protein-ssDNA Complex Research Articles

Experimental and computational studies of the effects of high hydrostatic pressure on biomolecular interactions.

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1,000 atmospheres at the bottom of the oceans (Ando N, et al. Annu. Rev. Biophys. 2021:50:343). Here I will summarize our recent experimental and computational work on various aspects of the effects of high hydrostatic pressure on biomacromolecules. Particular emphasis will be made on the volume changes, ΔV, that accompany conformational transitions in biomacromolecules such as proteins, protein-ssDNA complexes, and dsDNA duplexes. The importance of ΔV comes from the fact that it defines the pressure dependence of stability ΔV = (dΔG/dP)T. Thus, the response of the system to changes in pressure is driven by Le Chatelier's principle: if volume changes upon unfolding are positive, increase in hydrostatic pressure will lead to an increase in stability, whereas if the changes are negative, the stability will decrease with the increase in hydrostatic pressure. The results will be discussed in terms of relative contributions of volumes changes associated with voids and hydration to the net volume changed that accompany conformational transitions in biomacromolecules.

Unraveling the molecular basis of protein search on ssDNA track and binding specificity of the ssDNA sequences.

The genetic materials of cellular organisms are organized as double-stranded DNA (dsDNA), which transiently gets unwrapped and forms single-stranded DNA (ssDNA) intermediates. These intermediates serve as a template for the various cellular processes. In its isolated form, ssDNA is vulnerable to nucleolytic degradation and prone to adopt unwanted secondary structures. To avoid the mess, cells employ specialized ssDNA binding proteins (SSBs), which bind with high affinity and both specifically and non-specifically to ssDNA to stabilize these intermediates. To understand their biophysics, we have developed a computational model that has been extensively parametrized by studying ssDNA binding on replication protein A, a major eukaryotic SSB, which is later tested on numerous other protein-ssDNA complexes. Our study delineates the molecular mechanism of target search by SSBs on the ssDNA track, which is otherwise difficult to capture through experimental approaches due to their binding promiscuity. The SSB-ssDNA model is then used as a testbed to develop a hybrid simulation technique for calculating protein-ssDNA binding thermodynamics for many ssDNA sequences in a single run. The method explores both sequence specificity of ssDNA and conformational space simultaneously by simulating a joint probability distribution, which makes searching through ssDNA sequence space computationally efficient. Our results show that the method agrees well with the experiments in predicting the relative binding specificity of ssDNA sequences for a given SSB without performing a costly, time-consuming DNA microarray experiment.

Effects of Hydrostatic Pressure on the Thermodynamics of CspB-Bs Interactions with the ssDNA Template.

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.

Characterization of the T4 gp32-ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry.

Protein–DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32·ssDNA, with results that both support previous studies and simultaneously uncover novel insight into this non-covalent biological complex. Native mass spectrometry of the protein reveals the co-occurrence of Zn-bound monomers and homodimers, while addition of differing lengths of ssDNA generates a variety of protein:ssDNA complex stoichiometries (1 : 1, 2 : 1, 3 : 1), indicating sequential association of gp32 monomers with ssDNA. Ultraviolet photodissociation (UVPD) mass spectrometry allows characterization of the binding site of the ssDNA within the protein monomer via analysis of holo ions, i.e. ssDNA-containing protein fragments, enabling interrogation of disordered regions of the protein which are inaccessible via traditional crystallographic techniques. Finally, two complementary cross-linking (XL) approaches, bottom-up analysis of the crosslinked complexes as well as MS1 analysis of the intact complexes, are used to showcase the absence of ssDNA binding with the intact cross-linked homodimer and to generate two homodimer gp32 model structures which highlight that the homodimer interface overlaps with the monomer ssDNA-binding site. These models suggest that the homodimer may function in a regulatory capacity by controlling the extent of ssDNA binding of the protein monomer. In sum, this work underscores the utility of a multi-faceted mass spectrometry approach for detailed investigation of non-covalent protein-DNA complexes.

Pressure controllable aptamers picking strategy by targets competition

Selection of aptamers with high affinity and good specificity requires multiple rounds of alternating steps of separation and PCR amplification. Herein, we proposed a novel high-efficiency aptamers picking strategy: One-round pressure controllable selection (OPCS). OPCS integrates four types of screening superiority, high-efficiency separation, one-round selection and PCR amplification, synchronous negative selection and targets competition. The controllable screening pressure can be achieved through two approaches, balanced competition by the regulation of protein concentration, and dominant competition by introducing a predatory protein with high concentration. In OPCS process, two proteins were co-incubated with one ssDNA library, and each protein bound its favorable sequences specifically and formed protein-ssDNA complex respectively. Meanwhile, one protein could supply/suffer the picking pressure of affinity and specificity to/from another, which eliminated weakly bound or unbound sequences for each other. Two complexes could be separated and collected conveniently, and aptamers for two proteins obtained synchronously with high affinity and good specificity. This strategy not only provides a more effective way for aptamers selection, but shows great potential in other ligands or drugs selection.

Crystal structure of the Wheat dwarf virus Rep domain.

The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein-ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein-DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24 Å resolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.

Online reaction based single-step CE for Protein-ssDNA complex obtainment to assist aptamer selection

CE application in aptamer selection (CE-SELEX) shows more advantages than other selection methods. In this study, an online reaction based single-step CE (ssCE) mode was employed for fast obtaining protein-ssDNA complex. Using human thrombin (H-Thr) and its aptamer Apt29 as models, we accomplished the procedures of mixing, reaction, separation, detection and complex collection in single step online process, which took about 10 min to obtain the H-Thr/Apt29 complex. Important factors, affecting the aptamer and H-Thr interaction (buffer, ratio of aptamer and H-Thr amount), and complex separation and collection (voltage and temperature) were discussed. Later, the online reaction of H-Thr with an 80 nt ssDNA library was realized under optimized conditions, and the H-Thr/ssDNA complex was collected and subjected to PCR. By analyzing the PCR product through capillary gel electrophoresis, the resulting approximative 80 nt DNA length validated the ssDNA sequence in complex. To confirm the availability of ssCE mode, two ssDNA libraries with different lengths (56 nt and 82 nt ssDNA) and three proteins (platelet derived growth factor, PDGF-BB; lactoferrin protein, LF; and single-strand DNA binding protein, SSB) were utilized. Their complex peaks were also observed in electropherograms as expected. Additionally, the online incubation of ssDNA and H-Thr was achieved by stopping the separation voltage for some time when ssDNA passed the H-Thr zone. Our results show the ssCE mode has apparent merits of saving time and sample cost for aptamer selection against protein targets.

Trypsin enhances aptamer screening: A novel method for targeting proteins

A novel screening method for protein aptamer selection was developed in this study. Aptamers with high affinity and specificity to the surface recombinant antigen of Helicobacter pylori (HP-Ag) and to tumor markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 19-9(CA19-9) were screened using trypsin enhanced screening method. Briefly, the target proteins above were immobilized onto 96-well polystyrene plates and incubated with a single-stranded DNA (ssDNA) library for aptamer selection. Then, trypsin was introduced to digest the proteins and obtain ssDNA that bound to the target proteins with high specificity. The concentration of ssDNA that shed from protein-ssDNA complexes was detected. After sequencing, the enrichment of target-specific aptamers was monitored and the affinity of each aptamer was analyzed. Urea, which has been reported in other article, was used to compare with trypsin. The results revealed that trypsin was more effective than urea for protein aptamer selection. The protocol used in this study provided a novel method for generating aptamers.

Low pH capillary electrophoresis application to improve capillary electrophoresis-systematic evolution of ligands by exponential enrichment

In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein–ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein–ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE–UV are applied for interactive evaluation. By increasing injection time, larger amount protein–ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein–ssDNA complex in low pH CE separation is discussed.

Crystallographic analysis of an RNA polymerase σ-subunit fragment complexed with -10 promoter element ssDNA: quadruplex formation as a possible tool for engineering crystal contacts in protein-ssDNA complexes.

Structural studies of -10 promoter element recognition by domain 2 of the RNA polymerase σ subunit [Feklistov & Darst (2011), Cell, 147, 1257-1269] reveal an unusual crystal-packing arrangement dominated by G-quartets. The 3'-terminal GGG motif of the oligonucleotide used in crystallization participates in G-quadruplex formation with GGG motifs from symmetry-related complexes. Stacking between neighboring G-quadruplexes results in the formation of pseudo-continuous four-stranded columns running throughout the length of the crystal (G-columns). Here, a new crystal form is presented with a different arrangement of G-columns and it is proposed that the fortuitous finding of G-quartet packing could be useful in engineering crystal contacts in protein-ssDNA complexes.

Expanding the scope of CE reactor to ssDNA‐binding protein–ssDNA complexes as exemplified for a tool for direct measurement of dissociation kinetics of biomolecular complexes

CE reactor (CER), which was developed as a tool for direct measurement of the dissociation kinetics of metal complexes, was successfully applied to the complexes of Escherichia coli ssDNA-binding protein (SSB) with ssDNA. The basic concept of CER is the application of CE separation process as a dissociation kinetic reactor for the complex, and the observation of the on-capillary dissociation reaction profile of the complex as the decrease of the peak height of the complex with increase of the migration time. The peak height of [SSB-ssDNA] decreases as the migration time increases since the degree of the decrease of [SSB-ssDNA] through the on-capillary dissociation reaction is proportional to the degree of the decrease of the peak height of [SSB-ssDNA]. The dissociation degree-time profiles for the complexes are quantitatively described by analyzing a set of electropherograms with different migration times. Dissociation rate constants of [SSB-ssDNA] consisting of 20-mer, 25-mer and 31-mer ssDNA were directly determined to be 3.99x10(-4), 4.82x10(-4) and 1.50x10(-3)/s, respectively. CER is a concise and effective tool for dissociation kinetic analysis of biomolecular complexes.

Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture

Rad51, Rad52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. Here, we report that Rad52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPA(rfa1-t11)) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of Rad52 and RPA, also facilitated the same reaction, demonstrating its conserved nature. We also demonstrate that the two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture.

Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase

The rate of annealing of long linear complementary single-stranded (ss) DNAs can be increased greatly by certain DNA-binding proteins including the herpes simplex virus type 1 ICP8 SSB/recombinase. Using electron microscopy, we have investigated the DNA-protein structures involved in ICP8-mediated DNA annealing. We show that the formation of superhelical ICP8-ssDNA filaments is required for annealing. Two superhelices interact with each other to form a coiled-coil, which is the intermediate in annealing. In this process, the superhelices likely rotate and translocate relative to each other. Psoralen/UV photocrosslinking studies revealed that meta-stable contacts form at sites of limited sequence homology during the annealing. Partial proteolysis of ICP8 in the protein-ssDNA complexes showed that Mg2+ induces conformational changes in the N-terminal region (amino acid residues 1-305) of ICP8. In addition to Mg2+, Ca2+ and, to a significantly lesser extent, Cu2+ and Mn2+, were found to induce superhelix formation of the ICP8-ssDNA filament and to facilitate annealing. Mechanisms for how the coiled-coil structures facilitate annealing are discussed.

The interaction of the A and A* proteins of bacteriophage phi X174 with single-stranded and double-stranded phi X DNA in vitro.

The binding of the bacteriophage phi X 174-coded A and A* proteins to single-stranded (ssDNA) and double-stranded (dsDNA ) phi X DNA was studied by electron microscopy. The interaction of the A* protein with ssDNA and dsDNA was also studied by sedimentation velocity centrifugation. It was shown that the binding of the A and A* proteins to ssDNA occurs in a non-cooperative manner and requires no or very little sequence specificity under the conditions used here. Both protein-ssDNA complexes have the same compact structure caused by intrastrand cross-linking through the interaction of protein molecules with separate parts of the ssDNA molecule. The A protein does not bind to phi X dsDNA in the absence of divalent cations. The A* protein does bind to dsDNA, although it has a strong preference for binding to ssDNA. The structure of the A* protein-dsDNA complexes is different from that of the A* protein-ssDNA complexes, as the former have a rosette-like structure caused by protein-protein interactions. High ionic strengths favour the formation of large condensed aggregates.

Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics

Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, dϵA(pϵA) 19, that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer dϵA(pϵA) 9, which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. This mechanism is described by the equation: Helicase + ssDNA ↔ k −1 k 1 ( H-ssDNA ) 1 ↔ k −2 k 2 H-ssDNA ) 2 ↔ k −3 k 3 H-ssDNA ) 3 The value of the bimolecular rate constant, k 1, is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA) 1. These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix-projection operator technique, which provides a powerful method to address stopped-flow kinetics, particularly, the amplitudes. The method allowed us to determine the specific fluorescence changes accompanying the formation of all the intermediates. The sequential nature of the determined mechanism indicates the lack of the kinetically significant conformational equilibrium of the DnaB hexamer as well as a transient dissociation of the hexamer prior to the ssDNA binding. The significance of these results for the functioning of the DnaB helicase is discussed.

Conformational studies of the C-terminal domain of bacteriophage Pf1 gene 5 protein

The gene 5 protein (g5p) of the bacteriophage Pf1 is a 144 residue single-stranded (ss) DNA binding protein involved in replication and packaging of the viral DNA. Compared to the gene 5 proteins of other filamentous bacteriophages, such as fd, the Pf1 g5p has an additional C-terminal sequence (∼40 residues) with an unusual amino acid composition, being particularly rich in proline, glutamine and alanine. This C-terminal sequence is susceptible to limited proteolysis, in contrast to the globular N-terminal domain of the protein. The C-terminal sequence has been shown to play a role in the stabilisation of the protein–ssDNA complex. In the present study, the DNA sequence corresponding to the 38 amino acid residue C-terminal peptide has been cloned and expressed. A variety of biophysical techniques suggest that this peptide has a largely irregular conformation in solution, in contrast to the N-terminal globular domain that is principally β-sheet. However, circular dichroism (CD) spectroscopy indicates that the peptide can be induced to form a structure that resembles a left-handed polyproline-like (P II) helix, suggesting that the C-terminal tail of the protein may adopt a more structured conformation in the appropriate physiological environment.

Single-stranded DNA binding protein from bacteriophage cf: characterization, gene localization and protein-ssDNA complex

The single-stranded DNA binding protein from the filamentous bacteriophage cf has been purified and characterized. The first 12 amino acids, resulting from the N-terminal amino acid sequencing analysis of the protein, agree with an open reading frame (ORF) on the cf genome. The ORF contains 294 bp and codes for a 98 a.a. protein of molecular weight 10.8 kDa, consistent with the result from the denaturing protein gel analysis. The protein appears to be a homodimer as evident from the apparent molecular weight of about 22 kDa obtained from native protein gel analysis. The gene location of the protein has been identified as gene V of the cf single stranded genome, same as that from the M13 phage. The GVP of cf shows a strong sequence homology to the ssDNA binding proteins of Ff, IKe and Pf3 filamentous phages. The DNA binding wing of GVP, conserved among the filamentous phages, has been predicted for cf. To further characterize the protein, the GVP-ssDNA complex of cf has been purified from the infected host ( Xanthomonas campestris pv. citri) by density gradient centrifugation. Transmission electron microscopy (TEM) images of the complex showed that it is about 1200 nm in length and 9 nm in diameter and it has a highly regular morphology with a central groove shadow running along the entire structure, but without any apparent helical pattern seen in the M 13 complex. The GVP-ssDNA complex of cf seems more rigid than that of M13. Our computer modeling study suggested that this difference between the two complexes may be due to the additional 11 or 12 amino acids at the C-terminal end of the cf-GVP.

Crystal Structures of Y41H and Y41F Mutants of Gene V Protein from Ff Phage Suggest Possible Protein-Protein Interactions in the GVP-ssDNA Complex

Gene V protein (GVP) encoded by the filamentous phage Ff (M13, fl, fd) is a homodimeric protein of 87 amino acids that binds to single-stranded DNA (ssDNA) nonspecifically and cooperatively. The structure (monoclinic C2 form) of the wild-type protein has been determined and refined at 1.8-A resolution [Skinner et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2071-2075]. The monomer structure consists of a somewhat distorted five-stranded beta-barrel core with three prominent loops: a DNA-binding loop, a dyad loop, and a dimer contact loop. The amino acid residue at position 41 plays an important role in the dimer-dimer interactions of the protein-ssDNA complex. Two Y41 mutant structures have been studied by X-ray crystallography. The Y41F GVP structure has been refined to an R-factor of 0.180 at 2.2-A resolution and is very similar to the wild-type (wt) structure (rmsd of all C alpha atoms = 0.30 A). In contrast, Y41H GVP forms a new crystal lattice in the space group P2(1)2(1)2(1) with a = 77.18 A, b = 84.17 A, and c = 28.62 A. Its structure has been solved by the molecular replacement method and refined to an R-factor of 0.170 at 2.5-A resolution. The two monomers of Y41H are crystallographically independent, and their structures remain similar to wt-GVP but with significant differences, particularly in the DNA-binding hairpin region. In both crystals, the loop (residues 36-43) that contains the Y41 residue is involved in the crystal dimer packings but in a different manner. The dimer-dimer contacts found in the wt-GVP crystal may be important for GVP aggregation in the absence of DNA. In the presence of DNA, the dimer-dimer contacts may switch to the type found in the Y41H crystal, allowing the GVP-ssDNA complex to form cooperatively. A model of the complex, consistent with existing biochemical and biophysical data, has been constructed from those crystal packing data.

Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

The RecA803 protein suppresses the recombinational repair defect of recF mutations and displays enhanced joint molecule formation in vitro (Madiraju et al., 1988). To understand the physical basis for these phenomena, the biochemical properties of RecA803 protein were compared with those of the wild-type protein. The RecA803 protein shows greater DNA-dependent ATPase activity than the wild-type protein with either M13 single-stranded (ss) DNA, which contains secondary structure, or double-stranded DNA. This increased activity reflects an enhanced ability of the mutant protein to form active complexes with these DNA molecules rather than an enhanced catalytic turnover activity, because identical kcat values for ATP hydrolysis are obtained when DNA substrates lacking secondary structure are examined. In addition, the ssDNA-dependent ATPase activity of RecA803 protein displays greater resistance to inhibition by SSB (single-stranded DNA binding) protein. These properties of the RecA803 protein are not due to either an increased binding affinity for ssDNA or an increased kinetic lifetime of RecA803 protein-ssDNA complexes, demonstrating that altered protein-DNA stability is not the basis for the enhanced properties of RecA803 protein. However, the nucleation-limited rate of association with ssDNA is more rapid for the RecA803 protein than for wild-type RecA protein. Consequently, we suggest that altered protein-protein interactions may account for the differences between these two proteins. The implications of these results with regard to the partial suppression of recF mutations by recA803 are discussed (Madiraju et al., 1988).

The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition.

The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.