SSB Binding Research Articles

Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds to ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes that differ in occluded site size and cooperativity. High “unlimited” cooperativity is associated with the 35 site size ((SSB)35) mode at low [NaCl], whereas the 65 site size ((SSB)65) mode formed at higher [NaCl] (> 200mM), where ssDNA wraps completely around the tetramer, displays “limited” cooperativity forming dimers of tetramers. It was previously thought that high cooperativity was associated only with the (SSB)35 binding mode. However, we show here that highly cooperative binding also occurs in the (SSB)65/(SSB)56 binding modes at physiological salt concentrations containing either glutamate or acetate. Highly cooperative binding requires the 56 amino acid intrinsically disordered C-terminal linker (IDL) that connects the DNA binding domain with the 9 amino acid C-terminal acidic tip that is involved in SSB binding to other proteins involved in genome maintenance. These results suggest that high cooperativity involves interactions between IDL regions from different SSB tetramers. Glutamate, which is preferentially excluded from protein surfaces, may generally promote interactions between intrinsically disordered regions of proteins. Since glutamate is the major monovalent anion in E. coli, these results suggest that SSB likely binds to ssDNA with high cooperativity in vivo.

Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding

The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent polypeptides to assist protein folding. To reveal its working principle, we determined the nascent chain-binding pattern of Ssb atnear-residue resolution by invivo selective ribosome profiling. Ssb associates broadly with cytosolic, nuclear, and hitherto unknown substrate classes of mitochondrial and endoplasmic reticulum (ER) nascent proteins, supporting its general chaperone function. Ssb engages most substrates by multiple binding-release cycles to a degenerate sequence enriched in positively charged and aromatic amino acids. Timely association with this motif upon emergence at the ribosomal tunnel exit requires ribosome-associated complex (RAC) but not nascent polypeptide-associated complex (NAC). Ribosome footprint densities along orfs reveal faster translation at times of Ssb binding, mainly imposed by biases in mRNA secondary structure, codon usage, and Ssb action. Ssb thus employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.

The Role of Intrinsically Disordered Linker and Salt Type on Cooperativity of E. Coli SSB Binding to ssDNA

E. coli single strand DNA binding protein (SSB) is a key protein in DNA replication, recombination and repair. SSB functions as a homotetramer and binds ssDNA in different modes that differ in their excluded site size and cooperative binding to ssDNA. Two types of cooperativity have been identified previously: the high “unlimited”, nearest-neighbor type of cooperativity associated with the 35 site size ((SSB)35) binding mode at low [NaCl], and a lower, “limited” cooperativity associated with formation of dimers of tetramers in the 65 site size ((SSB)65) binding mode at [NaCl] > 200 mM. Using sedimentation velocity and equilibrium titration methods we have examined how salt concentration and type affects these cooperativities and binding mode transitions on long ssDNA, focusing on the effects of KGlu, since glutamate is the major monovalent anion in E. coli. Unexpectedly, we find that in contrast to NaCl, KGlu promotes highly cooperative binding to ssDNA even at high concentrations of 0.5 M, conditions that promote the ((SSB)65) mode, suggesting that highly cooperative binding is likely occurring in vivo. We also show that all cooperative binding behavior absolutely requires the unstructured C-terminal region (intrinsically disordered linker (IDL)) connecting the SSB DNA binding core and the 9 amino acid C-terminal end (tip) needed for SSB binding to other proteins involved in genome maintenance (supported by NIH GM030498 to TML).

Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction.

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction.

Structure and Function of the PriC DNA Replication Restart Protein

Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks.

Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant.

Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo.

Is a fully wrapped SSB-DNA complex essential for Escherichia coli survival?

Escherichia coli single-stranded DNA binding protein (SSB) is an essential homotetramer that binds ssDNA and recruits multiple proteins to their sites of action during genomic maintenance. Each SSB subunit contains an N-terminal globular oligonucleotide/oligosaccharide binding fold (OB-fold) and an intrinsically disordered C-terminal domain. SSB binds ssDNA in multiple modes in vitro, including the fully wrapped (SSB)65 and (SSB)56 modes, in which ssDNA contacts all four OB-folds, and the highly cooperative (SSB)35 mode, in which ssDNA contacts an average of only two OB-folds. These modes can both be populated under physiological conditions. While these different modes might be used for different functions, this has been difficult to assess. Here we used a dimeric SSB construct with two covalently linked OB-folds to disable ssDNA binding in two of the four OB-folds thus preventing formation of fully wrapped DNA complexes in vitro, although they retain a wild-type-like, salt-dependent shift in cooperative binding to ssDNA. These variants complement wild-type SSB in vivo indicating that a fully wrapped mode is not essential for function. These results do not preclude a normal function for a fully wrapped mode, but do indicate that E. coli tolerates some flexibility with regards to its SSB binding modes.

Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR)

Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact. This elevated temperature is used as the hybridization temperature. Practical implementation of this apparently trivial approach in DQAMmiR has two major challenges. First, melting temperatures of all mismatched hybrids should be similar to each other and should not reach the melting temperature of any of the matched hybrids. Second, the elevated hybridization temperature should not deteriorate CE separation of the hybrids from the excess probes and the hybrids from each other. The second problem is further complicated by the reliance of separation in DQAMmiR on single-strand DNA binding protein (SSB) whose native structure and binding properties may be drastically affected by the elevated temperature. These problems were solved by two approaches. First, locked nucleic acid (LNA) bases were incorporated into the probes to normalize the melting temperatures of all target miRNA hybrids allowing for a single hybridization temperature; binding of SSB was not affected by LNA bases. Second, a dual-temperature CE was developed in which separation started with a high capillary temperature required for proper hybridization and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids from excess probes and the hybrids from each other. The developed approach was sufficiently robust to allow its integration with sample preconcentration by isotachophoresis to achieve a limit of detection below 10 pM.

Basic and aromatic residues in the C-terminal domain of PriC are involved in ssDNA and SSB binding.

In bacterial organisms, the oriC-independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC-independent replication restart primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

Intrinsically Disordered C-Terminal Tails of E. coli Single-Stranded DNA Binding Protein Regulate Cooperative Binding to Single-Stranded DNA

The homotetrameric Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA replication, repair and recombination. E. coli SSB can bind to long single-stranded DNA (ssDNA) in multiple binding modes using all four subunits [(SSB)65 mode] or only two subunits [(SSB)35 binding mode], with the binding mode preference regulated by salt concentration and SSB binding density. These binding modes display very different ssDNA binding properties with the (SSB)35 mode displaying highly cooperative binding to ssDNA. SSB tetramers also bind an array of partner proteins, recruiting them to their sites of action. This is achieved through interactions with the last 9 amino acids (acidic tip) of the intrinsically disordered linkers (IDLs) within the four C-terminal tails connected to the ssDNA binding domains. Here, we show that the amino acid composition and length of the IDL affects the ssDNA binding mode preferences of SSB protein. Surprisingly, the number of IDLs and the lengths of individual IDLs together with the acidic tip contribute to highly cooperative binding in the (SSB)35 binding mode. Hydrodynamic studies and atomistic simulations suggest that the E. coli SSB IDLs show a preference for forming an ensemble of globular conformations, whereas the IDL from Plasmodium falciparum SSB forms an ensemble of more extended random coils. The more globular conformations correlate with cooperative binding.

Solid-State Nanopore Characterization of Single-Strand DNA-SSB Interactions

Single strand binding protein (SSB) plays a central role in genome replication by binding single-strand (ss) regions of DNA to prevent hybridization and nuclease digestion. Here, we study ssDNA-SSB interactions using solid-state nanopores. We show that a systematic increase in the molar ratio of SSB to ssDNA results in increased translocation event depth until DNA saturation is reached. These results are recapitulated in a bulk electromobility shift assay and indicate the weak cooperativity in SSB binding under high ionic strength conditions. We demonstrate that the strong selectivity of SSB for ssDNA over double-strand molecules can be used to differentiate a heterogeneous mixture and use comparisons of linearized and circular ssDNA data to suggest structural characteristics of SSB binding at high salt.

Ssb gene duplication restores the viability of ΔholC and ΔholD Escherichia coli mutants.

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.

SSB binding to single-stranded DNA probed using solid-state nanopore sensors.

Single-stranded DNA (ssDNA) binding protein plays an important role in the DNA replication process in a wide range of organisms. It binds to ssDNA to prevent premature reannealing and to protect it from degradation. Current understanding of SSB/ssDNA interaction points to a complex mechanism, including SSB motion along the DNA strand. We report on the first characterization of this interaction at the single-molecule level using solid-state nanopore sensors, namely without any labeling or surface immobilization. Our results show that the presence of SSB on the ssDNA can control the speed of nanopore translocation, presumably due to strong interactions between SSB and the nanopore surface. This enables nanopore-based detection of ssDNA fragments as short as 37 nt, which is normally very difficult with solid-state nanopore sensors, due to constraints in noise and bandwidth. Notably, this fragment is considerably shorter than the 65 nt binding motif, typically required for SSB binding at high salt concentrations. The nonspecificity of SSB binding to ssDNA further suggests that this approach could be used for fragment sizing of short ssDNA.

Mechanistic insights into PriA mediated DNA replication restart (924.2)

DNA replication restart is an essential genome maintenance process by which replication complexes (replisomes) are reloaded onto abandoned DNA replication forks. In Escherichia coli and related bacteria this process is orchestrated by the PriA DNA helicase, which binds to replication forks in a structure‐specific manner. PriA also binds to single‐stranded DNA‐binding protein (SSB), a protein that coats the lagging‐strand template at DNA replication forks. Complex formation with SSB stimulates PriA DNA unwinding DNA and, as we show here, this interaction is required for PriA remodeling of SSB‐DNA nucleoprotein complexes. Proteins that interact with SSB typically do so by binding directly to the evolutionarily conserved amphipathic C‐terminal tail of SSB (SSB‐CT). Although previous studies indicated that PriA also binds to SSB via the SSB‐CT, a lack of structural information on PriA has left the SSB binding site on PriA unknown. We determined the 4 Å resolution structure of PriA bound to the SSB‐CT. The electrostatic characteristics of the PriA SSB‐Ct binding site resemble those of other known SSB‐interacting proteins. Single‐molecule FRET studies show that a PriA variant that has lost the ability to bind the SSB‐CT fails to modulate SSB‐DNA complexes. Our studies point to the importance of the PriA/SSB interaction in allowing PriA to reload replisomes at damaged replication forks where SSB is bound to the lagging‐strand template.

Bound or Free: Interaction of the C-Terminal Domain of Escherichia coli Single-Stranded DNA-Binding Protein (SSB) with the Tetrameric Core of SSB

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Universal Drag Tag for Direct Quantitative Analysis of Multiple MicroRNAs

Using microRNA (miRNA) as molecular markers of diseases requires a method for accurate measurement of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed based on a classical hybridization approach. In DQAMmiR, miRNAs are hybridized with excess fluorescently labeled complementary DNA probes. Capillary electrophoresis (CE) is used to separate the unreacted probes from the hybrids and the hybrids from each other. The challenging separation is achieved by using two types of mobility modifiers. Single-strand DNA binding protein (SSB) is added to the running buffer to bind and shift the single-stranded unreacted probes from the double-stranded hybrids. Different drag tags are built into the probes to introduce significant differential mobility between their respective hybrids. For the method to be practical it requires a universal extendable drag tag. Polymers are a logical choice for making extendable drag tags. Our recent theoretical work suggested that short peptides could provide a sufficient mobility shift to facilitate DQAMmiR. Here, we experimentally confirm this prediction in the analysis of five miRNAs: mir10b, mir21, mir125b, mir145, and mir155. We conjugated four fluorescently labeled DNA molecules with peptides of 5, 10, 15, or 20 neutral amino acids in length; the fifth probe was peptide-free. The peptide tags showed no interference with SSB binding to the probes and facilitated separation of the five hybrids. The mobilities of the five hybrids were used to refine the previously suggested theory. The analysis was performed in both a pure buffer and in cell lysate. Our analysis of the experimental data suggests that using DNA-peptide probes can readily facilitate simultaneous analysis of more than 10 miRNAs.

Quantitative Analysis of MicroRNA in Blood Serum with Protein-Facilitated Affinity Capillary Electrophoresis

MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.

E. coli SSB tetramer binds the first and second molecules of (dT) 35 with heat capacities of opposite sign

We have previously shown that formation of a 1:1 fully wrapped complex of Escherichia coli SSB tetramer with (dT) 70 displays a temperature-dependent sign reversal of the binding heat capacity (ΔC P). Here we examine SSB binding to shorter oligodeoxynucleotides ((dX) 35) to probe whether this effect requires binding of one or two (dX) 35 molecules per SSB tetramer. We find that the ΔC P for the first molecule of (dX) 35 is always negative. However, a sign reversal of ΔC P from negative to positive occurs with increasing temperature for binding of the second (dX) 35. This striking behavior of ΔC P for the second (dX) 35 appears linked to conformational changes within the ssDNA–SSB complex that are required to form a fully wrapped (SSB) 65 binding mode. These results also underscore that binding heat capacities of macromolecular interactions have multiple origins that cannot be understood simply on the basis of examining static structures.

Displacement of SSB Protein from Single Stranded DNA by λ Phage Beta Protein

Genetic engineering in vivo by homologous recombination, also known as recombineering, is a well-established method allowing DNA alterations on any replicon in E. coli. Recombineering uses the bacteriophage λ Red recombination functions, Exo (a 5’ to 3’ double strand (ds)DNA exonuclease) and Beta (a single strand (ss)DNA binding and annealing protein). Recombineering with ss-oligonucleotides is more efficient than with dsDNA and requires only Beta activity. One of the two possible complementary oligonucleotides that can be used for any recombination gives a much higher frequency. The oligonucleotide of the same sequence as Okazaki fragments at the replication fork lagging strand shows the higher recombination frequency. This suggests a mechanism by which the oligonucleotide bound Beta is annealed directly to the complementary single strand region at the replication fork. For this to happen, single-strand DNA binding protein (SSB) would need to be displaced during annealing of the Beta-oligonucleotide complex. Recently Roy and colleagues (Nature (2009) 461, 1092-97) proposed that diffusion of SSB along a single-strand DNA allows its displacement by RecA. A 99 base oligo should be long enough to allow SSB to diffuse along it. We have investigated the binding of SSB to a 99 base oligonucleotide by surface plasmon resonance (SPR) spectroscopy in the presence of complementary or non-complementary oligos and without or with Beta. Beta only in the presence of complementary oligo is able to facilitate the displacement of SSB. We have evaluated the role of potassium glutamate and spermidine on Beta's ability to displace SSB.

Direct Evidence of the Role of ATPγS in the Binding of Single-Stranded Binding Protein (Escherichia coli) and RecA to Single-Stranded DNA

To gain insight into the influence of ATPγS on the competitive binding of RecA and single-stranded binding protein (SSB) on single-stranded DNA (ssDNA), AFM imaging was used to examine the three-dimensional structures of the different complexes formed by the binding of the two proteins on ssDNA in the presence and absence of ATPγS. In the presence of ATPγS, RecA attaches to ssDNA, displacing SSB, to form continuous binding regions that caused considerable elongation of the strand. When ATPγS is absent, RecA could not compete with SSB and only binds at a few sites that correspond to the vacancy in ssDNA left when SSB unbinds. These results provide direct evidence that, while SSB binding affinity to DNA is substantially higher than that of RecA, the presence of ATPγS is sufficient to alter the events and enable RecA coating of DNA.