Formation Of DNA-protein Complexes Research Articles

SmedOB1 is Required for Planarian Homeostasis and Regeneration.

The planarian flatworm is an emerging model that is useful for studying homeostasis and regeneration due to its unique adult stem cells (ASCs). Previously, planaria were found to share mammalian TTAGGG chromosome ends and telomerases; however, their telomere protection proteins have not yet been identified. In Schmidtea mediterranea, we identified a homologue of the human protection of telomeres 1 (POT1) with an OB-fold (SmedOB1). SmedOB1 is evolutionarily conserved among species and is ubiquitously expressed throughout the whole body. Feeding with SmedOB1 double-stranded RNAs (dsRNAs) led to homeostasis abnormalities in the head and pharynx. Furthermore, several ASC progeny markers were downregulated, and regeneration was impaired. Here we found that SmedOB1 is required for telomeric DNA-protein complex formation and it associates with the telomere TTAGGG sequence in vitro. Moreover, DNA damage and apoptosis signals in planarian were significantly affected by SmedOB1 RNAi. We also confirmed these phenotypes in Dugesia japonica, another flatworm species. Our work identified a novel telomere-associated protein SmedOB1 in planarian, which is required for planarian homeostasis and regeneration. The phylogenetic and functional conservations of SmedOB1 provide one mechanism by which planarians maintain telomere and genome stability to ensure their immortality and shed light on the regeneration medicine of humans.

Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.

Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato.

A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene

Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r2 > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility.

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures.

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.

Sequence-Specific Binding and Diffusion of TRF1 on Telomeric DNA Studied by Molecular Dynamics

Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes. Their protective and structural function depends on several protein factors that form the so-called shelterin complex, anchored to telomeric DNA via three different proteins: double-strand binding TRF1 and TRF2, and single-strand binding POT1. Homologous DNA-binding domains of TRF1 and TRF2 share the homeodomain fold commonly found among DNA-interacting factors, and both recognize the repeating telomeric motif (5'-TTAGGG-3') in a structurally similar fashion. Therefore, while both domains exhibit a high degree of similarity to other DNA-binding folds, they differ from them in the availability of target binding sites, which in the case of telomere-binding proteins are arranged in long (several kbp) tandem arrays. In such a system, diffusion between neighboring binding sites becomes a relevant aspect of protein dynamics at telomeres, facilitating e.g. the assembly of shelterin units on telomeric DNA.In this work, we present a comprehensive study of the sequence specific binding of TRF1 to telomeric DNA and the diffusion of TRF1 along the telomeric sequence. We try to identify the factors that facilitate the formation of sequence-specific protein-DNA complex, and explore the free energy landscape of TRF1 on telomeric DNA using both simulations of spontaneous complex formation and umbrella sampling along a helical path on DNA. The obtained results agree well with experimental data on the kinetics of diffusion on telomeric DNA, allowing us to extract the large-scale coarse grained picture of protein-DNA interaction in the telomeric region.

Transcriptional regulation of IER5 in response to radiation in HepG2.

Radiotherapy is one of the important treatments for patients with hepatocellular carcinoma. The treatment response (or efficacy), however, is limited in many patients due to acquired radiation resistance of cancer cells. Immediate-early response 5 (IER5) is one of the genes upregulated on radiation. The gene could modulate cell cycle checkpoint, leading to a decrease of cancer cell survival in response to radiation. To better understand how IRE5 expression is regulated on radiation, this study aims to identify transcription factors that interact with IER5 promoter region in liver cancer cell line. Using bioinformatic tool, we identified promoter region of IER5 gene. Subsequent luciferase reporter assay revealed two putative GC binding factor (GCF) binding sites. We found mutations of these binding sites increased the luciferase activity, suggesting a negative regulation of GCF on IER5 transcriptional activity. The physical interaction of GCF with the gene promoter was confirmed using chromatin immunoprecipitation and electrophoretic mobility shift assay assays. Different doses of radiation were also applied in these experiments, and we found the formation of protein-DNA complex reduced with the increasing dose of radiation. Together, we propose the GCF regulated transcriptional activity, at least in part, contributed to the upregulation of IER5 on radiation. The present findings provide insights into understanding the regulatory mechanisms of IER5.

The application of fluorescence-conjugated pyrrole/imidazole polyamides in the characterization of protein-DNA complex formation.

N-Methylpyrrole (Py)-N-methylimidazole-(Im) polyamides are sequence-specific DNA-binding modules that are widely used for gene regulation, as synthetic transcriptional factors and as sequence-specific DNA alkylating agents. Recently, Py-Im polyamides have been conjugated with fluorophores, resulting in conjugates that are useful for the detection of specific DNA sequences. A Förster resonance energy transfer has been observed between Cy3- and Cy5-conjugated Py-Im polyamides on the nucleosome, indicating that fluorescence-conjugated Py-Im polyamides could possibly be used to characterise protein-DNA complexes. In this minireview, we discuss recent reports regarding fluorescence-conjugated Py-Im polyamides and their future application in the characterization of protein-DNA complex formation.

Effects of protein-DNA complex formation on the intermolecular vibrational density of states of interfacial water.

Single-stranded DNAs (ss-DNAs) are formed as intermediates during DNA metabolic processes. ss-DNA binding (SSB) proteins specifically bind to the single-stranded segments of the DNA and protect it from being degraded. We have performed room temperature molecular dynamics simulations of the aqueous solution of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element (FUSE) binding protein (FBP) complexed with two ss-DNA oligomers. Efforts have been made to explore the influence of complex formation on low-frequency vibrational density of states of the surface water molecules. It is revealed that increased back scattering of water confined around the complexed structures leads to significant blue shifts of the band corresponding to the O···O···O bending or restricted transverse motions of water, the effect being more for the bridged water molecules. Importantly, it is demonstrated that the formation of such complexed structures of a similar type may often influence the transverse and longitudinal degrees of freedom of the surrounding water molecules in a nonuniform manner.

Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

Background/Aims: Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels. Methods: Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter. Results: Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation. Conclusion: NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.

Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach.

The TRAF-interacting protein (TRAIP) is a novel E2F target with peak expression in mitosis.

The TRAF-interacting protein (TRAIP) is an E3 ubiquitin ligase required for cell proliferation. TRAIP mRNA is downregulated in human keratinocytes after inhibition of the PI3K/AKT/mTOR signaling. Since E2F transcription factors are downstream of PI3K/AKT/mTOR we investigated whether they regulate TRAIP expression. E2F1 expression significantly increased the TRAIP mRNA level in HeLa cells. Reporter assays with the 1400 bp 5'-upstream promoter in HeLa cells and human keratinocytes showed that E2F1-, E2F2- and E2F4-induced upregulation of TRAIP expression is mediated by 168 bp upstream of the translation start site. Mutating the E2F binding site within this fragment reduced the E2F1- and E2F2-dependent promoter activities and protein-DNA complex formation in gel shift assays. Abundance of TRAIP mRNA and protein was regulated by the cell cycle with a peak in G2/M. Expression of GFP and TRAIP-GFP demonstrated that TRAIP-GFP protein has a lower steady-state concentration than GFP despite similar mRNA levels. Cycloheximide inhibition experiments indicated that the TRAIP protein has a half-life of around four hours. Therefore, the combination of cell cycle-dependent transcription of the TRAIP gene by E2F and rapid protein degradation leads to cell cycle-dependent expression with a maximum in G2/M. These findings suggest that TRAIP has important functions in mitosis and tumorigenesis.

GIP1 protein is a novel cofactor that regulates DNA-binding affinity of redox-regulated members of bZIP transcription factors involved in the early stages of Arabidopsis development.

In response to environmental light signals, gene expression adjustments play an important role in regulation of photomorphogenesis. LHCB2.4 is among the genes responsive to light signals, and its expression is regulated by redox-regulated members of G-group bZIP transcription factors. The biochemical interrelations of GBF1-interacting protein 1 (GIP1) and the G-group bZIP transcription factors have been investigated. GIP1, previously shown to enhance DNA-binding activities of maize GBF1 and Arabidopsis GBF3, is a plant specific protein that reduces DNA-binding activity of AtbZIP16, AtbZIP68, and AtGBF1 under non-reducing conditions through direct physical interaction shown by the yeast two-hybrid and pull-down assays. Fluorescence microscopy studies using cyan fluorescent protein (CFP)-fusion protein indicate that GIP1 is exclusively localized in the nucleus. Under non- reducing conditions, GIP1 exhibits predominantly high molecular weight forms, whereas it predominates in low molecular weight monomers under reducing conditions. While reduced GIP1 induced formation of DNA-protein complexes of G-group bZIPs, oxidized GIP1 decreased the amount of those complexes and instead induced its chaperone function suggesting functional switching from redox to chaperone activity. Finally analysis of transgenic plants overexpressing GIP1 revealed that GIP1 is a negative co-regulator in red and blue light mediated hypocotyl elongation. By regulating the repression effect by bZIP16 and the activation effect by bZIP68 and GBF1 on LHCB2.4 expression, GIP1 functions to promote hypocotyl elongation during the early stages of Arabidopsis seedling development.

Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

The DNA-binding protein HU has a regulatory role in the acid stress response mechanism in Helicobacter pylori.

Bacterial genomes are compacted by association with histone-like proteins to form a complex known as bacterial chromatin. The histone-like protein HU is capable of binding and bending the DNA molecule, a function related to compaction, protection, and regulation of gene expression. In Helicobacter pylori, HU is the only histone-like protein described so far. Proteomic analysis from our laboratory showed that this protein is overexpressed under acidic stress. We used a purified recombinant wild-type protein and two mutant proteins with the amino acid substitutions K3A/S27D and K62R/V63N/P64A to characterize the function of the N-terminal domain and the flexible arm of HU. In vitro assays for DNA protection, bending, and compaction were performed. We also designed a H.pylori hup::cat mutant strain to study the role of HU in the acid stress response. HUwt protein binds DNA and promotes its bending and compaction. Compared with the wild-type protein, both mutant proteins have less affinity for DNA and an impaired bending and compaction ability. By using qRT-PCR, we confirmed overexpression of two genes related to acid stress response (ureA and speA). Such overexpression was abolished in the hup::cat strain, which shows an acid-sensitive phenotype. Altogether, we have shown that HUwt -DNA complex formation is favored under acidic pH and that the complex protects DNA from endonucleolytic cleavage and oxidative stress damage. We also showed that the amino-terminal domain of HU is relevant to DNA-protein complex formation and that the flexible arm of HU is involved in the bending and compaction activities of HU.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

ObjectivesSystemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased...

VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus.

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator veA, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus Aspergillus flavus. We found that the expression of A. flavus homolog genes involved in the HOG signaling pathway is regulated by veA. Deletion of veA resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of cat1 and trxB indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the cat1 promoter, where the absence of VeA results in abnormally stronger complex formation with reduced cat1 expression and more sensitivity to ROS in a veA deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the cat1 promoter. Our study also revealed that veA positively influences the expression of the transcription factor gene atfB and that normal formation of DNA-protein complexes in the cat1 promoter is dependent on AtfB.

AB0187 Estrogen-Induction of STAT1 and STAT4 Expression by Estrogen Receptor Alpha Suggests a Novel IFN-Alpha-Independent Mechanism of Sex-Bias in Systemic Lupus Erythematosus Pathogenesis

BackgroundSystemic Lupus Erythematosus (SLE) is a devastating multi-organ autoimmune disease displaying an overwhelming female predilection. Estrogen (E2) influences gene expression by binding to estrogen receptors (ERα and ERβ) and has...

Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells

Galanin and its receptors have been shown to be expressed in undifferentiated mouse embryonic stem (ES) cells through transcriptome and proteomic analyses. Although transcriptional regulation of galanin has been extensively studied, the regulatory proteins that mediate galanin expression in mouse ES cells have not yet been determined. Through sequence alignments, we have found a high degree of similarity between mouse and human galanin upstream sequences at −146bp/+69bp (proximal region) and −2408bp/−2186bp (distal region). These regions could be recognized by ES cell nuclear proteins, and EMSA analysis suggests a specific functionality. Analysis of the proximal region (PR) using EMSA and ChIP assays showed that the CREB protein interacts with the galanin promoter both in vitro and in vivo. Additional EMSA analysis revealed that an SP1 consensus site mediated protein–DNA complex formation. Reporter assays showed that CREB is an activator of galanin expression and works cooperatively with SP1. Furthermore, analysis of the distal region (DR) using EMSA assays demonstrated that both HOX-F and PAX 4/6 consensus sites mediated protein–DNA complex formation, and both sites inhibited luciferase activity in reporter assays. These data together suggest that CRE and SP1 act as activators at the basal promoter, while HOX-F and PAX 4/6 act as silencers of transcription. The interplay of these transcription factors (TF) may drive regulated galanin expression in mouse ES cells.

In Vivo Studies of Active Processes in the Escherichia Coli Nucleoid

The cell is the site of actively motor-driven processes which drive the intracellular environment far from thermodynamic equilibrium. The dynamics of biological macromolecules such as DNA in such an environment are complex and subject to a multitude of constraints and forces. Inspired by our in vitro studies of DNA looping with optical tweezers that showed that additional non-thermal fluctuations in the DNA can substantially enhance the formation of regulatory DNA-protein complexes, we study the conformational fluctuations of chromosomal DNA in vivo in Escherichia coli by Fluorescence Correlation Spectroscopy (FCS).Conformational fluctuations of the DNA move the bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a confocal microscope. From the time correlation functions of the measured fluorescence intensity, we quantify the fluctuations of the DNA as measured by its time-dependent mean square displacement, and the viscoelastic moduli of the nucleoid. These quantities in live cells significantly differ from the ATP-depleted dead cells on longer time scales, indicating that the fluctuations on longer time scale may be driven by active processes involving molecular motors that generate forces by ATP hydrolysis. On shorter time scales, we see little difference between live and dead cells, suggesting that the processes on corresponding short length scales rely primarily on thermally-driven diffusive mechanisms. We also note that the rheological properties of E. coli nucleoid significantly change when the ATP hydrolysis in cells is inhibited.

Partial Unwrapping of SSB from SsDNA Facilitates RecA Filament Formation

DNA breaks can be repaired by homologous recombination, a process that maintains genetic stability in an organism. A protein that is essential for this mechanism in E. coli is RecA. During repair, RecA must bind and form nucleoprotein filaments on single-stranded DNA (ssDNA) in direct competition with single-stranded DNA binding protein (SSB). Despite extensive studies, the mechanism behind this competitive process remains unclear.Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the mechanical properties of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under a range of tensions. This single-molecule assay allows us to probe and visualize simultaneously the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution. Using a 70-nucleotide, poly-dT ssDNA construct designed to accommodate a single SSB, we investigated different scenarios of protein-DNA complex formation under a range of tensions between 0-10 pN.Individual SSBs on their own bind and condense ssDNA in discrete steps, the size of which depend on tension. Under low tensions (1-3 pN), an SSB wraps 50-70 nt of ssDNA in a single step. At higher tensions (>4 pN), SSB exhibit transient, partially wrapped states on ssDNA. In the absence of SSB, RecA filaments nucleate rapidly on ssDNA regardless of tension. In contrast, when RecA is added to ssDNA coated with an SSB, filament formation is inhibited at low tensions. At higher tensions, where the SSB can partially unwrap from ssDNA, we observe RecA filaments form after an extended lag time. Our results suggest a mechanism in which SSBs inhibit RecA by sequestering ssDNA. Tension-induced unwrapping of the SSB makes ssDNA available for RecA to bind and nucleate a filament.