Search

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR4O and RecR4O2 complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR4O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB) 35 ) mode favored at low [NaCl] and high SSB/DNA ratios displays high "unlimited" nearest-neighbor cooperativity (ω 35 ), forming long protein clusters, whereas the 65-site-size ((SSB) 65 ) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays "limited" cooperativity (ω 65 ), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB) 65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearest-neighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT) 70 and (dT) 140 . We find that the (SSB) 35 mode always shows extremely high "unlimited" cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-ΔL, bind in the (SSB) 65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (ω 65/35 ), with ω 65  ≪ ω 65/35  <ω 35 . The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearest-neighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer-DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain., Competing Interests: The authors declare no conflict of interest.

The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Bacteria encode homooligomeric single-stranded (ss) DNA-binding proteins (SSBs) that coat and protect ssDNA intermediates formed during genome maintenance reactions. The prototypical Escherichia coli SSB tetramer can bind ssDNA using multiple modes that differ by the number of bases bound per tetramer and the magnitude of the binding cooperativity. Our understanding of the mechanisms underlying cooperative ssDNA binding by SSBs has been hampered by the limited amount of structural information available for interfaces that link adjacent SSB proteins on ssDNA. Here we present a crystal structure of Bacillus subtilis SsbA bound to ssDNA. The structure resolves SsbA tetramers joined together by a ssDNA "bridge" and identifies an interface, termed the "bridge interface," that links adjacent SSB tetramers through an evolutionarily conserved surface near the ssDNA-binding site. E. coli SSB variants with altered bridge interface residues bind ssDNA with reduced cooperativity and with an altered distribution of DNA binding modes. These variants are also more readily displaced from ssDNA by RecA than wild-type SSB. In spite of these biochemical differences, each variant is able to complement deletion of the ssb gene in E. coli. Together our data suggest a model in which the bridge interface contributes to cooperative ssDNA binding and SSB function but that destabilization of the bridge interface is tolerated in cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000nt(2)/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold "tetramer". Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance., (© 2013.)

The tetrameric Escherichia coli single-stranded DNA (ssDNA) binding protein (Ec-SSB) functions in DNA metabolism by binding to ssDNA and interacting directly with numerous DNA repair and replication proteins. Ec-SSB tetramers can bind ssDNA in multiple DNA binding modes that differ in the extent of ssDNA wrapping. Here, we show that the structurally similar SSB protein from the malarial parasite Plasmodium falciparum (Pf-SSB) also binds tightly to ssDNA but does not display the same number of ssDNA binding modes as Ec-SSB, binding ssDNA exclusively in fully wrapped complexes with site sizes of 52-65 nt/tetramer. Pf-SSB does not transition to the more cooperative (SSB)(35) DNA binding mode observed for Ec-SSB. Consistent with this, Pf-SSB tetramers also do not display the dramatic intra-tetramer negative cooperativity for binding of a second (dT)(35) molecule that is evident in Ec-SSB. These findings highlight variations in the DNA binding properties of these two highly conserved homotetrameric SSB proteins, and these differences might be tailored to suit their specific functions in the cell., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fluorescence methods have proven to be extremely useful tools for quantitative studies of the equilibria and kinetics of protein-DNA interactions. If the protein contains tryptophan (Trp), as is often the case, and there is a change in intrinsic Trp fluorescence of the protein, one can use this change in signal (quenching/enhancement) to monitor binding. One can also attach an extrinsic fluorophore to either the protein or the DNA and monitor binding due to a change in fluorescence intensity or a change in fluorescence anisotropy. Such equilibrium studies can provide important quantitative information on stoichiometries (occluded site size, number of binding sites) and energetics (affinities and cooperativities) of the interactions. This information is needed to understand the mechanisms of protein-DNA interactions. A critical aspect of such approaches for systems that have non-unity stoichiometries (e.g., a protein that binds multiple ligands) is knowledge of the relationship between the change in fluorescence signal (intensity or anisotropy) and the average extent of binding. Here we describe procedures for using fluorescence approaches to examine the stoichiometries and equilibrium binding affinities of Escherichia coli single-stranded DNA-binding protein (SSB) and Deinococcus radiodurans SSB with long polymeric ssDNA to determine an occluded site size. We also provide examples of studies of SSB binding to shorter oligonucleotides to demonstrate analysis and fitting of the data to an appropriate model (monitoring fluorescence intensity or anisotropy) to obtain quantitative estimates of equilibrium binding parameters. We emphasize that the solution conditions (especially salt concentration and type) can influence not only the binding affinity, but also the mode by which an SSB oligomer binds ssDNA.

Isothermal titration calorimetry (ITC) is a powerful method for studying protein-DNA interactions in solution. As long as binding is accompanied by an appreciable enthalpy change, ITC studies can yield quantitative information on stoichiometries, binding energetics (affinity, binding enthalpy and entropy) and potential site-site interactions (cooperativity). This can provide a full thermodynamic description of an interacting system which is necessary to understand the stability and specificity of protein-DNA interactions and to correlate the activities or functions of different species. Here we describe procedures to perform and analyze ITC studies using as examples, the E. coli SSB (homotetramer with 4 OB-folds) and D. radiodurans SSB (homodimer with 4 OB-folds). For oligomeric protein systems such as these, we emphasize the need to be aware of the likelihood that solution conditions will influence not only the affinity and enthalpy of binding but also the mode by which the SSB oligomer binds ssDNA.

The advent of new technologies allowing the study of single biological molecules continues to have a major impact on studies of interacting systems as well as enzyme reactions. These approaches (fluorescence, optical, and magnetic tweezers), in combination with ensemble methods, have been particularly useful for mechanistic studies of protein-nucleic acid interactions and enzymes that function on nucleic acids. We review progress in the use of single-molecule methods to observe and perturb the activities of proteins and enzymes that function on flexible single-stranded DNA. These include single-stranded DNA binding proteins, recombinases (RecA/Rad51), and helicases/translocases that operate as motor proteins and play central roles in genome maintenance. We emphasize methods that have been used to detect and study the movement of these proteins (both ATP-dependent directional and random movement) along the single-stranded DNA and the mechanistic and functional information that can result from detailed analysis of such movement.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

SSB proteins bind to and control the accessibility of single-stranded DNA (ssDNA), likely facilitated by their ability to diffuse on ssDNA. Using a hybrid single-molecule method combining fluorescence and force, we probed how proteins with large binding site sizes can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unraveling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ∼10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) originates from a radiation-resistant bacterium and participates in DNA recombination, replication, and repair. Although it functions as a homodimer, it contains four DNA binding domains (OB-folds) and thus is structurally similar to the Escherichia coli SSB (EcoSSB) homotetramer. We examined the equilibrium binding of DrSSB to ssDNA for comparison with that of EcoSSB. We find that the occluded site size of DrSSB on poly(dT) is ∼45 nucleotides under low-salt conditions (<0.02 M NaCl) but increases to 50-55 nucleotides at ≥0.2 M NaCl. This suggests that DrSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB, although the site size difference between modes is not as large as for EcoSSB, suggesting that the pathways of ssDNA wrapping differ for these two proteins. The occluded site size corresponds well to the contact site size (52 nucleotides) determined by isothermal titration calorimetry (ITC). Electrophoretic studies of complexes of DrSSB with phage M13 ssDNA indicate the formation of stable, highly cooperative complexes under low-salt conditions. Using ITC, we find that DrSSB binding to oligo(dT)s with lengths close to the determined site size (50-55 nucleotides) is stoichiometric with a ΔH(obs) of approximately -94 ± 4 kcal/mol, somewhat smaller than that for EcoSSB (approximately -130 kcal/mol) under the same conditions. The observed binding enthalpy shows a large sensitivity to NaCl concentration, similar to that observed for EcoSSB. With the exception of the less dramatic change in occluded site size, the behavior of DrSSB is similar to that of EcoSSB protein (although clear quantitative differences exist). These common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.

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.

The Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions, we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the chi subunit of DNA polymerase III holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last approximately 9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over chi due primarily to a more favorable enthalpic component. PriA and chi also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both chi and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini.

Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein-DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.

When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems.

Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)(35) mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.

The Deinococcus radiodurans SSB protein has an occluded site size of 50 +/- 2 nucleotides on ssDNA but can form a stable complex with a 26-30-nucleotide oligodeoxynucleotide using a subset of its four ssDNA binding domains. Quantitative estimates of D. radiodurans SSB protein in the D. radiodurans cell indicate approximately 2500-3000 dimers/cell, independent of the level of irradiation. At biologically relevant concentrations, when bound at single-strand-double-strand DNA junctions in vitro, D. radiodurans SSB protein has a limited capacity to displace the shorter strand of the duplex, permitting it to bind to single-strand extensions shorter than 26-30 nucleotides. The capacity to displace the shorter strand of the duplex shows a pronounced bias for extensions with a free 3' end. The Escherichia coli SSB protein has a similar but somewhat less robust capacity to displace a DNA strand annealed adjacent to a single-strand extension. These activities are likely to be relevant to the action of bacterial SSB proteins in double-strand break repair, acting at the frayed ends created by ionizing radiation.

The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the approximately 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex.

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (DeltaH(obs)) and the negative heat capacity change (DeltaC(p,obs)) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined DeltaH(obs) over a much wider temperature range (5-60 degrees C) and as a function of monovalent salt concentration and type for SSB binding to (dT)(70) under solution conditions that favor the fully wrapped (SSB)(65) complex (monovalent salt concentration >or=0.20 M). Over this wider temperature range we observe a strongly temperature-dependent DeltaC(p,obs). The DeltaH(obs) decreases as temperature increases from 5 to 35 degrees C (DeltaC(p,obs) <0) but then increases at higher temperatures up to 60 degrees C (DeltaC(p,obs) >0). Both salt concentration and anion type have large effects on DeltaH(obs) and DeltaC(p,obs). These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 degrees C), the midpoint of which shifts to higher temperature (above 35 degrees C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br(-) > Cl(-) > F(-), and these anions are then released upon binding ssDNA, affecting both DeltaH(obs) and DeltaC(p,obs). We conclude that the experimentally measured values of DeltaC(p,obs) for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.

The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments. Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore [fluorescein (F) or Cy3]. The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA. When an initial 1:1 SSB-ssDNA complex is formed with (dT)(70) in the fully wrapped (SSB)(65) mode so that all four SSB subunits are bound to (dT)(70), the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k(2,app), ranging from 1.2 x 10(3) M(-1) s(-1) (0.2 M NaCl) to approximately 5.1 x 10(3) M(-1) s(-1) (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25 degrees C). These rate constants are approximately 7 x 10(5)- and approximately 7 x 10(4)-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex. However, when an initial SSB-ssDNA complex is formed with (dT)(35) so that only two SSB subunits interact with the DNA in an (SSB)(35) complex, the formation of the ternary intermediate occurs much faster with a k(2,app) ranging from >6.3 x 10(7) M(-1) s(-1) (0.2 M NaCl) to 2.6 x 10(7) M(-1) s(-1) (0.4 M NaBr). For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction. Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex. Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.

We have examined the kinetic mechanism for binding of the homotetrameric Escherichia coliSSB protein to single-stranded oligodeoxynucleotides [(dT)(70) and (dT)(35)] under conditions that favor the formation of a fully wrapped ssDNA complex in which all four subunits interact with DNA. Under these conditions, a so-called (SSB)(65) complex is formed in which either one molecule of (dT)(70) or two molecules of (dT)(35) bind per tetramer. Stopped-flow studies monitoring quenching of the intrinsic SSB Trp fluorescence were used to examine the initial binding step. To examine the kinetics of ssDNA wrapping, we used a single-stranded oligodeoxythymidylate, (dT)(66), that was labeled on its 3'-end with a fluorescent donor (Cy3) and on its 5'-end with a fluorescent acceptor (Cy5). Formation of the fully wrapped structure was accompanied by extensive fluorescence resonance energy transfer (FRET) from Cy3 to Cy5 since the two ends of (dT)(66) are in close proximity in the fully wrapped complex. Our results indicate that initial ssDNA binding to the tetramer is very rapid, with a bimolecular rate constant, k(1,app), of nearly 10(9) M(-1) s(-1) in the limit of low salt concentration (<0.2 M NaCl, pH 8.1, 25.0 degrees C), whereas the rate of dissociation is very low at all salt concentrations that were examined (20 mM to 2 M NaCl or NaBr). However, the rate of initial binding and the rate of formation of the fully wrapped complex are identical, indicating that the rate of wrapping of the ssDNA around the SSB tetramer is very rapid, with a lower limit rate of 700 s(-1). The implications of this rapid binding and wrapping reaction are discussed.