The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA

The crystal structure of full-length homotetrameric single-stranded DNA (ssDNA)-binding protein from Escherichia coli (SSB) has been determined to 3.3 A resolution and reveals that the entire C-terminal domain is disordered even in the presence of ssDNA. To our knowledge, this is the first experimental evidence that the C-terminal domain of SSB may be inherently disordered. The N-terminal DNA-binding domain of the protein is well ordered and is virtually indistinguishable from the previously determined structure of the chymotryptic fragment of SSB (SSBc) in complex with ssDNA. The absence of observable interactions with the core protein and the crystal packing of SSB together suggest that the disordered C-terminal domains likely extend laterally away from the DNA- binding domains, which may facilitate interactions with components of the replication machinery in vivo. The structure also reveals the conservation of molecular contacts between successive tetramers mediated by the L(45) loops as seen in two other crystal forms of SSBc, suggesting a possible functional relevance of this interaction.     

Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> AlkB interacts with single-stranded DNA binding protein SSB by an intrinsically disordered region of SSB

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152–169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.     

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure.

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

The structure of DdrB from Deinococcus: a new fold for single-stranded DNA binding proteins

Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 Å structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.     

Distinct properties of Mycobacterium tuberculosis single-stranded DNA binding protein and its functional characterization in Escherichia coli

Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions. Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world’s population and kills about 2–3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli. In this study, use of the yeast two-hybrid assay suggests that the EcoSSB and the MtuSSB are even capable of heterooligomerization. However, the MtuSSB failed to complement a Δssb strain of E.coli. The sequence comparison suggested that MtuSSB contained a distinct C-terminal domain. The C-terminal domain of EcoSSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MtuSSB and EcoSSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Δssb strain of E.coli. These data allude to the occurrence of a ‘cross talk’ between the N- and the C-terminal domains of the SSBs for their in vivo function. Further, compared with those of the EcoSSB, the secondary/tertiary interactions within MtuSSB were found to be less susceptible to disruption by guanidinium hydrochloride. Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.     

Regulation of Single-stranded DNA Binding by the C Termini of Escherichia coli Single-stranded DNA-binding (SSB) Protein

The homotetrameric Escherichia coli single-stranded DNA-binding (SSB) protein plays a central role in DNA replication, repair, and recombination. In addition to its essential activity of binding to transiently formed single-stranded (ss) DNA, SSB also binds an array of partner proteins and recruits them to their sites of action using its four intrinsically disordered C-terminal tails. Here we show that the binding of ssDNA to SSB is inhibited by the SSB C-terminal tails, specifically by the last 8 highly acidic amino acids that comprise the binding site for its multiple partner proteins. We examined the energetics of ssDNA binding to short oligodeoxynucleotides and find that at moderate salt concentration, removal of the acidic C-terminal ends increases the intrinsic affinity for ssDNA and enhances the negative cooperativity between ssDNA binding sites, indicating that the C termini exert an inhibitory effect on ssDNA binding. This inhibitory effect decreases as the salt concentration increases. Binding of ssDNA to approximately half of the SSB subunits relieves the inhibitory effect for all of the subunits. The inhibition by the C termini is due primarily to a less favorable entropy change upon ssDNA binding. These observations explain why ssDNA binding to SSB enhances the affinity of SSB for its partner proteins and suggest that the C termini of SSB may interact, at least transiently, with its ssDNA binding sites. This inhibition and its relief by ssDNA binding suggest a mechanism that enhances the ability of SSB to selectively recruit its partner proteins to sites on DNA.     

Phage Orf Family Recombinases: Conservation of Activities and Involvement of the Central Channel in DNA Binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.     

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.     

The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding

Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70–protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70_1–326). Correlation analysis of the amide ¹H and ¹⁵N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70_1–326 with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70_1–326, indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70_1–326 and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70_1–168). We also examined the structure of hRPA70_1–326 after addition of three different ssDNA substrates. Each of these substrates induced specific amide ¹H and/or ¹⁵N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide ¹H and ¹⁵N chemical shift changes of hRPA70_1–168 after addition of ssDNA. The same amide ¹H and ¹⁵N chemical shift changes were observed for the NTD in hRPA70_1–168 and hRPA70_1–326. The NTD residues with the largest amide ¹H and/or ¹⁵N chemical shift changes were localized to a basic cleft that is important for hRPA70–protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70–protein interactions.     

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2

BackgroundHuman Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown.MethodsRecombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC.ResultsBoth hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1.ConclusionsBiochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism.General significanceThis is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.     

DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery

Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the χ subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between χ and an EcoSSB/ssDNA complex when compared to that of χ to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of χ, showing that χ interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that χ specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to χ at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III.     

SSB protein limits RecOR binding onto single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.     

A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.     

Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair.     

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

     

Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

BackgroundSSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.ResultsThis paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (T_m) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis.ConclusionNeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.     

3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis–trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the χ subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli.     

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.