DNA. The experiments show that when the levels of salt increase, single-stranded DNA that is coated with SSB proteins becomes compacted and the length of the DNA molecules decreases, a process referred to as ‘intramolecular condensation’. Bell et al. found that condensation occurred because two SSB tetramers that are associated with different regions of the single-stranded DNA interact to form stable ‘octamers’. In the presence of RecO and RecR, the single-stranded DNA compacted even further. Bell et al. propose that these recombination proteins act as a scaffold to bring together distant partner sites of single-stranded DNA. This condensation allows two DNA sequences that can be far apart in the cell to find one another more quickly. The next challenge is to understand how the matching regions of single-stranded DNA are identified, and what causes the SSBs to move to allow other repair proteins to gain access to the DNA. https://doi.org/10.7554/eLife.08646.002 Introduction Single-stranded DNA (ssDNA) binding protein (SSB) binds rapidly and avidly to ssDNA generated during the normal processes of DNA replication, recombination, and repair (Meyer and Laine, 1990). In doing so, SSB protects ssDNA from chemical damage and exonucleolytic degradation, removes secondary structure, and enhances the enzymatic activity of many proteins involved in DNA metabolism (Shereda et al., 2008). The extent to which ssDNA is wrapped around a tetramer of SSB is often referred to as a binding mode, defined by the apparent site size or footprint (i.e. nucleotides bound per tetramer). These binding modes are sensitive to salt, temperature, pH, and binding density (Lohman and Ferrari, 1994). The cooperativity, i.e. nearest neighbor interactions, of SSB is also altered when SSB binds ssDNA in different binding modes (Lohman et al., 1986; Bujalowski and Lohman, 1987; Ferrari et al., 1994). At low salt concentrations, where ssDNA is partially wrapped around SSB, cooperativity is very high or ‘unlimited’. As such, proteins crowd very close to each other along the ssDNA. At higher more physiological salt concentrations, SSB binds in the fully wrapped binding mode and exhibits ‘limited’ cooperativity, where SSB forms dimers of tetramers (i.e. octamers) along the ssDNA (Bujalowski and Lohman, 1987; Lohman and Ferrari, 1994). Early electron microscopic visualization of SSB-coated ssDNA revealed a beads-on-string structure similar to those observed for nucleosomes bound to dsDNA (Chrysogelos and Griffith, 1982). These structures are observed at a low binding density of SSB; however, at higher binding densities, the structures form smooth, contoured nucleoprotein complexes that are condensed relative to the contour length of the corresponding dsDNA (Griffith et al., 1984; Hamon et al., 2007). High-resolution atomic force microscopy (AFM) imaging of spread SSB-coated ssDNA formed in low and high salt, measured approximately a twofold difference between the contour lengths of the nucleoprotein complexes. This difference in contour length was proposed to reflect the partially wrapped SSB35 and fully wrapped SSB65 binding modes, corresponding to a site size of 35 and 65 nucleotides, respectively (Hamon et al., 2007). It is worth noting that an additional, intermediate binding mode, SSB55, was also observed in direct binding experiments (Lohman and Overman, 1985; Bujalowski and Lohman, 1986). SSB has been studied extensively using single-molecule FRET on short oligonucleotide substrates (Roy et al., 2007, 2009; Zhou et al., 2011); however, relatively little is known about the more complex dynamics of the SSB-coated ssDNA nucleoprotein fiber that forms on the extensive regions of ssDNA during DNA unwinding, resection, and replication. These ssDNA regions can range from a few hundred to tens of thousands of nucleotides in length. More than a dozen proteins interact directly with SSB via its short, unstructured C-terminal tail (Shereda et al., 2008; Wessel et al., 2013; Bhattacharyya et al., 2014). In the absence of interaction partners or ssDNA, this unstructured peptide tail interacts with the subunits within the SSB tetramer (Kozlov et al., 2010a). This inter-subunit allostery contributes to the complex, cooperative nature of SSB binding to ssDNA. It has been proposed that the binding modes of SSB might be modulated in vivo for differential roles during ssDNA processing. Direct evidence for such modulation remained elusive for many years (Shereda et al., 2008); however, recent work has shown that PriC remodels the SSB-ssDNA complex to create a DNA structure competent for DnaB loading during replication restart (Wessel et al., 2013) and that PriA modulates the SSB-ssDNA complex to expose a potential replication initiation site (Bhattacharyya et al., 2014). RecO catalyzes the annealing of complementary strands of ssDNA even in the presence of SSB, which otherwise kinetically blocks annealing (Kantake et al., 2002); in this regard, perhaps RecO is mimicking the action of PriA (Bhattacharyya et al., 2014). This annealing activity is essential for RecA-independent, homology-directed DNA repair that proceeds through the single-strand annealing (SSA) pathway (Kantake et al., 2002). RecO also stimulates RecA-dependent homologous recombination by acting with RecR and RecF to promote RecA filament assembly (Umezu et al., 1993; Morimatsu and Kowalczykowski, 2003; Handa et al., 2009). RecR, which does not bind to ssDNA, dsDNA, or SSB, binds to RecO and enhances the affinity of RecO for ssDNA-bound SSB (Umezu et al., 1993; Umezu and Kolodner, 1994); however, neither RecO nor RecOR are capable of physically displacing SSB from ssDNA (Umezu and Kolodner, 1994; Ryzhikov et al., 2011). When RecR is bound to RecO, it partially inhibits the annealing activity of RecO but stimulates both the rate of RecA nucleation and filament growth on SSB-coated ssDNA (Kantake et al., 2002; Bell et al., 2012; Morimatsu et al., 2012). As RecA does not interact with SSB, RecO, or RecR (Umezu et al., 1993), this activity must proceed through a RecOR-induced conformational change in the SSB-ssDNA complex (Ryzhikov et al., 2011; Zhou et al., 2011). Using both direct visualization of SSB-coated ssDNA and single-molecule force spectroscopy, we observed the reversible intramolecular condensation of single SSB-coated ssDNA fibers. The extent of this intramolecular condensation increases with salt concentration, but exceeds the expected extent of condensation based on most previous measurements of SSB-ssDNA complexes (Chrysogelos and Griffith, 1982; Hamon et al., 2007). We also observe RecO-induced condensation of the SSB-ssDNA complex, as well as long-range intramolecular bridging in the presence of both RecO and RecR. We propose that the nature of this condensation is due to the ability of SSB to interact with distant sites along the ssDNA—either through dimerization of SSB tetramers or through the partial wrapping of distant ssDNA sites on a single SSB protomer—and that one role of RecOR is to enhance these distant interactions, which in turn would facilitate annealing of complementary strands. Our observations raise the possibility that the microscopic changes in ssDNA-binding modes observed for SSB cause macroscopic condensation (or de-condensation) of the nucleoprotein fiber that, in turn, might regulate access to ssDNA. Results Single molecules of SSB-coated ssDNA reversibly condense in response to increasing salt concentration We previously described a fluorescent biosensor for ssDNA derived from an engineered mutant, SSBG26C, wherein a fluorophore was conjugated to the protein using Alexa Fluor 488 maleimide to produce SSBAF488 (Dillingham et al., 2008). This protein maintains a high, albeit attenuated, affinity for ssDNA (Bell, 2011; Bell et al., 2012). SSBAF488-ssDNA nucleoprotein complexes were formed by first denaturing bacteriophage λ genomic dsDNA that had been biotinylated at the 3′-terminated ends using DNA polymerase (Figure 1A). The denatured DNA was mixed with buffer containing SSBAF488, attached to a glass surface functionalized with streptavidin, extended using flow within a microfluidic chamber, and visualized using total internal reflection fluorescence (TIRF) microscopy (Figure 1B). When the concentration of sodium acetate (NaOAc) was increased during buffer exchange at a constant flow rate and a constant concentration of fluorescent SSB, the length of single molecules of SSBAF488-coated ssDNA shortened (Figure 1C, Video 1, and Figure 1D); however, the fluorescent intensity of individual molecules remained constant (Figure 1E, Video 1, and Figure 1—figure supplement 1), indicating that the protein had not dissociated, but rather redistributed, along the ssDNA molecule. In contrast, when the SSBAF488 was exchanged for unmodified wild type SSB, which has a higher affinity for ssDNA, the fluorescence rapidly decreased as SSBAF488 was displaced from the ssDNA (Figure 1F). Figure 1 with 1 supplement see all Download asset Open asset Visualization of salt-induced intramolecular condensation of single molecules of SSBAF488-ssDNA complexes. (A) Bacteriophage λ dsDNA (48.5 kbp) was biotinylated, denatured, coated with SSBAF488, and then (B) attached to a streptavidin-coated glass coverslip of a microfluidic chamber where it was extended by buffer flow for direct imaging using total internal reflection fluorescence (TIRF) microscopy. (C) A montage of frames from a video recording the change in length of a single molecule of SSBAF488-coated single-stranded DNA (ssDNA) upon increasing [NaOAc] from 0 to 100 mM. The frames were rendered into a topographical intensity map. Time zero corresponds to the time at which the pump was turned on. The dead time of the experiment was ∼25 s due to the liquid volume in the lines between the syringe valve and the microfluidic chamber. (D) The length of the molecule in panel C during the change in salt from 0 mM to 100 mM NaOAc was measured for each frame and is plotted as a function of time. The dotted line represents the injection of the buffer into the microfluidic flow chamber. (E) The fluorescence intensity of the molecule in panel C was also measured for each frame and is plotted as a function of time. (F) The fluorescence intensity of a single molecule of SSBAF488-coated ssDNA is plotted as function of time during a similar experiment where SSBAF488 was exchanged for wild-type, unlabeled SSB. The decrease in fluorescence intensity corresponds to the displacement by wild-type SSB, which has higher affinity for ssDNA than SSBAF488. The fluorescence intensity (green circles) was determined by the mean pixel intensity of region of interest (ROI), and the gray error bars are the standard deviation of the pixels within the ROI. https://doi.org/10.7554/eLife.08646.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Salt-induced intramolecular condensation of SSBAF488-ssDNA. Video recording of a single molecule of SSBAF488-coated ssDNA, imaged using TIRF microscopy, upon increasing [NaOAc] from 0 to 100 mM. The video frames were rendered into a topological intensity map. Time zero corresponds to the time at which the pump was turned on. The dead time of the experiment was approximately 25 s due to the volume in the lines between the syringe valve and the microfluidic chamber. The molecule in the video corresponds to the molecule presented in Figure 1, panels C–E https://doi.org/10.7554/eLife.08646.005 High resolution imaging of SSB-coated ssDNA, using electron microscopy (EM) and AFM, had previously observed that the length of the nucleoprotein fiber is dependent on the buffer condition in which the complex is formed (Chrysogelos and Griffith, 1982; Hamon et al., 2007). However, we were perplexed by the observation that the amount of protein—indicated by the total fluorescent intensity—along the ssDNA remained essentially unchanged during each salt-jump transition, despite the fact that the length had changed substantially. Stopped-flow kinetic studies have previously demonstrated that SSB tetramers can transfer between ssDNA molecules without proceeding through a free protein intermediate (Kozlov and Lohman, 2002a, b) and single-molecule experiments have directly demonstrated that SSB tetramers diffuse rapidly on ssDNA and can ‘hop or jump’ across long distances of ssDNA via intersegmental transfer (Roy et al., 2009; Zhou et al., 2011; Lee et al., 2014). To distinguish between the intramolecular redistribution of SSB along the ssDNA in cis vs dissociation balanced with rebinding during the transition, we asked whether the salt-induced condensation of single molecules might be reversible in the absence of free protein. To address this possibility, SSB-coated ssDNA was tethered in a flow cell and extensively washed with buffer to remove free protein (∼100–200 volumes of the flow chamber). An injection loop was then used to transiently pulse the tethered molecules with buffer containing either 100 or 400 mM NaOAc, followed by a sufficient volume of buffer to remove the injected salt. When the salt concentration was raised to 100 mM NaOAc, the flow-extended molecules compacted to ∼60% of the length in the absence of salt (Figure 2A, Video 2), and then returned to the previously extended length when the salt was removed from the flow chamber. Similarly, this condensation was also observed when 400 mM NaOAc was used (Figure 2B, Video 3); however, the extent of the condensation was greater, wherein the molecules shortened to ∼12% of the flow-extended length in the absence of salt. In both experiments, the molecules were dimmer at the end of the experiment (Figure 2—figure supplement 1), where approximately 20% of the SSB dissociated in the 0→100 transition, and ∼60% dissociated in the 0→400 transition. Figure 2 with 1 supplement see all Download asset Open asset The length change upon salt-induced condensation of SSBAF488-coated ssDNA is nearly reversible in the absence of free SSB protein. (A) A montage of frames from a video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 100 mM NaOAc, and then subsequently reduced back to zero, conducted in the absence of free SSBAF488. The flow cell was extensively washed with buffer to remove free SSB protein from the flow cell before beginning the experiment. Video recording began when the pump was turned on, requiring ∼40-50 s for the dead volume to be flushed from the lines to the flow chamber. (B) Same as in (A), except the salt concentration was increased from 0 mM NaOAc to 400 mM NaOAc and then back to zero. Each frame of the montages is one micron wide. SSBAF488 was omitted from both of the high salt washes and from the 0 mM wash. Flow is from top to bottom in each image. https://doi.org/10.7554/eLife.08646.006 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Condensation of SSBAF488 in the absence of free protein during a transient increase from 0 to 100 mM NaOAc. Video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 100 mM NaOAc, and then subsequently reduced back to zero mM, conducted in the absence of free SSBAF488. The flow cell was extensively washed with buffer to remove free SSB protein before beginning the experiment. Video recording began when the pump was turned on, requiring ∼40-50 s for the dead volume to be flushed from the lines to the flow chamber. SSBAF488 was omitted from both of the high-salt washes and from the 0 mM wash. The video corresponds to the molecule presented in Figure 2A and Figure 2—figure supplement 1, panels B, C. https://doi.org/10.7554/eLife.08646.008 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Condensation of SSBAF488 in the absence of free protein during a transient increase from 0 to 400 mM NaOAc. Video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 400 mM NaOAc, and then subsequently reduced back to zero mM, conducted in the absence of free SSBAF488. The flow cell was extensively washed with buffer to remove free SSB protein before beginning the experiment. Video recording began when the pump was turned on, requiring ∼40-50 s for the dead volume to be flushed from the lines to the flow chamber. SSBAF488 was omitted from both of the high-salt washes and from the 0 mM wash. The video corresponds to the molecule presented in Figure 2B and Figure 2—figure supplement 1, panels D, E. https://doi.org/10.7554/eLife.08646.009 The extent of intramolecular condensation of SSB-coated ssDNA exceeds expectations based on simple wrapping or binding-mode transitions SSBAF488 is particularly suitable for single molecule measurements due to its relative photostability, whereas an alternative biosensor derived from fluorescein-5-maleimide, SSBf, is particularly suitable as an ensemble ssDNA-biosensor due to the large, linear increase in fluorescence upon ssDNA binding (Dillingham et al., 2008; Bell, 2011). To determine whether the measured lengths of individual SSBf-coated ssDNA complexes were correlated with the DNA-binding modes of SSB, we measured the stoichiometry of SSBf binding to poly(dT) using ensemble fluorescence measurements. Poly(dT) was titrated into a fixed concentration of SSBf at various concentrations of NaOAc, and the data were fit to a two-segment line to determine the apparent site size, which reflects the extant binding mode at each salt concentration (Figure 3A). The observed site size increased from ∼43 to ∼70 nucleotides per SSB tetramer over the range of salt concentrations tested as expected; however, we noted that the amplitude of the fluorescence enhancement increased dramatically with salt concentration, indicating the molecular environment of the fluorophore was altered (Figure 3B). In addition to the stoichiometric titrations performed by adding ssDNA to a fixed concentration of SSBf, we performed so-called ‘salt back-titrations’ to determine the concentration at which SSBf dissociates from ssDNA. When pre-formed complexes of SSBf-poly(dT) were titrated with an increasing concentration of salt (Figure 3—figure supplement 1), we observed an initial sharp increase in the fluorescence corresponding to the amplitudes from our direct titrations shown in Figure 3A. The fluorescence peaked between 200 and 400 mM NaOAc, and was followed by a shallow, linear decrease until the concentration reaches approximately 2M NaOAc, where the fluorescence intensity exhibited a sharp decrease due to dissociation (Figure 3—figure supplement 1). The midpoint of this sharp transition corresponds to the so-called, ‘salt-titration midpoint’, where ∼50% of the complex is dissociated (Kowalczykowski et al., 1981; Newport et al., 1981). The salt-titration midpoint for this experiment shows that ∼2 M NaOAc in the presence of 5 mM Mg(OAc)2 is required to dissociate half of the protein from the DNA. Figure 3 with 6 supplements see all Download asset Open asset The extent of SSB-ssDNA condensation is greater than anticipated based on known ssDNA-wrapping transitions. (A) Poly(dT) was titrated into 100 nM SSBf (tetramer) and the average fluorescence enhancement of SSBf from three titrations was plotted as a function of ratio of poly(dT) to SSB tetramer. The data were fit to a two-segment line, where the breakpoint is the stoichiometric endpoint of the titration corresponding to the site size of SSBf. (B) The amplitude of the fluorescence enhancement from the titrations performed in Figure 3A was plotted as a function of [NaOAc]. The error is smaller than the symbols. A larger number of titrations are shown here than in panel A to prevent panel A from being overcrowded; each fold-increase was determined by a full stoichiometric titration where each titration was completely and fully saturated. (C) Representative images of single molecules of SSBf-coated ssDNA at increasing [NaOAc] indicated. (D) The apparent binding site size (black circles, ± error of the fits from panel A determined from the titrations performed in panel A were plotted as a function of salt concentration. (E) Length of SSBf-coated ssDNA molecules plotted as a function [NaOAc] (N = 213). (F) Length of SSBf-coated ssDNA plotted as a function of [Mg(OAc)2] (N = 156) and (G) as a function of [NaGlu] in the absence (black, closed circles, N = 205) and presence (blue, open circles, N = 214) of 1 mM Mg(OAc)2. Unless otherwise indicated, all error bars represent standard deviation and when not visible were smaller than the symbols. https://doi.org/10.7554/eLife.08646.010 Individual SSBf-ssDNA complexes were also visualized with TIRF microscopy at increasing concentration of NaOAc, and it was evident that the length of the nucleoprotein fibers decreased as the NaOAc concentration increased (Figure 3C). Because we initially considered that the change in length might simply correspond to the change in the salt-dependent binding mode, we plotted the apparent site size determined from the titrations shown in Figure 3A, as a function of increasing [NaAOc] (Figure 3D) and compared this to the average length of SSBf-ssDNA complexes (Figure 3E), measured from images such as those in Figure 3C and more thoroughly represented in Figure 3—figure supplement 2. This comparison shows that the site size changes approximately ∼1.7-fold (43 nts to 70 nts) over this range whereas the length of the SSB-ssDNA molecules changes approximately ∼13-fold (from ∼6.5 μm to ∼0.5 μm). We note that the apparent site size of SSB can vary depending on the ssDNA used owing to exclusion of SSB from regions capable of forming stable secondary structure (Lohman and Overman, 1985); however, the reported change in site size for natural M13 ssDNA is only ∼2.2-fold (from 35 to 77) over a comparable range (1 mM to 300 mM NaCl), which is insufficient to account for our observations. If SSBf dissociated from ssDNA during the salt transitions, then formation of secondary structure could explain the additional compaction; however, when we measured the intensity of individual SSBf-ssDNA fibers at each salt concentration (Figure 3—figure supplement 3), we see an increase in fluorescence intensity similar to––and in good agreement with––the titration performed with poly(dT) in Figure 3A,B, and Figure 3—figure supplement 1. We note that the increase in fluorescence observed in Figure 3—figure supplement 3 is due to the environmental sensitivity of SSBf, and should not be confused with the results from Figure 1, where we used SSBAF488. Owing to the complex changes in fluorescence upon ssDNA binding, we cannot completely rule out the possibility that protein partially dissociates from the SSBf-ssDNA; however, we see no evidence of significant net dissociation in our assay in the presence of free protein. In the absence of free protein in solution, dissociation is apparent during the salt transitions (Figure 2—figure supplement 1), indicating that the net constant intensity we observe in Figure 1 and Figure 1—figure supplement 1 is maintained by mass action and rapid re-binding and redistribution of SSB along the ssDNA polymer. To further assess the condensation state of SSB-coated ssDNA at approximately physiological ionic conditions, we measured the SSBf-ssDNA lengths in the presence of the divalent cation magnesium, which is known to affect SSB binding modes (Bujalowski et al., 1988). Mg(OAc)2 induces a large condensation of nucleoprotein fibers that plateaus between 1 and 2 mM, and results in complexes that are as short as those produced with the much higher monovalent salt concentrations (Figure 3F and Figure 3—figure supplement 4). Escherichia coli maintains its intracellular osmolality by adjusting the intracellular concentration of glutamate, which ranges from ∼30 to 260 mM when cells are grown in media containing from ∼100 to 1100 mM solute (Richey et al., 1987). Therefore, we also titrated sodium glutamate (NaGlu) in the presence and absence of 1 mM Mg(OAc)2, which is within the range of the measured intracellular free magnesium ion concentration (1–2 mM) (Alatossava et al., 1985). At low concentrations of NaGlu (i.e. below 100 mM), the condensation was dominated by the presence of 1 mM Mg(OAc)2; however, the observed condensation became dominated by NaGlu at higher concentrations (Figure 3G and Figure 3—figure supplement 5 and 6). In the absence of Mg(OAc)2, there was a log-linear decrease in length with increasing concentrations of NaGlu, similar to our observation for NaOAc. Extrapolating from this data, the shorter, more condensed molecules that we observe likely represent the physiologically relevant condensation state of the SSB-ssDNA complex as estimated by the in vivo concentration and composition of salts. Force spectroscopy of single molecules of ssDNA and SSB-coated ssDNA reveals a nearly complete relief of hysteresis in SSB-ssDNA unfolding transitions To further explore the intramolecular condensation of SSB-coated ssDNA, we used a magnetic tweezer instrument to generate force-extension curves (Gosse and Croquette, 2002; Meglio et al., 2009). In particular, we reasoned that force spectroscopy would enable us to distinguish between intramolecular collapse owing to secondary structure formation and exclusion of SSB versus intrinsic, protein-mediated folding of the SSB-ssDNA molecule. We further reasoned that because SSBf is a modified variant of SSB (Dillingham et al., 2008; Bell, 2011), we could not exclude the possibility that a component of the intramolecular condensation might be due to the fluorescent adduct. This concern prompted us to assess the condensation state of single molecules of ssDNA coated with wild-t
Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy. Rad51 assembly extends the DNA by approximately 65%. Nucleoprotein filament formation occurs via rapid nucleation followed by growth from these nuclei. Growth does not continue indefinitely, however, and nucleoprotein filaments terminate when approximately 2 mum in length. The dependence of nascent filament formation on Rad51 concentration suggests that 2-3 Rad51 monomers are involved in nucleation. Rad51 nucleoprotein filaments are stable and remain extended when ATP hydrolysis is prevented; however, when permitted, filaments decrease in length as a result of conversion to ADP-bound nucleoprotein complexes and partial protein dissociation. Dissociation of Rad51 from dsDNA is slow and incomplete, thereby rationalizing the need for other proteins that facilitate disassembly.
