Regulation of Nearest-Neighbor Cooperative Binding of E. coli SSB Protein to DNA

Abstract

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 (ω₃₅), 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 (ω₆₅), 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 ω₆₅ ≪ ω_65/35 < ω₃₅. 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.

Significance

Single-stranded DNA-binding (SSB) proteins are involved in all aspects of genome maintenance. E. coli SSB protein binds single-stranded DNA in multiple modes and displays a range of positive cooperativity, both of the nearest-neighbor (nn) and the non-nn type, depending on the mode. Because of these complexities, it has been difficult to examine cooperative binding quantitatively. Here, we combine fluorescence and sedimentation velocity studies using defined-length oligodeoxythymidylates to quantitatively examine nn cooperativity within each binding mode and intermode cooperativity. Cooperativity is dependent on the binding mode and [NaCl] and is regulated by the intrinsically disordered C-terminal region of SSB. Glutamate, the major monovalent anion in bacteria, enhances non-nn cooperativity but has no effect on nn cooperativity.

Introduction

Single-stranded (ss) DNA-binding proteins (SSBs) play essential roles in most aspects of DNA replication, recombination, and repair. They bind selectively and with high affinity to ssDNA intermediates formed transiently during genome maintenance, protecting them from degradation and inhibiting DNA secondary structures (1, 2, 3, 4). SSB proteins also serve as hubs for interactions with other proteins involved in genome maintenance. This is exemplified by E. coli SSB, which binds at least 15 proteins referred as SSB-interacting proteins, involved in replication, recombination, and repair (5).

E. coli SSB (EcSSB) functions as a homotetramer (Fig. 1 C; (3,6)), with each subunit (177 amino acids) composed of two domains (Fig. 1 A): an N-terminal DNA-binding domain (DBD) (residues 1–112) containing an oligonucleotide/oligosaccharide binding fold, referred to here as a core, and a C-terminal domain (residues 113–177) composed of a flexible, intrinsically disordered linker (IDL) (56 aa (Fig. 1 A)) and a nine-residue “acidic tip,” which is conserved among many bacterial SSBs and is the primary site of interaction with the SSB-interacting proteins (5,7, 8, 9, 10, 11, 12, 13). EcSSB binds ssDNA in several binding modes, differing in the number of subunits used to contact ssDNA. Two of the major binding modes observed in vitro are referred to as (SSB)₃₅ and (SSB)₆₅, where the subscripts denote the average number of nucleotides occluded upon binding ssDNA (14,15). The transitions between binding modes are reversible, and their relative stabilities depend on solution conditions, primarily salt concentration and type and protein/DNA ratio (binding density) (14,16, 17, 18, 19, 20, 21, 22), as well as applied force (23,24). A salt-dependent intratetramer negative cooperativity contributes to regulating the binding mode transition (25, 26, 27).

Figure 1.

EcSSB constructs and SSB binding modes. (A) An SSB subunit (177 aa) is composed of an N-terminal DBD (oligonucleotide/oligosaccharide binding fold) (residues 1–112) and a C-terminal tail (residues 113–177) that contains a 56 aa intrinsically disordered linker (IDL) and a conserved 9 aa acidic tip. (B) An SSB variant lacking the IDL (SSB Δ115–168 deletion), SSB-ΔL. (C) A cartoon of the SSB-ssDNA complex in the (SSB)₆₅ binding mode, with 65 nts of DNA (orange ribbon) wrapped around each SSB tetramer (6); the IDLs (gray) with the acidic tips (red letters) are depicted emanating from the dimer-dimer interface as an extension of the C-termini visible in the crystal structure. (D) A cartoon of a proposed model for SSB-ssDNA binding in the (SSB)₃₅ binding mode (6) in which two SSB tetramers interact with ∼70-nt-long DNA (orange tube) using an average of only two subunits per tetramer. To see this figure in color, go online.

In the (SSB)₆₅ mode, favored at [NaCl] > 0.20 M or [Mg²⁺] > 10 mM, the ssDNA wraps around all four subunits of the tetramer (6) (see Fig. 1 C), with an occluded site size of ∼65 nucleotides. The topology of ssDNA wrapping in the (SSB)₆₅ binding mode is such that ssDNA enters and exits the tetramer in close proximity. On long ssDNA, the (SSB)₆₅ mode displays “limited” cooperativity between adjacent tetramers (16,28), as depicted in Fig. 1 C. In this mode, SSB can diffuse along ssDNA, transiently destabilizing DNA hairpins and promoting RecA filament formation (23,29). ssDNA translocases are able to actively push SSB tetramers bound in the (SSB)₆₅ mode along ssDNA, providing a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA (30).

In the (SSB)₃₅ mode, favored at [NaCl] < 10 mM (Fig. 1 D) or [MgCl₂] < 1 mM, and high SSB/DNA ratios (14,15,18), ssDNA wraps around only two subunits on average, with an occluded site size of ∼35 nucleotides. In this mode, SSB binds ssDNA with unlimited nearest-neighbor cooperativity, favoring formation of long protein clusters (17,18,21,31, 32, 33) as depicted in Fig. 1 D. A structural model for the (SSB)₃₅ binding mode has been proposed, suggesting direct interactions of adjacent tetramers through the L₄₅ loops within the tetrameric core of the protein (Fig. 1 D; (6)). In this mode, SSB can diffuse along ssDNA (23,24) and undergo direct or intersegment transfer between separate ssDNA molecules (34) or between distant sites on the same DNA molecule (35). The ability to undergo direct transfer appears to play a role in SSB recycling during replication (34,36).

Previous studies suggested that highly cooperative SSB binding to ssDNA occurred only in the (SSB)₃₅ mode at low [NaCl] (18,32). However, we showed recently that highly cooperative binding can also occur in the (SSB)₆₅ mode at higher salt concentrations (22,33). This high cooperativity is promoted by the IDL region of the C-terminus, as well as by glutamate or acetate anions (22). This high cooperativity differs from the nearest-neighbor high cooperativity in the (SSB)₃₅ mode, appears to be associated with non-nearest-neighbor interactions among SSB tetramers bound to distant regions of long ssDNA, and results in an intramolecular “condensation” or collapse of the nucleoprotein complex (22,37). This non-nearest-neighbor cooperativity requires the IDL regions of SSB (22,33). However, the relationship between nearest-neighbor and non-nearest-neighbor cooperativities remains unclear.

Quantitative estimates of nearest-neighbor SSB cooperativity on ssDNA is difficult for several reasons and thus has only been measured under a few conditions. First, it is difficult to isolate only one particular binding mode for study. Second, even when one can isolate a particular binding mode, the solution conditions needed to study each binding mode are generally different. As such, estimates of the limited cooperativity parameter in the (SSB)₆₅ mode (ω₆₅ = 420 ± 80) have only been obtained for SSB binding to the ssRNA poly(U) at [NaCl] > 0.20 M (28,38). Estimates of the unlimited cooperativity parameter in the (SSB)₃₅ mode (ω₃₅ ∼ 10⁵) have only been made on (dA)₇₀ at 0.125 M NaCl (32). No studies have examined whether any cooperativity exists between modes.

In this study, we combine equilibrium fluorescence titrations and sedimentation velocity methods using the homo-oligodeoxynucleotides, (dT)₇₀ and (dT)₁₄₀, to obtain estimates of both ω₃₅ and ω₆₅, as well as the intermode cooperativity, ω_65-35, for both wild-type (wt)SSB and a variant, SSB-ΔL, missing the IDL linker. Fluorescence titrations provide information on the SSB binding mode, whereas sedimentation velocity can directly determine how the distribution of the various SSB-DNA species in the population changes throughout the titration. By combining these methods with the approaches of Epstein (39) to model large ligand binding to finite lattices, we have obtained quantitative estimates of all nearest-neighbor cooperativities at both low and high [NaCl].

Materials and Methods

Reagents and buffers

Buffers were prepared with reagent grade chemicals and distilled water treated with a Milli Q (Millipore, Bedford, MA) water purification system. Buffer T is 10 mM Tris (pH 8.1), 0.1 mM Na₃EDTA.

DNA, wtSSB, and SSB-ΔL tail variant

The oligodeoxythymidylates (dT)₇₀ and (dT)₁₄₀ and (dT)₆₈ doubly labeled with Cy3 and Cy5 fluorophores (5′-Cy5-(dT)₆₈-Cy3-dT-3′) were synthesized and purified to >99% homogeneity as described (32,40). DNA concentrations were determined spectrophotometrically in buffer T + 0.10 M NaCl, using ε₂₆₀ = 8.1 × 10³ M⁻¹ (nucleotide) cm⁻¹ for (dT)₇₀ and (dT)₁₄₀ (41) (70 or 140 × ε₂₆₀, molecule) and ε₂₆₀ = 5.74 × 10⁵ M⁻¹ (molecule) for 5′-Cy5-(dT)₆₈-Cy3-T-3′ (molecule) (40).

EcSSB protein and SSB-ΔL (previously referred to as SSB-GG (33)) in which the IDL (residues 115–168) was deleted were expressed and purified as described (33). Both proteins form stable tetramers under all solution conditions used in this study, as determined by sedimentation velocity (33). Protein concentrations were determined spectrophotometrically (14) (buffer T, 0.20 M NaCl), using ε₂₈₀ = 1.13 × 10⁵ M⁻¹ cm⁻¹ (tetramer) for wtSSB and ε₂₈₀ = 8.98 × 10⁴ M⁻¹ cm⁻¹ (tetramer) for SSB-ΔL.

Fluorescence measurements

Fluorescence titrations were performed in buffer T, 25°C, at the NaCl concentrations indicated in the text and figure legends using a QM-4 spectrofluorometer (Photon Technology International/Horiba Scientific, Edison, NJ). Reverse titrations of SSB and SSB-ΔL (0.3 μM) with (dT)₁₄₀ in Figs. 3, E and F, 4, E and F, and 5 were performed by monitoring quenching of the intrinsic SSB tryptophan fluorescence and analyzed as described (33,42). Normal titrations of Cy5-(dT)₆₈ Cy3dT (0.1–0.3 μM) with wtSSB or SSB-ΔL were performed by exciting Cy3 donor (515 nm) while monitoring sensitized emission from Cy5 acceptor at 665 nm and analyzed as described (20,42). The SSB Trp fluorescence measurements were judged to have reached equilibrium by waiting until the fluorescent signal no longer changed after each addition of the titrant (42). For all titrations with (dT)₇₀, this generally required 2–5 min. However, for titrations of wtSSB with (dT)₁₄₀ at 10 mM NaCl at r < 4 ([(dT)₁₄₀]_tot/[wtSSB]_tot > 0.25; see Fig. 5 A), longer times (15–20 min) were required, indicating that this system equilibrates much more slowly. A final measurement made 3–4 h after finishing a titration showed no further changes. As such, the total time required for a titration of SSB with (dT)₁₄₀ was ∼10 h.

Figure 3.

Fluorescence and sedimentation velocity titrations for wtSSB binding to (dT)₇₀. (A and B) Results of equilibrium titrations of Cy5-(dT)₆₈-Cy3dT (0.1 μM) with wtSSB monitoring Cy5 fluorescence enhancement (excitation: 515 nm, emission: 665 nm) in 10 mM NaCl and 0.30 M NaCl (buffer T, 25°C), respectively, are plotted as normalized Cy5 fluorescence (F_n = (F_obs – F₀)/F₀) versus the ratio of concentrations of total SSB tetramer/DNA (where F₀ is the Cy5 fluorescence of DNA alone and F_obs is the Cy5 fluorescence measured at each point of the titration). Black lines are simulated binding isotherms for the data using Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), and 5 and the parameters (A) K₆₅ = 3 × 10¹⁴ M⁻¹, K₃₅ = 10¹⁰ M⁻¹, ω₃₅ = 10⁵, F_65,1 = 9.0, F_35,1 = 0.09, and F_35,2 = 4.1 and (B) K₆₅ = 3 × 10¹⁴ M⁻¹ (minimal estimate), K₃₅ = 10¹⁰ M⁻¹, ω₃₅ = 1, F_65,1 = 4.5, F_35,1 = 0.05, and F_35,2 = 2.05 (see Table S2). The cartoons depict the (SSB)₃₅ and (SSB)₆₅ complexes and where they form during the course of the titrations. (C and D) c(s) profiles from sedimentation velocity experiments (buffer T, 25°C) for wtSSB-(dT)₇₀ complexes formed at different ratios, r = [SSB]_tot/[dT₇₀]_tot ([dT₇₀]_tot = 0.7 μM), are shown: (C) 10 mM NaCl, r = 0 (green), 1.0 (blue), 2.0 (red), 4.0 (dashed black); (D) 0.30 M NaCl: r = 0 (green), 0.5 (cyan), 1.0 (blue), 2 (red). (E and F) Results of equilibrium titrations of wtSSB (0.30 μM) monitoring intrinsic Trp fluorescence quenching (excitation: 296 nm, emission: 350 nm) in 10 mM NaCl and 0.30 M NaCl (buffer T, 25°C), respectively, are plotted as normalized Trp fluorescence quenching (Q_n = (Q₀ − Q_obs)/Q₀) versus the ratio of total DNA/SSB tetramer concentrations (where Q₀ is the fluorescence intensity of wtSSB alone and Q_obs is the Trp fluorescence measured at each point in the titration). Black lines are simulated binding isotherms for the data using Eqs. 1, 2, 3, 6, and (7a), (7b), (7c), (7d) and the binding parameters as in (A) and (B): (E) K₆₅ = 3 × 10¹⁴ M⁻¹, K₃₅ = 10¹⁰ M⁻¹, ω₃₅ = 10⁵ (Q₆₅ = 0.9, Q_35,1 = 0.5) and (F) K₆₅ = 3 × 10¹⁴ M⁻¹, K₃₅ = 10¹⁰ M⁻¹, ω₃₅ = 1 (Q₆₅ = 0.9, Q_35,1 = 0.5). The cartoons schematically depict (SSB)₃₅ and (SSB)₆₅ protein-(dT)₇₀ complexes and expected transitions in the course of titrations. To see this figure in color, go online.

Figure 4.

Fluorescence and sedimentation velocity titrations for SSB-ΔL binding to (dT)₇₀. (A and B) Results of equilibrium titrations of Cy5-dT₆₈-Cy3dT (0.1 μM) with SSB-ΔL monitoring Cy5 fluorescence enhancement (excitation: 515 nm, emission: 665 nm) in 10 mM NaCl and 0.3 M NaCl (buffer T, 25°C), respectively, are plotted as normalized Cy5 fluorescence (F_n = (F_obs − F₀)/F₀) versus the ratio of total concentrations of SSB tetramer/DNA (where F₀ is the Cy5 fluorescence of DNA alone and F_obs is the Cy5 fluorescence measured at each point of the titration). Black lines show simulated binding isotherms using Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), and 5 and the parameters (A) K₃₅ = 5 × 10⁹ M⁻¹, ω₃₅ = 40, F_35,1 = 0.09, F_35,2 = 4.1 (these are best-fit parameters for titration shown in Fig. S4) and (B) K₆₅ = 3 × 10¹² M⁻¹ (minimum estimate), K₃₅ = 5 × 10⁹ M⁻¹, ω₃₅ = 1, F_65,1 = 4.5, F_35,1 = 0.05, F_35,2 = 2.05 (see Table S2 and text for more details). The cartoons depict the different SSB-(dT)₇₀ complexes (with corresponding position of the Cy3/Cy5 dyes) formed at each region of the titrations. (C and D) c(s) profiles from sedimentation velocity experiments for SSB-ΔL-(dT)₇₀ complexes formed at different ratios, r = [SSB-ΔL]_tot/[dT₇₀]_tot ([dT₇₀]_tot = 0.7 μM): (C) 10 mM NaCl, r = 0, 0.5, 1.0, 1.5, 2.0, 3.0 (c(s) profiles for r = 0, 1, 2, and 3 are shown in thick lines of light green, blue, red, and dashed black, respectively; distributions for r = 0.5 and 1.5 are shown in thin cyan and magenta lines); (D) 0.30 M NaCl, r = 0, 0.5, 1.0, 2.0 (color scheme as in C). (E and F) Results of equilibrium titrations of SSB-ΔL (0.3 μM) with (dT)₇₀ monitoring intrinsic Trp fluorescence quenching (excitation: 296 nm, emission: 350 nm) in 10 mM NaCl and 0.3 M NaCl (buffer T, 25°C), respectively, are plotted as normalized Trp fluorescence (Q_n = (Q₀ − Q_obs)/Q₀) versus the ratio of total DNA/protein tetramer concentrations (where Q₀ is the Trp fluorescence of SSB-ΔL alone and Q_obs is the Trp fluorescence measured at each point of the titration). Black lines are simulated binding isotherms for the data using Eqs. 1, 2, 3, 6, and (7a), (7b), (7c), (7d) and the binding parameters as in (A) and (B): (E) K₃₅ = 5 × 10⁹ M⁻¹, ω₃₅ = 40 (Q₃₅ = 0.6) and (F) K₆₅ = 3 × 10¹² M⁻¹, K₃₅ = 5 × 10⁹ M⁻¹, ω₃₅ = 1 (Q₆₅ = 0.9, Q₃₅ = 0.6). The cartoons show the (SSB)₃₅ and (SSB)₆₅ complexes that are formed in the different salt conditions. To see this figure in color, go online.

Figure 5.

wtSSB and SSB-ΔL can form both (SSB)₃₅ and (SSB)₆₅ binding modes on (dT)₁₄₀ depending on salt concentration and binding density. Reverse equilibrium titrations of wtSSB (0.30 μM tetramer) (blue circles, readings taken at 15- to 20-min intervals; open circles, readings taken at 3-min intervals) and SSB-ΔL (0.30 μM tetramer) (orange circles, readings taken at 15-min intervals) with (dT)₁₄₀ (monitoring SSB Trp fluorescence quenching), buffer T, 25°C. (A) 10 mM NaCl and (B) 0.20 M NaCl are plotted as normalized Trp fluorescence quenching (Q_n = (F₀ − F_obs)/F₀) versus the ratio of total (dT)₁₄₀/SSB tetramer concentration, where F₀ is the free SSB Trp fluorescence intensity and F_obs is the fluorescence measured at each point in the titration (lines are drawn to represent the data but are not fits to the data). To see this figure in color, go online.

Analytical sedimentation

Sedimentation velocity experiments were performed as described (22) with an Optima XL-A analytical ultracentrifuge and An50Ti rotor (Beckman Instruments, Fullerton, CA) at 42,000 rpm in buffer T, 25°C, with the salt concentrations and type indicated in the text and figure legends. Constant concentrations of (dT)₇₀ (in the range from 0.5 to 0.7 μM) or (dT)₁₄₀ (in the range from 0.2 to 0.5 μM) were used while adding SSB to make solutions with different protein/DNA ratios (r = [SSB]_tot/[(dT)n]_tot. The absorbance was monitored at 260 nm, reflecting predominantly the ssDNA. The contribution of SSB to the absorbance at 260 nm is small compared to the DNA at most protein/DNA ratios used in this study (maximal ∼15% for (dT)₁₄₀ having four wtSSB tetramers bound). Data were analyzed using SEDFIT (www.analyticalultracentrifugation.com) to obtain c(s) distributions and estimate molecular weights for the species described by single peaks (43). The c(s) refers to the continuous sedimentation distribution that results from a SEDFIT analysis of the absorbance traces obtained in a sedimentation velocity AUC (analytical ultracentrifugation) experiment using the Lamm equation (43). c(s) represents the concentration (in absorbance units in this case) of species with a given sedimentation coefficient range.

The densities and viscosities at 25°C were calculated using SEDNTERP for NaCl solutions and from van Holst et al. (44) for KGlu solutions. Partial specific volumes for protein-DNA complexes were calculated using the formula υ = (υ_DNAM_DNA + nυ_nMi)/(M_DNA + nMi), where υ_DNA and M_DNA are the partial specific volume and molecular weight of DNA, υ_i and M_i are the partial specific volume and molecular weight of the protein, and n is the number of SSB molecules bound to DNA. In all experiments, the absorbance estimated from integrated c(s) profiles was the same as the absorbance of loaded solutions, indicative of no precipitation/aggregation occurring during the experimental runs. Species distributions in Figs. 6, C and D and 7 D were calculated from the corresponding c(s) profiles by estimating the area under the peaks that relates to the populations of the different SSB-DNA complexes by fitting to multiple Gaussian functions and then dividing by the total integrated area with corresponding corrections for contributions from SSB absorbance. In Fig. S10, c(s) profiles obtained for experiments performed in 0.2 M NaCl and 0.2 M KGlu were converted to c(s_20,w) profiles using SEDFIT and corresponding viscosities and densities.

Figure 6.

Sedimentation velocity c(s) profiles for wtSSB binding to (dT)₁₄₀ at low (10 mM) and high (0.20 M) NaCl concentrations. c(s) profiles for wtSSB-(dT)₁₄₀ complexes formed at different protein/DNA ratios, r = [SSB]_tot/[(dT)₁₄₀]_tot: (A) 10 mM NaCl, [(dT)₁₄₀]_tot = 0.5 μM; r = 0, 0.6, 1.0, 1.6, 1.5, 2.0, 2.5, 3.0, 3.2, 3.5, 3.8, and 4.0. Profiles obtained for r = 1.0 after 1 and 10 h of equilibration time are shown in thin and thick blue lines, respectively; (B) 0.20 M NaCl, [(dT)₁₄₀]_tot = 0.2 μM; r = 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2.0, 3.0, 4.0, and 5.0. Both panels show c(s) profiles for r = 0 (light green), 1 (blue), 2 (red), 3 (gray), 4 (dark green), and 5 (dashed black); representative profiles for noninteger r-values are shown as thin dashed lines. (C and D) Distributions of the (dT)₁₄₀ species: free DNA (yellow circles), one SSB tetramer bound (blue circles), two SSB tetramers bound (red circles), three SSB tetramers bound (gray circles), and four SSB tetramers bound (green circles), estimated from the c(s) profiles in (A) and Fig. S5 (10 mM NaCl) and (B), r ≤ 2 (0.20 M NaCl). The solid lines in (C) represent the best fit of the species distributions to the Epstein-140 model with K₆₅ = 10¹⁶ M⁻¹ (fixed), K₃₅ = 10¹⁰ M⁻¹ (fixed), and ω₆₅ = 38 ± 2, ω_m = (5.0 ± 1.4) × 10⁴, and ω₃₅ = (4.4 ± 1.4) × 10⁵ (see Fig. S6 for alternative fits). Solid lines in (D) represent the best fit or simulation for Epstein-140 model for r ≤ 2 ([(dT)₁₄₀]_tot = 0.2 μM), assuming that binding in this range occurs exclusively in the (SSB)₆₅ binding mode. The species distributions are best described with K₆₅ > 1 × 10¹¹ M⁻¹ and ω₆₅ = 1. The dashed blue lines were simulated for ω₆₅ = 10² and ω₆₅ = 10³ and show the predicted suppression of the species with one tetramer bound as cooperativity increases. The cartoons show the expected SSB-DNA complexes (rectangle, (SSB)₆₅ mode and circles, (SSB)₃₅ mode). To see this figure in color, go online.

Figure 7.

Sedimentation velocity c(s) profiles for SSB-ΔL tetramers binding to (dT)₁₄₀ at low (10 mM) and high (0.20 M) NaCl concentrations. (A) c(s) profiles for SSB-ΔL-(dT)₁₄₀ complexes formed in 10 mM NaCl at different SSB-ΔL/DNA ratios, r = [SSB]_tot/[dT₁₄₀]_tot, [dT₁₄₀]_tot = 0.5 μM: r = 0, 1.0, 2.0, 3.0, 4.0, and 5.0 (profiles for r = 0 (light green), 1 (blue), 2 (red), 3 (gray), 4 (dark green), and 5 (dashed black)). (B) c(s) profiles for SSB-ΔL-dT₁₄₀ complexes formed in 0.20 M NaCl at different SSB-ΔL/DNA ratios, r = [SSB]_tot/[dT₁₄₀]_tot, [dT₁₄₀]_tot = 0.20 μM: r = 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2.0, 3.0, and 4.0 (c(s) profiles for r = 0, 1, 2, 3, and 4 are shown as thick solid lines of light green, blue, red, gray, and dark green, respectively, whereas the profiles for the noninteger values of r are shown as thin dashed lines). (C) Simulations of the species distributions of (dT)₁₄₀ with 0, 1, 2, 3, and 4 SSB-ΔL tetramers bound at 10 mM [NaCl] predicted by the Epstein-140 model (Eqs. 8, 9, 10, and (11a), (11b), (11c), (11d), (11e)), assuming that SSB-ΔL binds to (dT)₁₄₀ exclusively in the (SSB)₃₅ binding mode with K₃₅ = 5 × 10⁹ M⁻¹ and ω₃₅ = 40 ([dT₁₄₀]_tot = 0.5 μM). (D) Species distributions of (dT)₁₄₀ with zero (yellow circles), 1 (blue circles), and 2 (red circles) SSB-ΔL tetramers bound are estimated for the range 0 ≤ r ≤ 2 from the c(s) profiles in (B), as described in the text. The solid lines (yellow, blue, and red) describe the best fit of the data to the Epstein-140 model (Eqs. 8, 9, 10, and (11a), (11b), (11c), (11d), (11e)) for SSB-ΔL binding in the (SSB)₆₅ mode in the range r ≤ 2 with K₆₅ > 1 × 10¹¹ M⁻¹ and ω₆₅ = 212 ± 28. Species distributions in gray are predicted for mixed 3:1 complexes (solid line, total; dashed lines, fractions of SSB bound cooperatively; ω_m = 25) using Eqs. 8, 9, 10, and (11a), (11b), (11c), (11d), (11e) and the following parameters: K₆₅ = 3.3 × 10¹² M⁻¹, K₃₅ = 5 × 10⁹ M⁻¹, ω₆₅ = 212, ω_m = 25, and ω₃₅ = 1 (minimal estimates; see text and Fig. S9 for alternative simulations). These parameters also provide a good description of the data for r < 2 (yellow, blue, and red lines). To see this figure in color, go online.

To determine the time required for the SSB-DNA systems to reach equilibrium, we performed a series of experiments in which the solutions were allowed to equilibrate for ∼2, 6, or 10 h before starting the sedimentation velocity run. We found that for most experiments, 2 h was sufficient for the species to equilibrate, judged by the fact that the c(s) distributions did not change after this time. The one exception was for the wtSSB-(dT)₁₄₀ system in 10 mM NaCl at SSB/(dT)₁₄₀ ratios r ≤ 2 (see Fig. 6 A), which required ∼10 h to reach equilibrium. Experiments performed at longer times (up to 4 weeks) did not show further changes. Therefore, c(s) distributions in Fig. 6 A and Fig. S5, which we used for analysis, are for experiments performed after the solutions were allowed to equilibrate for >10 h.

Modeling of SSB binding to (dT)₇₀ and (dT)₁₄₀ in the (SSB)₆₅ and (SSB)₃₅ binding modes with nearest-neighbor cooperativities

Epstein-70: Model for SSB binding to a finite homogeneous DNA lattice of 70 nts, (dT)₇₀, in two modes with site sizes of 35 and 65 nts

The three possible SSB-(dT)₇₀ species configurations and their associated statistical weights are shown schematically in Fig. 2 A, in which the rectangles represent (SSB)₆₅ complexes and circles represent the (SSB)₃₅ complexes. The partition function for this model, Z₇₀, is given by Eq. 1,

$$Z_{\mathit{70}}=\mathit{1}+S_{\mathit{65}}{K}_{\mathit{65}}X+S_{\mathit{35},\mathit{1}}{K}_{\mathit{35}}X+S_{\mathit{35},\mathit{2}}{\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}},$$ (1)

where X is the free SSB tetramer concentration; K₆₅ and K₃₅ are the intrinsic association equilibrium constants for SSB binding in the (SSB)₆₅ and (SSB)₃₅ modes, respectively; ω₃₅ is the nearest-neighbor cooperativity; and S₆₅ = 6 and S_35,1 = 36 are the statistical weights associated with a single SSB tetramer bound to (dT)₇₀ in the 65 and 35 modes, respectively (S_35,2 = 1 for two SSB tetramers each bound in the 35 mode). The cooperativity parameter, ω₃₅, represents the process of bringing two isolated SSB tetramers bound to DNA in the (SSB)₃₅ mode into nearest-neighbor contiguous contact, as depicted in Fig. 2 Aiii. The statistical weights were calculated according to Epstein (39). The resulting expression for the average number of SSB tetramers bound per (dT)₇₀ (binding density) is given in Eq. 2.

$$\langle X\rangle =\mathit{dln}{Z}_{\mathit{70}}/\mathit{dlnX}=\left[\mathit{6}{K}_{\mathit{65}}X+\mathit{36}{K}_{\mathit{35}}X+\mathit{2}{\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}\right]/Z_{\mathit{70}}$$ (2)

Figure 2.

Statistical models for SSB binding to (dT)₇₀ (A) and (dT)₁₄₀ (B). The two SSB binding modes are indicated as circles ((SSB)₃₅) and rectangles ((SSB)₆₅). (A) The three possible SSB-(dT)₇₀ species (i) and (ii) that can form are shown along with the statistical weights for each species. The nearest-neighbor cooperativity parameter, ω₃₅ (iii), is the equilibrium constant for moving two SSB tetramers bound in (SSB)₃₅ mode from isolated to single contiguous positions. (B) The 17 possible (SSB)-(dT)₁₄₀ species (i) and (iii)–(v) are shown along with the statistical weights for each species. The nearest-neighbor cooperativities, ω₆₅ (ii) and ω_m (vi), represent the equilibrium constants for the process of moving two SSB tetramers from isolated positions in the (SSB)₆₅ and (SSB)₆₅-(SSB)₃₅ modes, respectively, to single contiguous positions. The numerical statistical factors representing the number of ways of obtaining each species were calculated using Epstein’s model (39).

The mass conservation Eq. 3 is required to calculate the free concentration of SSB tetramers (X), where D_tot is the total concentration of (dT)₇₀ and X_tot is the total SSB tetramer concentration.

$$X_{\mathit{tot}}=X+D_{\mathit{tot}}\langle X\rangle$$ (3)

When Z (Eq. 1) and <X> (Eq. 2) are introduced into Eq. 3, this yields a third-degree polynomial in X (free ligand concentration). This equation is then solved numerically for the one real root (X) for any set of parameters and DNA_tot and X_tot. This value of X is then used to calculate the distribution of DNA or SSB species (free or bound, using (4a), (4b), (4c), (4d) and (7a), (7b), (7c), (7d) below) and related signals (Eqs. 5 and 6 below). By varying DNA_tot or SSB_tot (titrations), we can then fit or simulate the data.

Equations for the fractions of free (dT)₇₀, f_D0, and (dT)₇₀ with one SSB tetramer bound in each mode, f_D35,1 and f_D65,1, and two tetramers bound cooperatively in the (SSB)₃₅ binding mode, f_D35,2, are given in (4a), (4b), (4c), (4d).

$$f_{D\mathit{0}}=\mathit{1}/Z_{\mathit{70}}$$ (4a)

$$f_{D\mathit{65},\mathit{1}}=\mathit{6}{K}_{\mathit{65}}X/Z_{\mathit{70}}$$ (4b)

$$f_{D\mathit{35},\mathit{1}}=\mathit{36}{K}_{\mathit{35}}X/Z_{\mathit{70}}$$ (4c)

$$f_{D\mathit{35},\mathit{2}}={\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}/Z_{\mathit{70}}$$ (4d)

The expression for the binding isotherm for titrations of (dT)₇₀ labeled with Cy3 and Cy5, used when we monitor the fluorescent signal from DNA as a function of SSB_tot, is given in Eq. 5,

$$F_{\mathit{obs}}=F_{\mathit{65},\mathit{1}}{f}_{D\mathit{65},\mathit{1}}+F_{\mathit{35},\mathit{1}}{f}_{D\mathit{35},\mathit{1}}+F_{\mathit{35},\mathit{2}}{f}_{D\mathit{35},\mathit{2}},$$ (5)

where F_obs is the experimentally observed fluorescence signal normalized by the fluorescence of free DNA and F_65,1, F_35,1 and F_35,2 are the normalized fluorescent signals associated with each SSB-DNA complex. The free [SSB], X in (4a), (4b), (4c), (4d) and 5, is obtained by solving the mass conservation Eq. 3.

For the reverse titrations in which we monitor the quenching of the intrinsic Trp fluorescence of SSB upon addition of (dT)₇₀, the binding isotherm is given in Eq. 6 (45),

$${\mathrm{Q}}_{obs}={\mathrm{Q}}_{\mathit{65}}f{\mathrm{x}}_{\mathit{65},\mathit{1}}+{\mathrm{Q}}_{\mathit{35}}\left(f{\mathrm{x}}_{\mathrm{35},\mathrm{1}}+f{\mathrm{x}}_{\mathrm{35},\mathrm{2}}\right),$$ (6)

where Q_obs is the observed Trp fluorescence quenching and Q₆₅ and Q₃₅ are the fluorescence quenching associated with SSB bound in the corresponding binding modes and fx₀, fx_65,1, fx_35,1, and fx_35,2 are the fractions of free SSB tetramers, tetramers bound in the 65 mode, and one or two tetramers bound in the 35 mode, respectively, as defined in (7a), (7b), (7c), (7d):

$$f{\mathrm{x}}_{\mathit{0}}=X/X_{tot},$$ (7a)

$$f{\mathrm{x}}_{\mathit{65},\mathit{1}}=\left(D_{tot}/X_{tot}\right)\mathit{6}{K}_{\mathit{65}}X/Z_{\mathit{70}},$$ (7b)

$$f{\mathrm{x}}_{\mathit{35},\mathit{1}}=\left(D_{tot}/X_{tot}\right)\mathit{36}{K}_{\mathit{35}}X/Z_{\mathit{70}},$$ (7c)

$$f{\mathrm{x}}_{\mathit{35},\mathit{2}}=\mathit{2}\left(D_{tot}/X_{tot}\right){\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}/Z_{\mathit{70}},$$ (7d)

where X is obtained by solving Eq. 3. We assume that Q₃₅ is the same for SSB bound to (dT)₇₀ with and without cooperativity.

Epstein-140: Model for binding of SSB tetramers to a finite homogeneous DNA lattice of 140 nucleotides, (dT)_14, in two modes with site sizes of 35 and 65 nts

The 17 SSB-(dT)₁₄₀ species distributions possible for SSB binding to (dT)₁₄₀ and their associated statistical weights were calculated as in Epstein (39) and are shown in Fig. 2 B, yielding the partition function for this model, Z₁₄₀, given in Eq. 8:

$$\begin{array}{l}{Z}_{\mathit{140}}=\mathit{1}+\mathit{76}{K}_{\mathit{65}}X+\mathit{55}{\left(K_{\mathit{65}}X\right)}^{\mathit{2}}+\mathit{11}{\omega }_{\mathit{65}}{\left(K_{\mathit{65}}X\right)}^{\mathit{2}}+\mathit{106}{K}_{\mathit{35}}X+\mathit{2485}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}+\mathit{71}{\omega }_{\mathit{35}}\\ {\left(K_{\mathit{35}}X\right)}^{\mathit{2}}+\mathit{7140}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+\mathit{1260}{\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+\mathit{36}{\omega }_{\mathit{35}}^{\mathit{2}}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+{\omega }_{\mathit{35}}^{\mathit{3}}{\left(K_{\mathit{35}}X\right)}^{\mathit{4}}+\mathit{1640}{K}_{\mathit{65}}\\ K_{\mathit{35}}{\left(X\right)}^{\mathit{2}}+\mathit{82}{\omega }_m{K}_{\mathit{65}}{K}_{\mathit{35}}{\left(X\right)}^{\mathit{2}}+\mathit{60}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}+\mathit{30}{\omega }_{\mathit{35}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}+\mathit{60}{\omega }_m{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}\\ +\mathit{6}{\omega }_m^{\mathit{2}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}+\mathit{12}{\omega }_m{\omega }_{\mathit{35}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}},\end{array}$$ (8)

where X, K₆₅, K₃₅, and ω₃₅ are as defined above and ω₆₅ and ω_m (ω_65-35) are the corresponding nearest-neighbor cooperativity parameters for two tetramers bound contiguously in the (SSB)₆₅ mode and between two tetramers bound contiguously in the (SSB)₆₅ and (SSB)₃₅ modes, as defined in Fig. 2, Bii and vi, respectively. The expression for the binding density (average number of SSB tetramers bound per (dT)₁₄₀) is obtained from Eq. 9, and the corresponding mass conservation Eq. 10, where D_tot = [(dT)₁₄₀]_tot.

$$\langle X\rangle =\mathit{dln}{Z}_{\mathit{140}}/\mathit{dlnX}$$ (9)

$$X_{\mathit{tot}}=X+D_{\mathit{tot}}\langle X\rangle$$ (10)

As described above for (dT)₇₀, introduction of Eqs. 8 and 9 into Eq. 10 yields a fifth-degree polynomial in X that can be solved numerically for the only real root, providing the value of the free ligand concentration (X) for any particular set of parameters (K₆₅, K₃₅, ω₆₅, ω_m, and ω₃₅) and total species concentrations (D_tot and SSB_tot), which is then used in further calculations (see below).

Expressions for the fractions of (dT)₁₄₀, f_D, having 0 and 1, 2, 3, and 4 SSB tetramers bound are given in (11a), (11b), (11c), (11d), (11e).

$$f_{D\mathit{0}}=\mathit{1}/Z_{\mathit{140}}$$ (11a)

$$f_{D\mathit{1}}=\left(\mathit{76}{K}_{\mathit{65}}X+\mathit{106}{K}_{\mathit{35}}X\right)/Z_{\mathit{140}}$$ (11b)

$$f_{D\mathit{2}}=\left(\mathit{55}{\left(K_{\mathit{65}}X\right)}^{\mathit{2}}+\mathit{11}{\omega }_{\mathit{65}}{\left(K_{\mathit{65}}X\right)}^{\mathit{2}}+\mathit{2485}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}+\mathit{71}{\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{2}}++\mathit{1640}{K}_{\mathit{65}}{K}_{\mathit{35}}{\left(X\right)}^{\mathit{2}}+\mathit{82}{\omega }_m{K}_{\mathit{65}}{K}_{\mathit{35}}{\left(X\right)}^{\mathit{2}}\right)/Z_{\mathit{140}}$$ (11c)

$$f_{D\mathit{3}}=\left(\mathit{7140}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+\mathit{1260}{\omega }_{\mathit{35}}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+\mathit{36}{\omega }_{\mathit{35}}^{\mathit{2}}{\left(K_{\mathit{35}}X\right)}^{\mathit{3}}+\mathit{60}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}++\mathit{30}{\omega }_{\mathit{35}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}+\mathit{60}{\omega }_m{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}+\mathit{6}{\omega }_m^{\mathit{2}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}++\mathit{12}{\omega }_m{\omega }_{\mathit{35}}{K}_{\mathit{65}}{K}_{\mathit{35}}^{\mathit{2}}{\left(X\right)}^{\mathit{3}}\right)/Z_{\mathit{140}}$$ (11d)

$$f_{D\mathit{4}}={\omega }_{\mathit{35}}^{\mathit{3}}{\left(K_{\mathit{35}}X\right)}^{\mathit{4}}/Z_{\mathit{140}}$$ (11e)

(11e), (11a), (11b), (11c), (11d)a–e were used to fit or simulate the species distributions obtained from the sedimentation velocity data. To simulate the data for reverse titrations, we used Eq. 12 (45),

$$\mathrm{Q}={\mathrm{Q}}_{\mathit{65}}\sum fx{,}_{\mathit{65}}+{\mathrm{Q}}_{\mathit{35}}\sum fx{,}_{\mathit{35}},$$ (12)

where Q₆₅ and Q₃₅ are the values of SSB Trp fluorescence quenching in the (SSB)₆₅ and (SSB)₃₅ modes, respectively, and ∑fx,₆₅ and ∑fx,₃₅ represent the sum of the fractions of SSB bound to (dT)₁₄₀ in the 65 or 35 mode, respectively. Those are determined in the same way as in (7a), (7b), (7c), (7d)a–d, considering all possible configurations in Fig. 2 B and using the formula fx_i = n_if_Di(D_tot/X_tot), where n_i is the number of SSB tetramers bound to (dT)₁₄₀ in configuration i and f_Di is given by each term in Eq. 8 divided by Z₁₄₀.

Fitting and simulations

Fitting/simulations of the experimental data were performed using the Epstein-70 and Epstein-140 models described above and using Scientist (MicroMath Scientist Software, St. Louis, MO) and Mathematica (Wolfram Research, Champaign, IL).

Results

Use of defined-length oligodeoxythymidylates to study nearest-neighbor cooperativities

To study the energetics of nearest-neighbor cooperative binding of SSB to ssDNA, one needs to either isolate a particular binding mode for study or be able to identify the modes in which the SSB tetramers are bound to the DNA. For this purpose, we used two monodisperse oligodeoxythymidylates, (dT)₇₀ and (dT)₁₄₀. (dT)₇₀ can accommodate either one tetramer bound in the fully wrapped (SSB)₆₅ mode or two tetramers bound in the (SSB)₃₅ mode (Fig. 2 A) and thus allows us to probe nearest-neighbor (SSB)₃₅ cooperativity. (dT)₁₄₀ can accommodate two tetramers bound in the (SSB)₆₅ mode (Fig. 2 Bi), up to four tetramers bound in the (SSB)₃₅ mode (Fig. 2 Biii), or two tetramers bound in the (SSB)₃₅ mode and one tetramer bound in the (SSB)₆₅ mode (see Fig. 2 Bv). Hence, (dT)₁₄₀ allows us to probe nearest-neighbor cooperativity in the (SSB)₃₅ mode and the (SSB)₆₅ mode, as well as any intermode cooperativity. Two types of fluorescence titrations are performed, one in which the DNA is unlabeled and binding is monitored by SSB Trp fluorescence quenching and a second in which the DNA is labeled with Cy3 on the 3′-end and Cy5 on the 5′-end and SSB binding is monitored by changes in Cy3/Cy5 Förster resonance energy transfer (FRET). Both of these fluorescence methods provide signals that are sensitive to the particular SSB binding mode. In parallel experiments, we use sedimentation velocity to determine the distributions of SSB tetramers on (dT)₇₀ and (dT)₁₄₀ as a function of SSB/DNA ratio throughout a titration, which provides essential information to estimate cooperativity.

Analysis of these experiments also relies on Epstein’s models (39) to calculate the statistical weights associated with nonspecific binding of a large ligand (protein) to a finite-length DNA lattice. The statistical weights for each SSB-DNA species considered in our study are shown in Fig. 2. The studies with (dT)₇₀ are analyzed using three parameters: K₆₅, the equilibrium constant for isolated binding in the (SSB)₆₅ mode; K₃₅, the equilibrium constant for isolated binding in the (SSB)₃₅ mode; and ω₃₅, the nearest-neighbor cooperativity parameter describing bringing two isolated tetramers in the (SSB)₃₅ mode into contiguous contact (Fig. 2 Aiii). The studies with (dT)₁₄₀ require two additional parameters, ω₆₅ and ω_65-35 (hereafter designated ω_m (mixed mode)), describing two SSB tetramers bound cooperatively in the (SSB)₆₅ mode or in a mixed (SSB)₆₅/(SSB)₃₅ mode (Fig. 2, Bii and vi).

The Epstein model was developed for a large ligand (protein) binding to a homogeneous DNA (e.g., all dT) lattice of finite length. The model does not allow for partial binding of the protein to ssDNA ends that are less than the site size of the protein. Because all DNAs used in this study are composed of homo-oligodeoxythymidylates, the intrinsic binding parameters for SSB should be independent of ssDNA length if the statistical factors have been calculated correctly. Furthermore, the lengths of the DNA that we use in the study were chosen to exactly correspond to integer values of the occluded site sizes for SSB binding in its two modes. The fact that the same sets of binding parameters describe the data for SSB binding to (dT)₇₀ and (dT)₁₄₀ supports the assumption of no end binding.

Cooperative binding of wtSSB to (dT)₇₀ in the (SSB)₃₅ mode

Previous studies of SSB binding to (dT)₇₀ showed that an increase in [NaCl] promotes a transition from the (SSB)₃₅ to the (SSB)₆₅ binding mode (26,33). However, binding constants and cooperativities could not be estimated in NaCl buffers because the binding affinities are too high to measure accurately.

In this study, to check whether both modes can coexist and be characterized quantitatively at low salt conditions (10 mM NaCl), we use (dT)₆₈ labeled at the ends with the fluorophores Cy5 and Cy3, which can undergo FRET. As the two fluorophores come closer together, the Cy5 fluorescence increases, reflecting an increase in FRET (20,40). This enables us to determine the SSB binding mode that is being populated because high FRET will be observed for the fully wrapped (SSB)₆₅ mode, in which the two ends of (dT)₆₈ are in close proximity (6), whereas a lower FRET value is observed for the less wrapped (SSB)₃₅ mode (see Fig. 1, C and D). The results of titrations with wtSSB at 10 mM NaCl are shown in Fig. 3 A. The titrations start at low SSB/DNA ratios r (binding densities) at which the (SSB)₆₅ binding mode is favored (19,22). From inspection, it is clear that at low SSB/DNA ratios (r ≤ 1), one SSB tetramer binds to (dT)₆₈ in the (SSB)₆₅ mode (high Cy5 intensity, ∼9 at r = 1.0), whereas upon further addition of SSB (r > 1), a transition to the (SSB)₃₅ mode occurs until the DNA is saturated with two tetramers in the (SSB)₃₅ mode (lower Cy5 intensity, ∼4 at r ≥ 2). It is important to note that the initial linear increase (up to r = 1) and then linear decrease in Cy5 fluorescence (up to r = 2) are indicative of very high affinity (stoichiometric) binding of SSB to the DNA in both modes (all protein added binds to the DNA until saturation is reached at r = 2). This is further supported by sedimentation velocity experiments performed under identical solution conditions at wtSSB/(dT)₇₀ ratios of r = 1 and 2 monitoring absorbance at 260 nm. These results are shown in Fig. 3 C in the form of c(s) distributions (22,33,43). Only one c(s) peak is observed at each ratio, with corresponding sedimentation coefficients of 5.6S and 7.4S (Fig. 3 C, blue (r = 1) and red (r = 2)). Moreover, c(s) analysis predicts molecular masses for these complexes of ∼97 kDa (r = 1) and ∼172 kDa (r = 2), corresponding to (dT)₇₀ with one and two SSB tetramers bound, respectively.

Upon performing equilibrium titrations at 0.30 M NaCl, we observe one wtSSB tetramer binds to (dT)₇₀ in the fully wrapped (SSB)₆₅ mode (Fig. 3 B) as expected (26,33). We note that the observed Cy5 normalized fluorescence for the (SSB)₆₅ 1:1 complex is lower at 0.30 M NaCl than at 10 mM NaCl (∼4.5 vs. ∼9). This is due to the ∼2-fold increase in Cy5-FRET fluorescence of the free Cy5-(dT)₆₈-Cy3-dT (which is used to normalize the observed signal) caused by the higher compaction of the free ssDNA at 0.30 M NaCl. The titration results also agree with the results of sedimentation velocity experiments showing a single c(s) peak at ∼5.6S, corresponding to a stable 1:1 complex even at a twofold excess of SSB protein (Fig. 3 D).

To determine whether the SSB-(dT)₇₀ system is at equilibrium under the conditions of our experiments, we also performed reverse equilibrium titrations, monitoring SSB Trp fluorescence quenching upon addition of (dT)₇₀ to SSB at the same 10 mM and 0.30 M NaCl concentrations (Fig. 3, E and F, respectively). Comparison of Fig. 3 A versus Fig. 3 E and Fig. 3 B versus Fig. 3 F shows that the same complexes are formed at the same SSB/(dT)₇₀ ratios, indicating that the systems are at equilibrium. Binding in the fully wrapped (SSB)₆₅ mode displays high Trp fluorescence quenching of ∼90% (Q₆₅ = 0.9) because the fluorescence of all Trp residues that contact DNA (Trp 40, 54 and 88) within each subunit are partially quenched, whereas binding in the (SSB)₃₅ mode displays only ∼50% Trp fluorescence quenching (Q₃₅ = 0.5) because only the Trp residues in two of the subunits of the tetramer are involved in DNA binding (14,15,26,33). In low salt conditions (Fig. 3 E), a biphasic linear change in Trp fluorescence quenching is observed with transition points at DNA to protein ratios of 0.5 and then 1.0. The characteristic values of normalized Trp quenching at these ratios (Q₃₅ = 0.5 and Q₆₅ = 0.9) are indicative of the initial formation of a complex with two SSB tetramers bound to (dT)₇₀ in the (SSB)₃₅ mode (SSB in excess), which then transitions to a fully wrapped 1:1 (SSB)₆₅ complex upon further titration with (dT)₇₀ (DNA in excess). In contrast, at 0.30 M NaCl, a simple linear change in Trp fluorescence quenching indicates that only the (SSB)₆₅ complex is formed (Fig. 3 F).

We analyzed all fluorescence and sedimentation velocity data using Epstein’s approach (39) (see Epstein-70 model and Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), 5, 6, and (7a), (7b), (7c), (7d)). Because binding is stoichiometric, it is not possible to directly fit these data to obtain the equilibrium constants K₆₅, K₃₅, and ω₃₅; thus, we simulated the expected results for a series of titrations using a minimal estimate of K₃₅ = 10¹⁰ M⁻¹ while varying K₆₅ and ω_35. This minimal estimate of K₃₅ is based on previous studies of the binding of two molecules of (dT)₃₅ to an SSB tetramer at different NaCl concentrations (26). First, we show in Fig. S1 that for K₃₅ = 10¹⁰ M⁻¹ and ω₃₅ = 1, the observed isotherms cannot be reproduced for any value of K₆₅, indicating that cooperativity must exist between two SSB tetramers bound in the (SSB)₃₅ mode. We next varied both K₆₅ and ω₃₅ and found that to reproduce the data in Fig. 3 (10 mM NaCl) requires a minimal estimate of K₆₅ = 1 × 10¹³ M⁻¹ and a range of values for ω₃₅ from 1 × 10³ to 5 × 10⁴ (see Fig. S2, Aiii and Biii). If a higher estimate of K₆₅ = 10¹⁶ M⁻¹ is used, a higher range of values of ω₃₅ (10⁶–10¹⁰) are required to reproduce the experimental isotherms (Fig. S3, Aiv and Biv). Based on this analysis for SSB binding to (dT)₇₀, our initial estimate of ω₃₅ ranges from 10³ to 10¹⁰ (see Table S1, column 5). This range can be narrowed further based on analysis of the data for SSB binding to (dT)₁₄₀, as shown below.

We next tried to simulate the experimental isotherms in Fig. 3, A and E (10 mM NaCl) using a value of K₆₅ = 3 × 10¹⁴ M⁻¹, which is our best estimate of the lower limit of K₆₅ at 0.30 M NaCl (see below). We use this as a lower estimate of K₆₅ at 10 mM NaCl because the affinities of SSB binding to ssDNA only increase with decreasing [salt] (26,38). In this case, fixing K₆₅ = 3 × 10¹⁴ M⁻¹ and K₃₅ = 10¹⁰ M⁻¹, we find that values of ω₃₅ ranging from 10⁴ to 5 × 10⁵ can reproduce the experimental isotherms in Fig. 3, A and E. The solid lines superimposed on the experimental data in Fig. 3, A and E are simulations for ω₃₅ ≈ 10⁵. Therefore, the data in 10 mM NaCl indicate that wtSSB is able to form the (SSB)₆₅ binding mode on (dT)₇₀ at low binding densities (r ≤ 1), whereas at higher binding densities, the (SSB)₃₅ binding mode is promoted because of a very high nearest-neighbor cooperativity of ω₃₅ = 10⁵. This is the same value for ω₃₅ reported for SSB binding to (dA)₇₀ in the (SSB)₃₅ mode (32).

At a higher [NaCl] (0.30 M), formation of the (SSB)₆₅ mode is favored (Fig. 3, B, D, and F), as expected, although the slight decrease in Cy5 fluorescence observed at high SSB to DNA ratios, r = 4 (Fig. 3 B), indicates that a large excess of SSB shows only a weak ability to begin formation of an (SSB)₃₅ complex, indicating a much lower value of ω₃₅. Hence, we first tried to estimate a value for K₆₅ in these conditions by fitting the data in Fig. 3 B to the Epstein-70 model (Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), and 5) with ω₃₅ = 1 (no cooperativity), K₃₅ = 10¹⁰ M⁻¹ (still the same low limit estimate as at low salt conditions (26)), and the fluorescence values of F_35,2 = 2.0 and F_35,1 = 0.05 for binding of two SSB tetramers or one SSB tetramer in the (SSB)₃₅ mode (estimated from the data in Figs. 3 A and 4 A (see below)) while floating K₆₅ and F₆₅. The data are fitted best with K₆₅ = (3.1 ± 1.4) × 10¹⁴ M⁻¹ and F₆₅ = 4.38 ± 0.01 (Fig. 3 B; Table S2). These same binding parameters also provide a good description of the reverse equilibrium titration data in Fig. 3 F. We also tried fitting these data using fixed values of ω₃₅ (10 or 30) while floating K₆₅ (Table S2) and found that ω₃₅ cannot exceed ∼30 at 0.30 M NaCl even for a value of K₆₅ = 10¹⁶ M⁻¹, which is an upper limit for K₆₅. This indicates that ω₃₅ value decreases from ∼(10⁴–5 × 10⁵) at 10 mM to a much lower range of ∼(1–30) at 0.30 M NaCl.

Deletion of the IDL favors the (SSB)₃₅ binding mode on (dT)₇₀ but with a much lower cooperativity

We next examined the SSB variant, SSB-ΔL, in which the 56-amino-acid IDL has been deleted so that the DNA-binding core is connected directly to the 9-amino-acid acidic tip (Fig. 1 B). Deletion of the IDL promotes the (SSB)₃₅ binding mode on poly(dT) (22,33) and eliminates the high non-nearest-neighbor SSB cooperativity on M13ssDNA (22,33). Here, we investigate whether removal of the IDL affects nearest-neighbor cooperativity in the (SSB)₃₅ binding mode. The results of a titration of SSB-ΔL into Cy5-(dT)₆₈-Cy3-dT in 10 mM NaCl, shown in Fig. 4 A, indicate a sigmoidal binding character with transitions at SSB-ΔL/(dT)₇₀ ratios of r ∼ 1 and r ∼ 2. The Cy5 intensity at r ≥ 2 is identical to the intensity observed for the wtSSB binding at r ≥ 2 (Fig. 3 A), indicating that two SSB-ΔL tetramers bind to (dT)₇₀ at saturation in the (SSB)₃₅ mode. However, at r = 1, the Cy5 intensity is much lower (∼1) compared to the intensity of wtSSB bound at the same ratio (∼9). This indicates that SSB-ΔL does not bind in the fully wrapped (SSB)₆₅ mode but rather binds only in the (SSB)₃₅ mode, even at low binding densities. Furthermore, the transitions at r = 1 and r = 2 are not sharp, indicating that binding of SSB-ΔL is weaker than wtSSB.

To confirm the stoichiometry of the SSB-ΔL-Cy5-(dT)₆₈-Cy3-dT complexes and to determine the fluorescence characteristics associated with one and two tetramers of SSB-ΔL bound in the (SSB)₃₅ mode, we performed titrations at two DNA concentrations, 0.10 and 0.30 μM (see Fig. S4 A), and analyzed these using the binding-density-function method (42,45). This method allows us to obtain a model-independent estimate of the binding density (<X> = SSB bound/DNA)) at any point in the titration and thus determine the relationship of <X> to the experimental fluorescence signal (shown as the inset in Fig. S4 A). This analysis indicates a stoichiometry at saturation of 2.1 SSB-ΔL/Cy5-(dT)₆₈-Cy3-dT and estimated values of Cy5 fluorescence for one SSB-ΔL bound (F_Cy5,1 ≈ 0.9) and two SSB-ΔL bound (F_Cy5,2 ≈ 3.7). These fluorescence values were then used as initial guesses to perform a global nonlinear least-squares fit of the two isotherms in Fig. S4 A to the Epstein-70 model (Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), and 5), assuming that only the (SSB)₃₅ mode forms with a stoichiometry of 2 at saturation. The isotherms are fit well by this model with F_35,1 = 0.87 ± 0.04, F_35,2 = 4.09 ± 0.01, K₃₅ = (5.1 ± 4.1) × 10⁹ M⁻¹, and ω₃₅ = 44 ± 15 (see Fig. S4 A). The simulated isotherm based on these parameters is shown as a continuous black line in Fig. 4 A. The predicted distributions of free DNA and DNA having one or two SSB-ΔL tetramers bound are shown in Fig. S4 B. These distributions agree well with the results of sedimentation velocity experiments performed at different SSB-ΔL to (dT)₇₀ ratios (see Fig. 4 C). Therefore, SSB-ΔL forms only the (SSB)₃₅ mode at low salt concentrations and displays a very low nearest-neighbor cooperativity. Hence, the IDL of SSB is essential for the high nearest-neighbor cooperativity that wtSSB displays in the (SSB)₃₅ mode.

The results of the reverse equilibrium titrations shown in Fig. 4 E also demonstrate that in these conditions, SSB-ΔL binds to (dT)₇₀ exclusively in the (SSB)₃₅ binding mode. In fact, the isotherm shown as a solid line in Fig. 4 E, using the parameters determined in Fig. 4 A, describes the experimental data quite well. We note that the characteristic Trp quenching in the (SSB)₃₅ mode (Q_35,2 = 0.6) is slightly higher for SSB-ΔL than for wtSSB (Q_35,2 = 0.5) because of the fact that deletion of the IDL to form SSB-ΔL removes four Trp residues (22,33).

The results of titrations of SSB-ΔL into Cy5-(dT)₆₈-Cy3-dT at 0.30 M NaCl (Fig. 4 B) indicate that SSB-ΔL first binds stoichiometrically in the (SSB)₆₅ mode, forming a 1:1 complex similar to wtSSB (note that at r = 1, the Cy5 normalized fluorescence (F ∼ 4.5) is identical to that of wtSSB (Fig. 3 B)). However, as the SSB-ΔL concentration is increased (r > 1), the Cy5 fluorescence starts to decrease, indicating partial formation of a DNA complex with two SSB-ΔL tetramers bound in the (SSB)₃₅ mode. This is also reflected in sedimentation velocity experiments (Fig. 4 D), which show a continuous shift of the c(s) distribution peak position as r increases. Compared to wtSSB (Fig. 3 B), this indicates that SSB-ΔL is more prone to form the (SSB)₃₅ binding mode at 0.30 M NaCl, which is consistent with observations made for poly(dT) binding (22). As in the case of wtSSB, we fit the isotherms to Eqs. 1, 2, 3, (4a), (4b), (4c), (4d), and 5, floating F₆₅ and K₆₅ and varying ω₃₅ in the range from 1 to 30 while fixing K₃₅ = 5 × 10⁹ M⁻¹ (low estimate, based on the best-fit value in 10 mM NaCl) and using F_35,2 = 2.0 and F_35,1 = 0.05. These parameters are defined based on the comparison of titrations with wtSSB and SSB-ΔL shown in Figs. 3, A and B and 4, A and B. The normalized Cy5 fluorescence for a 1:1 (SSB)₆₅ complex at low and high [salt] (F₆₅ = 9 and F₆₅ = 4.5, respectively) are twofold different because of a ∼2-fold difference in Cy5 fluorescence of free DNA, which is used to normalize the signal. As such, it is expected that the values of F_35,2 = 4.1 and F_35,1 = 0.09 observed at low salt should also be twofold lower in high salt conditions. Using F_35,2 = 2.0 and F_35,1 = 0.05, we fit the data in Fig. 4 B and obtain estimates of K₆₅ ranging from (3.3 ± 0.5) × 10¹² to (1.0 ± 0.1) × 10¹⁴ M⁻¹ and for ω₃₅ ranging from 1 to 30 with F₆₅ = 4.48 ± 0.03 (see also Table S2). The simulations using ω₃₅ = 1, K₃₅ = 5 × 10⁹ M⁻¹, and K₆₅ = 3 × 10¹² M⁻¹ shown in Fig. 4, B and F describe the experimental isotherms very well.

In summary, SSB-ΔL does not form the (SSB)₆₅ binding mode at low (10 mM) [NaCl] but binds to (dT)₇₀ exclusively in the (SSB)₃₅ binding mode; however, with much lower cooperativity (ω₃₅ = 44 ± 15). At higher [NaCl] (0.30 M), SSB-ΔL can form a fully wrapped (SSB)₆₅ mode but still shows some tendency to form the (SSB)₃₅ mode, in contrast to wtSSB, which forms the (SSB)₆₅ mode almost exclusively at 0.30 M NaCl.

Cooperative binding of wtSSB to (dT)₁₄₀

Fluorescence titrations

Use of the longer DNA, (dT)₁₄₀, enables us to examine nearest-neighbor cooperativity between two SSB tetramers bound in the (SSB)₆₅ mode. In addition, (dT)₁₄₀ can also bind four tetramers in the (SSB)₃₅ mode or three tetramers in a mixture of modes (one (SSB)₆₅ and two (SSB)₃₅). Hence, the experiments with (dT)₁₄₀ provide additional constraints on the cooperativity values that were estimated from the (dT)₇₀ experiments. The statistical weights (39) for the various SSB species that can form on (dT)₁₄₀ are given in Fig. 2 B (Epstein-140 model). With (dT)₁₄₀, three nearest-neighbor cooperativity parameters are needed: ω₆₅, ω₃₅, and ω_65-35 (hereafter designated ω_m (for mixed mode)). The development of the Epstein-140 model is presented in Materials and Methods (see Eqs. 8, 9, 10, (11a), (11b), (11c), (11d), (11e), and 12).

We first studied wtSSB-(dT)₁₄₀ binding using reverse titrations monitoring SSB Trp fluorescence quenching. Recall that binding in the fully wrapped (SSB)₆₅ mode displays 90% Trp fluorescence quenching (Q₆₅ = 0.9), whereas binding in the (SSB)₃₅ mode displays 50% Trp fluorescence quenching (Q₃₅ = 0.5) (14,15,26,33). Titrations of wtSSB with (dT)₁₄₀ at 10 mM NaCl and 0.20 M NaCl are presented in Fig. 5, A and B, respectively. At 0.20 M NaCl (Fig. 5 B), two tetramers bind stoichiometrically to (dT)₁₄₀ (saturation occurs at 1/r = [dT₁₄₀]_tot/[wtSSB]_tot = 0.5) in the fully wrapped (SSB)₆₅ mode (Q_max ≈ 0.9). However, at 10 mM NaCl (Fig. 5 A), binding is more complicated, showing a transition from the (SSB)₃₅ to the (SSB)₆₅ mode as the concentration of (dT)₁₄₀ increases. At the beginning of the titration (wtSSB ≫ (dT)₁₄₀), binding occurs exclusively in the (SSB)₃₅ mode until (dT)₁₄₀ is saturated with four tetramers at 1/r = [(dT)₁₄₀]_tot/[wtSSB]_tot ≈ 0.25 (Fig. 5 A, cyan and open circles). As more (dT)₁₄₀ (1/r > 0.25) is added, a transition to the (SSB)₆₅ mode occurs, reflected by the further increase in Trp fluorescence quenching similar to the transition observed with (dT)₇₀ (Fig. 3 E). However, the kinetics of this transition are slow, as indicated by comparison of the two titrations in Fig. 5 A in which data points were collected at 3-min intervals (open circles) versus 15- to 20-min intervals (cyan circles). The higher Trp quenching observed for the titration using 15- to 20-min intervals indicates a slow transition to the (SSB)₆₅ mode, consistent with previous observations with poly(dT) (14,19,33).

Therefore, wtSSB can form both binding modes on (dT)₁₄₀ even at low salt (10 mM NaCl) concentrations, although the kinetics of the transition from four tetramers bound in the (SSB)₃₅ mode to two tetramers bound in the (SSB)₆₅ mode is slow. This is in stark contrast with the behavior on (dT)₇₀, where the transition from two tetramers bound in the (SSB)₃₅ mode to one tetramer in the (SSB)₆₅ mode is much faster (Fig. 3 E). This suggests that disruption of the cooperative interactions involving more than two tetramers in the (SSB)₃₅ mode is energetically more difficult and thus slower. In contrast, SSB-ΔL binds exclusively in the (SSB)₃₅ mode on (dT)₁₄₀ at 10 mM NaCl (Fig. 5 A, orange circles), consistent with SSB-ΔL-(dT)₇₀ binding (Fig. 4, A and E). However, at higher salt (0.20 M NaCl), SSB-ΔL regains its ability to bind in the (SSB)₆₅ mode (Fig. 5 B).

In summary, wtSSB can bind to (dT)₁₄₀ in both modes at low salt concentrations; however, removal of the linker favors the (SSB)₃₅ mode. Nevertheless, as salt concentration increases, both proteins favor formation of the (SSB)₆₅ mode.

Sedimentation of wtSSB-(dT)₁₄₀ complexes at low [NaCl] (10 mM)

To facilitate analysis of the fluorescence titrations, we used sedimentation velocity to obtain independent information on the distributions of SSB-(dT)₁₄₀ species throughout the titration. In these experiments, the (dT)₁₄₀ concentration was maintained constant and the wtSSB concentration was varied. The c(s) distributions for 10 mM and 0.20 M NaCl are shown in Fig. 6 A ([(dT)₁₄₀]_tot = 0.5 μM) and Fig. 6 B ([(dT)₁₄₀]_tot = 0.2 μM), respectively. The c(s) distributions at 10 mM NaCl are unchanged when examined at lower concentrations ([(dT)₁₄₀]_tot = 0.25 μM) (Fig. S5).

The c(s) distributions in Fig. 6 A (10 mM NaCl) show the formation of four distinct species with corresponding peaks at ∼5.5, 7.8, 9.4, and 11.0 S (the peak at ∼2.9 S corresponds to free DNA). We note that the complexes formed at low protein/DNA ratios (r < 2) require ∼10 h to reach equilibrium but remain unchanged at longer times. This is exemplified for the distributions at r = 1 in Fig. 6 A. The c(s) profile after 1 h of mixing (thin blue line) differs from the profile at equilibrium (>10 h; thick blue line). However, the c(s) profiles for the complexes formed at r ≥ 2 do not change after a 1 h incubation time. This indicates that equilibrium for the wtSSB-(dT)₁₄₀ system is established within ∼10 h, and these are shown as the c(s) profiles in Fig. 6 (thick lines for r = 1, 2, 3, and 4 and dashed lines for noninteger values of r; see figure legend). Additional sedimentation velocity runs performed at lower DNA concentrations (Fig. S5) show that the peaks in the c(s) distribution profiles do not change position, indicating high affinity (stoichiometric binding) with slow exchange kinetics on the sedimentation timescale. Hence, these c(s) peaks represent distinct wtSSB-(dT)₁₄₀ complexes that differ in the number of wtSSB tetramers bound. The c(s) profiles at SSB/DNA ratios r = 2.0 (thick red lines), 3.0 (thick gray lines), and 4.0 (thick dark green lines) show single peaks with estimated molecular weights consistent with (dT)₁₄₀ bound with 2, 3, and 4 wtSSB tetramers, whereas the c(s) profile at r = 1.0 (thick blue line) shows three well-separated peaks corresponding to free (dT)₁₄₀ (∼18%), (dT)₁₄₀ with one SSB tetramer bound (∼64%), and (dT)₁₄₀ with two SSB tetramers (∼18%).

Fig. 6 C shows the distribution of (dT)₁₄₀ species calculated from the c(s) profiles in Figs. 6 A and S5 as a function of wtSSB/(dT)₁₄₀ ratio (see Materials and Methods for details). The distributions in Fig. 6 C suggest that at low wtSSB/(dT)₁₄₀ ratios (r ≤ 2), (dT)₁₄₀ binds one and then two tetramers in the (SSB)₆₅ binding mode. This suggestion is corroborated first by the (dT)₇₀ binding data (see Fig. 3 A), in which the first SSB molecule binds in the (SSB)₆₅ mode, and second by the reverse titration data (Fig. 5 A), showing that the (SSB)₆₅ mode is formed at low binding densities [wtSSB]_tot/[(dT)₁₄₀]_tot < 2. The fraction of (dT)₁₄₀ having one tetramer bound in Fig. 6 C is lower (64%) than expected if ω₆₅ = 1 (∼82% is expected for ω₆₅ = 1, as shown in the simulations in Fig. 6 D), suggesting that some cooperativity exists between two tetramers bound in the (SSB)₆₅ mode at 10 mM NaCl. As the wtSSB to (dT)₁₄₀ ratio increases above r = 2, a complex with three wtSSB tetramers bound is formed, with ∼95% of the DNA forming this species at r = 3. Based on these data alone, we cannot determine if this three-tetramer complex represents three molecules bound in the (SSB)₃₅ mode or two in (SSB)₃₅ and one SSB₆₅ (a mixture of both). However, we show below that it is the latter. As more wtSSB is added, a complex with four wtSSB tetramers begins to form, and this is the exclusive species at r ≈ 4. This complex represents four wtSSB tetramers bound to (dT)₁₄₀ in the (SSB)₃₅ mode.

To resolve the ambiguities related to formation of the different (dT)₁₄₀ complexes, we used the Epstein-140 model (Eqs. 8, 9, 10, and (11a), (11b), (11c), (11d), (11e)) to fit and simulate the species distributions in Fig. 6 C. We first verified that the (SSB)₆₅ mode is involved in wtSSB binding to (dT)₁₄₀ at low salt conditions by showing that the data in Fig. 6 B cannot be described by a model that considers only binding in the (SSB)₃₅ mode (see Fig. S6, Ai and Bi). We next used the complete Epstein-140 model (which includes the (SSB)₆₅ mode) but constrained ω_m = 1 (no cooperativity between adjacent tetramers bound in different modes) and found that although the fit is better (see Fig. S6 Aii), the resulting fitted parameters (K₆₅ = (1.7 ± 1.0) × 10¹⁷ M⁻¹, ω₆₅ = 38 ± 2, K₃₅ = (1.0 ± 0.4) × 10¹⁶ M⁻¹, and ω₃₅ = 0.29 ± 0.05) are not reasonable because these indicate negative cooperativity in the (SSB)₃₅ mode and similar affinities for the different binding modes. These parameters also do not provide a good description of the observed titration isotherm in Fig. 5 A (compare with simulated isotherm in Fig. S6 Bii).

Finally, we performed fits by floating ω₆₅, ω_m, and ω₃₅ while constraining K₃₅ = 1 × 10¹⁰ M⁻¹ and varying K₆₅ from 1 × 10¹³ to 1 × 10¹⁶ M⁻¹, as we did for (dT)₇₀ (Table S1), and found that the best fit is achieved when the value of K₆₅ is constrained at 1 × 10¹⁶ M⁻¹ with fitted parameters of ω₆₅ = 38 ± 2, ω_m = (5.0 ± 1.4) × 10⁴, and ω₃₅ = (4.4 ± 1.4) × 10⁵ (see Fig. S6, Aiii–vi; Table S1). The simulated species distributions using these parameters are shown in Fig. 6 C (lines superimposed on the data points). Importantly, we note that these parameters also provide a good description of the reverse titrations in Fig. 5 A (compare to Fig. S6 Bvi). Therefore, these results indicate that there is cooperativity between modes at 10 mM NaCl with the following ranking: ω₆₅ ≪ ω_m < ω₃₅ (see Table S1). We also note that for the corresponding values of K₃₅ and K₆₅, the values of ω₃₅ (lower bound) determined for the binding to (dT)₇₀ are consistent with the values of ω₃₅ determined for the binding to (dT)₁₄₀ (compare columns 4 and 5 in Table S1).

Therefore, at low salt conditions (10 mM NaCl), the transition from the (SSB)₆₅ to the (SSB)₃₅ mode on (dT)₁₄₀ is dependent on binding density and regulated by nearest-neighbor cooperativities. The new, to our knowledge, important finding is that wtSSB tetramers bound to ssDNA in different binding modes also display a high nearest-neighbor cooperativity. This cooperativity promotes formation of mixed-mode complexes during the transition from the (SSB)₆₅ to the (SSB)₃₅ binding mode as the binding density increases. We note that at this low [NaCl], the high cooperativity values for ω_m and ω₃₅ largely suppress the formation of any noncooperatively bound (isolated) SSB tetramers. The only isolated SSB tetramers bound in the (SSB)₆₅ mode are observed only at the lowest binding densities (r < 2), whereas all other species exist as cooperatively bound complexes, as depicted by the cartoons above the curves in Fig. 6 C.

Sedimentation of wtSSB-(dT)₁₄₀ complexes at moderate [NaCl] (0.20 M)

Equilibrium titrations of wtSSB with (dT)₇₀ and (dT)₁₄₀ (Figs. 3, B and F and 5 B), as well as sedimentation velocity data for (dT)₇₀ (Fig. 3 D), indicate that at moderate NaCl concentrations, wtSSB binds exclusively in the (SSB)₆₅ mode at a ratio of r = 1. Sedimentation velocity data for wtSSB binding to (dT)₁₄₀ in 0.20 M NaCl at different protein/DNA ratios are shown in Fig. 6 B. At r ≤ 2.0, three distinct c(s) peaks are observed at s ∼ 3.2, s ∼ 6.1, and s ∼ 8.2, reflecting free (dT)₁₄₀ and (dT)₁₄₀ with one and two SSB tetramers bound, respectively, with molecular weights of 44, 123, and 195 kDa, estimated from fits of the c(s) peaks at r = 0 (light green), r = 1 (blue), and r = 2 (red). These results indicate stoichiometric binding of one and then two tetramers in the (SSB)₆₅ mode. However, at higher SSB/DNA ratios (r > 2), only single c(s) peaks are observed, which continue to shift to higher s-values as r increases. The molecular weight estimate for the peak at r = 5 is ∼245 kDa, which is close to that expected for (dT)₁₄₀ with three SSB tetramers bound (265 kDa), suggesting that weaker binding of a third SSB tetramer occurs in the range r > 2. This is also supported by the appearance in Fig. 6 B of a c(s) peak at ∼4.5S, indicating some free wtSSB for r > 3.

Because deconvolution of the c(s) peaks for r > 2 is difficult, we first evaluated the species distributions for the range r ≤ 2, in which binding is stoichiometric (as described for the 10 mM NaCl data (Fig. 6, A and C)). The resulting species distribution plot is shown in Fig. 6 D and indicates that at r = 2, all of the (dT)₁₄₀ has two SSB tetramers bound, whereas at r = 1, three species exist: free (dT)₁₄₀ (∼10%), one tetramer per (dT)₁₄₀ (∼80%), and two tetramers per (dT)₁₄₀ (∼10%). These distributions are in good agreement with simulations based on the Epstein-140 model (see Materials and Methods), assuming that SSB binds exclusively in the (SSB)₆₅ mode with no cooperativity (ω₆₅ ≈ 1), as shown by the solid lines in Fig. 6 D. The simulated distributions in Fig. 6 D are unchanged for any value of K₆₅ > 10¹¹ M⁻¹. Therefore, the increase in [NaCl] to 0.20 M eliminates the modest nearest-neighbor (SSB)₆₅ cooperativity (ω₆₅ ∼38) observed at 10 mM NaCl.

We next attempted to describe the weak binding of a third wtSSB tetramer to (dT)₁₄₀ in 0.20 M NaCl that we observe for r > 2 (Fig. 6 B). We performed a series of simulations (Eqs. 8, 9, 10, and 11) constraining K₃₅ = 10¹⁰ M⁻¹ because wtSSB still binds to (dT)₃₅ stoichiometrically at this NaCl concentration (26,46) and ω₆₅ = 1 while varying K₆₅, ω_m, and ω₃₅. We also assumed that at r = 5, (dT)₁₄₀ is nearly saturated with three SSB tetramers (∼90%). These simulations are shown in Fig. S7, Ai–iii. To be consistent with the parameters determined for SSB binding to (dT)₇₀ under similar conditions (Fig. 3 B; Table S2), we started with the minimal estimate of K₆₅ = 3 × 10¹⁴ M⁻¹ and ω₃₅ = 1 and found that ∼90% saturation of (dT)₁₄₀ is achieved when ω_m ≈ 20 (Fig. S7 Ai). Then, we increased K₆₅ to 3 × 10¹⁵ M⁻¹ and ω₃₅ = 10 and finally K₆₅ to 1 × 10¹⁶ M⁻¹ and ω₃₅ = 30 (as in Table S2) and found that values of ω_m ≈ 80 and ω_m ≈ 140 are needed to reproduce the experimental distributions (see Fig. S7, Aii and iii, respectively). These values also provide a good description of the observed reverse titrations in Fig. 5 B (compare with simulated isotherms in Fig. S7, Bi–iii).

Therefore, in 0.20 M NaCl, we conclude that two wtSSB tetramers bind stoichiometrically to (dT)₁₄₀ in the (SSB)₆₅ mode with no cooperativity for r ≤ 2; however, at higher wtSSB concentrations, a transition occurs such that at r = 5, (dT)₁₄₀ is ∼90% saturated with one tetramer bound in the (SSB)₆₅ mode and two tetramers bound in the (SSB)₃₅ mode, although with lower intermode cooperativity than at the lower salt conditions. At this higher [NaCl], a complex of (dT)₁₄₀ that is fully saturated with four tetramers bound in the (SSB)₃₅ mode is not populated because of the significant decrease in ω_m and ω₃₅ compared to the much higher values at 10 mM NaCl.

The IDL is required for SSB nearest-neighbor cooperativity

Binding of SSB-ΔL to (dT)₁₄₀ in 10 mM NaCl

We showed that at low salt (10 mM NaCl), two molecules of SSB-ΔL bind to (dT)₇₀ exclusively in the (SSB)₃₅ mode but with very little to no cooperativity (Fig. 4, A, C, and E; Fig. S4). At moderate salt concentrations (0.30 M NaCl), one SSB-ΔL tetramer binds stoichiometrically to (dT)₇₀ in the (SSB)₆₅ mode; however, at higher protein concentrations, there is some evidence for the formation of 2:1 complexes (Fig. 4 B). At 10 mM NaCl, SSB-ΔL saturates (dT)₁₄₀ with four tetramers in the (SSB)₃₅ mode but does not show any transition to the (SSB)₆₅ mode with increasing (dT)₁₄₀, in contrast to wtSSB (Fig. 5 A), although at moderate salt (0.20 M NaCl), SSB-ΔL favors the (SSB)₆₅ mode, similar to wtSSB (Fig. 5 B).

We next used sedimentation velocity to obtain SSB-ΔL-(dT)₁₄₀ species distributions. c(s) profiles at different SSB-ΔL/(dT)₁₄₀ ratios r are shown in Fig. 7 A. These profiles differ significantly from those for wtSSB (Fig. 6 A), with broader peaks that are less resolved. This suggests that the SSB-ΔL-(dT)₁₄₀ species are in rapid exchange on the sedimentation timescale and thus cannot be analyzed as was done for wtSSB (Fig. 6, A and C). This is supported by the much weaker, low cooperative binding of SSB-ΔL to (dT)₇₀ under similar conditions (Fig. 4, A, C, and E). Assuming that SSB-ΔL also binds to (dT)₁₄₀ in the (SSB)₃₅ mode and with similar parameters as for (dT)₇₀ (K₃₅ = 5 × 10⁹ M⁻¹ and ω₃₅ = 40), we first simulated the predicted species distributions (Fig. 7 C) with the Epstein-140 model, using only the terms for the (SSB)₃₅ mode. These predicted distributions are clearly very different from those formed by wtSSB (Fig. 6 C). Inspection of Fig. 7 C indicates a much broader species distribution and, in particular, a significant suppression of the species with one and two tetramers bound. To check whether the c(s) profiles in Fig. 7 A are consistent with SSB-ΔL binding in the (SSB)₃₅ mode, we simulated sedimentation velocity experiments for the same SSB-ΔL/(dT)₁₄₀ ratios using a model of sequential binding of four SSB-ΔL tetramers to (dT)₁₄₀ using SedAnal (47), as described in Supporting Materials and Methods and in the legend for Fig. S8. The simulated sedimentation velocity traces were then reanalyzed using SEDFIT. The resulting c(s) profiles shown in Fig. S8 are in reasonable agreement with the experimental profiles in Fig. 7 A, suggesting that the parameters determined for SSB-ΔL binding to (dT)₇₀ (K₃₅ = 5 × 10⁹ M⁻¹ and ω₃₅ = 40) adequately describe the binding of SSB-ΔL to (dT)₁₄₀, with no evidence for formation of the (SSB)₆₅ mode.

Binding of SSB-ΔL to (dT)₁₄₀ in 0.20 M NaCl

The titrations of SSB-ΔL with (dT)₇₀ (Fig. 4 F) and (dT)₁₄₀ (Fig. 5 B) provide evidence that at moderate [NaCl] (0.20 M NaCl), SSB-ΔL binds stoichiometrically in the (SSB)₆₅ mode. However, titrations of Cy5-(dT)₆₈-Cy3-dT with SSB-ΔL (Fig. 4 B), as well as sedimentation velocity data (Fig. 4 D), indicate that at high SSB-ΔL/(dT)₇₀ ratios (r > 1), a second SSB-ΔL tetramer can bind weakly to (dT)₇₀, presumably transitioning both tetramers to the (SSB)₃₅ mode. We next performed sedimentation velocity experiments for SSB-ΔL binding to (dT)₁₄₀ in 0.20 M NaCl. In the range from r = 1 to r = 2, the c(s) profiles in Fig. 7 B suggest three species corresponding to free (dT)₁₄₀ (s ∼ 3.2) and (dT)₁₄₀ with one (s ∼ 6) and two (s ∼ 8.3) SSB-ΔL tetramers bound, similar to what was observed for wtSSB (Fig. 6 B). However, direct comparison of the c(s) distributions at r = 1.0 (blue lines) for SSB-ΔL (Fig. 7 B) and wtSSB (Fig. 6 B) suggests some cooperative binding in the (SSB)₆₅ mode for SSB-ΔL. This is reflected by the suppression of the dominant peak (s ∼ 6) representing the 1:1 complex and the corresponding increase in the populations of free (dT)₁₄₀ and the 2:1 complex. In fact, this distribution is qualitatively similar to the distributions observed for wtSSB at low salt at the same r value (Fig. 6 A) for which a cooperativity parameter ω₆₅ ≈ 40 was determined; however, the species representing the 1:1 complex is more suppressed for SSB-ΔL, suggesting a higher cooperativity. To quantify the cooperativity for SSB-ΔL binding to (dT)₁₄₀ in the (SSB)₆₅ mode at 0.20 M NaCl, we first estimated the species distributions in the range r ≤ 2 from the areas of the c(s) peaks. Those are plotted as a function of r in Fig. 7 D (circles). A fit of these distributions (solid dark yellow, blue, and red lines) to the (SSB)₆₅ version of the Epstein-140 model (excluding all terms for (SSB)₃₅) yields an estimate of ω₆₅ = 212 ± 28 for all K₆₅ ≥ 1 × 10¹¹ M⁻¹.

As the SSB-ΔL/(dT)₁₄₀ ratio increases (r > 2), we observe only single c(s) peaks (Fig. 7 B) that shift to higher s-values, as was the case for wtSSB (Fig. 6 B). An estimate of the molecular weight from the c(s) peak at r = 4 (dark green) is ∼204 kDa, suggesting a (dT)₁₄₀ complex bound with three SSB-ΔL tetramers (209 kDa predicted). Therefore, it appears that at higher salt, a complex with three SSB-ΔL tetramers bound to (dT)₁₄₀ can form at saturation with two tetramers bound in the (SSB)₃₅ mode and one bound in the (SSB)₆₅ mode, as was observed for wtSSB.

We next optimized the parameters describing binding of SSB-ΔL to (dT)₁₄₀ in 0.20 M NaCl, following the same simulation procedure as for wtSSB (Fig.S7, Ai–iii) but using the parameters (K₆₅, K₃₅ and ω₃₅) that best describe binding of SSB-ΔL to (dT)₇₀ under similar conditions (Fig. 4 B; Table S2) with ω₆₅ = 212 fixed. We start with the minimal estimate of K₆₅ = 3 × 10¹² M⁻¹ (ω₃₅ = 1) and then increase these to K₆₅ = 1 × 10¹⁴ M⁻¹ (ω₃₅ = 30) and find that ω_m needs to be in the range from 25 to ∼145 to reproduce the distributions (see simulations in Fig. S9, Ai–iii). This is the same range as found for wtSSB. This striking similarity suggests that the IDL has no effect on intermode cooperativity ω_m and ω₃₅ when SSB binds to ssDNA at moderate salt concentrations. However, under these conditions, cooperativity is clearly already quite low.

Therefore, we conclude that at low salt conditions (10 mM NaCl), SSB-ΔL binds exclusively in the (SSB)₃₅ mode but with much lower, almost negligible cooperativity compared to wtSSB (ω₃₅ ∼ 40 vs. ω₃₅ = 5 × 10⁴–5 × 10⁶, Table 1). On the other hand, SSB-ΔL regains some cooperativity when binding in the (SSB)₆₅ mode in 0.20 M NaCl (ω₆₅ ∼ 200 vs. ω₆₅ ∼ 1 for wtSSB, Table 1). These observations indicate that the IDL plays a role in regulating all cooperative binding of SSB to ssDNA.

Table 1.

Estimated Ranges of Nearest-Neighbor Cooperativities for wtSSB and SSB-ΔL Binding to (dT)₇₀ and (dT)₁₄₀ at Different NaCl Concentrations

Protein	[NaCl] (M)	K65 (M−1)a	K35 (M−1)b	ω65	ω65-35	ω35
wtSSB	0.01	1 × 1015–1016	1 × 1010	38 ± 3	(1–5) × 104	5 × 104–1 × 106
	0.2–0.3	3 × 1014–1016	1 × 1010	1	20–140	1–30
SSB-ΔL	0.01	–	(5 ± 4) × 109	–	–	44 ± 15
	0.2–0.3	3 × 1012–1014	5 × 109	210 ± 30	25–145	1–30

^aRanges for K₆₅ variation (lower and upper bound).

^bLower bound estimates for K₃₅ (the values presented with standard deviation are the rigorously estimated parameters).

KGlu (versus NaCl) has no effect on cooperativity of wtSSB and SSB-ΔL binding to (dT)₁₄₀

We have shown previously that physiological concentrations of KGlu (0.10–0.20 M) promote the formation of highly cooperative (collapsed or condensed) SSB-M13ssDNA complexes (22). We suggested that the formation of such collapsed complexes is due to non-nearest-neighbor cooperative interactions among SSB tetramers bound to ssDNA (22), consistent with the single-molecule experiments of Bell et al. (37). To probe whether KGlu also affects nearest-neighbor cooperativity, we performed sedimentation velocity runs with wtSSB and SSB-ΔL binding to (dT)₁₄₀ in 0.20 M KGlu, and the results are shown in Fig. S10, C and D. For comparison, we also show the data obtained at 0.20 M NaCl (Fig. S10, A and B). To eliminate the effects due to differences in buffer densities and viscosities, these distributions are plotted vs. s_20,w. Fig. S10 shows that the c(s) profiles in 0.20 M KGlu are very similar to the profiles in 0.20 M NaCl for both proteins. This suggests that KGlu has little effect on the nearest-neighbor cooperativity in the (SSB)₆₅ mode. However, we expect SSB to bind with higher affinity in KGlu versus NaCl (38). Indeed, this is reflected by the fact that for r > 2, at which SSB binds nonstoichiometrically, (dT)₁₄₀ has more SSB bound in KGlu than in NaCl at the same protein/DNA ratio (compare c(s) peak positions for r = 4 in Fig. S10). Therefore, the increase in cooperativity promoted by KGlu for wtSSB binding to M13ssDNA in its (SSB)₆₅ mode results from an effect only on non-nearest-neighbor cooperative interactions. Importantly, those non-nearest-neighbor interactions also require the IDLs (22).

Discussion

Sigal et al. (31) first identified and characterized EcSSB protein and showed by electron microscopy that it can bind ssDNA cooperatively to form protein clusters at less than saturating SSB/DNA ratios at low salt concentrations. Ruyechan and Wetmur (48), also using electron microscopy, reported the first quantitative estimate of ∼10⁵ for a nearest-neighbor cooperativity parameter at 150 mM NaCl. However, both of those studies were performed before it was recognized that SSB can bind ssDNA in different modes (14,15,17), depending primarily on the salt concentration and type and protein/DNA ratio, and that the different modes differ in their cooperative binding properties (18). Early studies of SSB binding to M13ssDNA using agarose gel experiments indicated that high cooperativity is associated with the (SSB)₃₅ binding mode because it is lost at higher [NaCl], which favors the (SSB)₆₅ mode (18). The nearest-neighbor cooperativity in the (SSB)₃₅ mode was first estimated using (dA)₇₀ and found to be ω₃₅ ∼ 10⁵, but this estimate was made only under one set of solution conditions (0.125 M NaCl (pH 8.1), 25°C) (32). SSB binds to ssDNA exclusively in the fully wrapped (SSB)₆₅ mode at monovalent salt concentrations ≥0.20 M and displays a “limited” cooperativity that appears to be restricted to forming dimers of tetramers (octamers) (16,28). Quantitative estimates of this limited nearest-neighbor cooperativity have previously only been made with the ssRNA, poly(U), at salt concentrations >0.15 M, yielding ω₆₅ = 420 ± 80 (28,38).

It is now clear that SSB-ssDNA cooperativity is even more complicated than we had previously known. Most recently, we have shown that the (SSB)₆₅ mode also displays high cooperativity at low salt concentrations (10 mM NaCl) (22,33). These highly cooperative interactions require the presence of the SSB IDL (22,33). This high cooperativity in the (SSB)₆₅ mode is inhibited at NaCl or KCl concentrations >0.20 M but is facilitated by glutamate or acetate anions, persisting even up to 0.50 M KGlu (22). A recent single-molecule study has shown that ssDNA collapse or condensation of long SSB-ssDNA complexes can occur at high Na acetate concentrations (37), and we observe highly cooperative binding in the (SSB)₆₅ mode under these same conditions (22). However, high [NaCl] does not appear to facilitate ssDNA collapse (49). This high cooperativity/condensation appears to involve non-nearest-neighbor interactions between SSB tetramers (22,37). A recent study has identified residues within the DNA-binding core of SSB that also contribute to cooperativity, likely nearest-neighbor (50).

In this study, we examined SSB binding to oligonucleotides of fixed length ((dT)₇₀ and (dT)₁₄₀) to probe the linkage between nearest-neighbor cooperativity and SSB binding mode and the effects of the SSB IDL and salt concentration. The combined use of oligodeoxynucleotides of these lengths allowed us to obtain a range of estimates of nearest-neighbor cooperativity in both the (SSB)₃₅ and (SSB)₆₅ binding modes and to examine whether cooperativity exists between modes. The use of (dT)₇₀ enabled us to examine cooperative interactions between two tetramers bound in the (SSB)₃₅ mode in competition with one tetramer bound in the (SSB)₆₅ mode, as was done previously with (dA)₇₀ (32). The use of (dT)₁₄₀ enabled us to examine cooperativity between two tetramers bound in the (SSB)₆₅ mode and multiple tetramers bound in the (SSB)₃₅ mode. To probe binding and cooperativity, we combined fluorescence titrations and sedimentation velocity methods. The fluorescence titrations provided information on the particular SSB binding mode, whereas sedimentation velocity provided quantitative information on the number of SSB tetramers bound to (dT)₇₀ and (dT)₁₄₀ during a titration, which was needed to evaluate cooperativity. Quantitative analysis of these data required the statistical mechanical models developed by Epstein (39) that account for the statistical effect of a large ligand (protein) binding to a homogeneous finite-length linear lattice.

Nearest-neighbor cooperativity is influenced by SSB binding mode and salt concentration

We show that the (SSB)₆₅ mode can be formed on (dT)₁₄₀ at low salt concentration (10 mM NaCl), but only at low binding densities. Moreover, the binding of two tetramers in the (SSB)₆₅ mode occurs with low cooperativity, ω₆₅ ≈ 38 ± 3 (Table 1). Upon increasing SSB concentration further (r > 2), the system first transitions to a state with three tetramers bound to (dT)₁₄₀ in a mixture of modes (one tetramer bound in (SSB)₆₅ mode with high intermode cooperativity (ω_m ∼ 10⁴) and two tetramers bound in (SSB)₃₅ mode (see these species distributions in dashed gray lines in Figs. 6 C and S6, Aiii–vi)), then finally to a state with four tetramers bound in the (SSB)₃₅ mode with very high cooperativity (ω₃₅ ∼10⁴–10⁶). Because the intrinsic affinity in the (SSB)₆₅ mode is much higher than in the (SSB)₃₅ mode under these conditions (K₆₅ ∼ 10¹⁵–10¹⁶ M⁻¹ vs. K₃₅ ∼ 10¹⁰ M⁻¹), high nearest-neighbor cooperativities are required in the mixed mode and the (SSB)₃₅ mode for these transitions to occur. This is the first estimate of intermode SSB cooperativity (ω_m ∼ 10⁴).

The situation changes at higher salt concentration (0.20–0.30 M NaCl). As expected, one and two wtSSB tetramers bind stoichiometrically to (dT)₇₀ and (dT)₁₄₀, respectively, in the (SSB)₆₅ mode (see Figs. 3, B–F and 5 B), although binding under these conditions is noncooperative (ω₆₅ = 1). However, upon further increasing the SSB concentration (r > 2), a mixed-mode complex can still form (r > 2) such that 80–90% of (dT)₁₄₀ can eventually bind a third SSB tetramer at r = 5. However, the transition to four tetramers bound in the (SSB)₃₅ mode does not occur at the higher [NaCl] because of the significantly lower cooperativities for this mode, ω₃₅ ≈1–30 (Table 1). The suppression of all cooperativities at higher [NaCl] suggests that these cooperative interactions have a significant electrostatic component.

We speculate that the low cooperativity observed for wtSSB binding in the (SSB)₆₅ mode at low salt (ω₆₅ ∼ 40, Table 1) is associated with interactions between the DBDs of adjacent tetramers. Among the likely candidates are R72, S75 (loop 3–4) (51) and Y22 (loop 1–2), K73 (loop 3–4) (50). A mutation, R72A, has been shown to weaken the interaction of SSB with poly(U) and affects the binding mode distributions on poly(dT) (51), whereas the double mutation Y22A-K73E affects formation of the (SSB)₆₅ mode on poly(dT) and the highly cooperative binding to M13ssDNA (50). This suggests that nearest-neighbor cooperativity in the (SSB)₆₅ mode could also be affected by these mutations.

As the concentration of SSB increases, the fully wrapped (SSB)₆₅ complexes on (dT)₁₄₀ have to be partially unraveled to provide free ssDNA to bind additional SSB tetramers (ultimately four in the (SSB)₃₅ mode at low [NaCl]). This transition to the (SSB)₃₅ mode results in unoccupied (by ssDNA) SSB subunits and opens the possibility for new intertetramer interactions via residues previously involved in DNA binding in the (SSB)₆₅ mode. For example, K73, which appears to be involved in ssDNA binding (6,52), has also been implicated in intertetramer interactions (50). Moreover, the DNA wrapping path in a postulated (SSB)₃₅ binding mode (6) (see also Fig. 1 D) suggests that intertetramer interactions in this mode might also be promoted by interactions between the L₄₅ loops of neighboring tetramers. Those interactions may include multiple loop-loop and loop-core contacts, including aromatic and charged residues (6,53). We have previously suggested that an additional electrostatic component that could contribute to cooperativity in (SSB)₃₅ complexes might involve interactions of the negatively charged tips of the C-termini with the unoccupied DNA-binding sites (33,54,55). Because all of these interactions have electrostatic contributions, they would be expected to be mitigated at increased salt concentrations.

Nearest-neighbor cooperativity is regulated by the intrinsically disordered C-terminal linker

We find that deletion of the IDL affects both the binding mode transitions and nearest-neighbor cooperativities. At low salt, SSB-ΔL binds to both (dT)₇₀ and (dT)₁₄₀ exclusively in the (SSB)₃₅ mode but with quite low cooperativity (ω₃₅ ≈ 40), Table 1). This is consistent with observations made on long ssDNA (poly(dT) and M13ssDNA) (22,33), although as the [NaCl] increases, SSB-ΔL regains its ability to form the (SSB)₆₅ mode. Surprisingly, we find that cooperativity of SSB-ΔL in this mode (ω₆₅ ≈ 210) is higher than for wtSSB (ω₆₅ ∼ 1) under identical solution conditions and is still higher than for wtSSB at low salt (ω₆₅ ∼ 40). We also find that similar to wtSSB, three SSB-ΔL tetramers can bind to (dT)₁₄₀ in mixed modes at higher SSB-ΔL concentrations (r > 2) but with very low or no cooperativity, ω_m ∼ 25 and ω₃₅ ∼ 1, similar to wtSSB in these conditions (see Table 1). This suggests that these cooperative interactions result from interactions between the N-terminal DNA-binding cores of adjacent tetramers as discussed above.

We note that the SSB-ΔL variant still retains the 9-amino-acid acidic tip that is forced to be adjacent to the DNA-binding core because of deletion of the IDL. As such, we expect there to be a significant electrostatic effect on DNA binding that may contribute to the inability of SSB-ΔL to form the (SSB)₆₅ mode and its loss of high cooperativity in the (SSB)₃₅ mode at low salt conditions. It has been shown that the high negative charge of the last nine amino acids of the C-terminus (the tip) can affect ssDNA binding. At low salt concentrations, the tip can compete with ssDNA for the positively charged core, weakening DNA-binding affinity and inhibiting formation of the fully wrapped (SSB)₆₅ mode (54,55). We have suggested that interactions of the tip of one tetramer with the unoccupied ssDNA-binding site of an adjacent tetramer, both in the (SSB)₃₅ mode, may contribute to cooperativity (33). However, an increase in salt concentration should attenuate these electrostatic interactions and therefore promote the (SSB)₆₅ mode. We note that these electrostatic effects are still present, although to a lesser extent, at moderate salt concentrations (0.10–0.20 M NaCl) but are totally eliminated at high salt concentrations (0.6 M NaBr) (54).

The transition from the (SSB)₃₅ to the (SSB)₆₅ mode for SSB-ΔL on poly(dT) is shifted to higher NaCl concentrations (22), consistent with removal of the IDL promoting the (SSB)₃₅ binding mode. We have shown previously that wtSSB displays a strong intratetramer negative cooperativity for binding of (dT)₃₅ (second molecule binds more weakly than the first), which increases as the salt concentration decreases, correlating with the binding mode transition (26,46). This negative cooperativity appears to be much greater upon removal of the IDL. At 10 mM NaCl, a second molecule of (dT)₃₅ can bind to wtSSB with low but measurable affinity (K_2,35 ∼ 10⁵ M⁻¹), whereas we find no evidence that a second (dT)₃₅ molecule can bind to SSB-ΔL under these conditions even at a 20-fold excess of (dT)₃₅ (33). This indicates that SSB-ΔL has a much higher negative cooperativity that stabilizes the (SSB)₃₅ mode. We speculate that the inability of SSB to form the (SSB)₆₅ mode upon removal of the IDL may be related to the fact that the high negative charge of the acidic tips is concentrated at the dimer-dimer interface (where the C-termini emanate from the core; Fig. 1 B) and precludes the ssDNA from crossing this interface to form the fully wrapped (SSB)₆₅ binding mode. We also observe a significant decrease in ω₃₅ for SSB-ΔL. This might also relate to the electrostatic repulsions between the acidic tips at the tetramer-tetramer interface.

It is also possible that SSB-ΔL uses an alternative wrapping path in its (SSB)₃₅ mode. In the (SSB)₃₅ mode proposed by Raghunathan et al. (6) (see Fig. 1 D) and supported by single-molecule studies (24), the ssDNA crosses the dimer-dimer interface. However, if this path is blocked, the ssDNA could instead wrap around two SSB subunits within a single dimer without crossing the dimer-dimer interface while still occluding only 35 nucleotides of DNA, essentially following the path of ssDNA wrapping in the (SSB)₆₅ binding mode (Fig. 1 C). This alternate path would eliminate forming any contacts between the L_4–5 loops of adjacent tetramers and decrease intertetramer cooperativity. In fact, the existence of an alternative (SSB)₃₅ mode was suggested based on single-molecule studies of wtSSB binding to (dT)₇₀ (20) in low salt conditions, although the population of such alternative complexes was low.

Glutamate enhances only non-nearest-neighbor SSB-DNA cooperativity

We had shown previously that SSB displays highly cooperative binding to long ssDNA (e.g., ss M13 phage DNA) at low [NaCl], as indicated by a bimodal distribution of SSB-ssDNA complexes in the presence of less than saturating ratios of SSB/DNA (18,33). This bimodal distribution is eliminated at higher [NaCl], conditions that favor the (SSB)₆₅ binding mode. However, this bimodal distribution can be restored at high salt concentrations by replacing chloride with acetate or glutamate (22). This is interesting in light of the fact that glutamate is the major monovalent anion in bacterial cells, ranging in concentration from 0.03 to 0.25 molal, and is preferentially excluded from protein surfaces (56, 57, 58). This effect of glutamate on SSB-ssDNA cooperativity is lost when the IDL is deleted, indicating that this high cooperativity involves the IDL (22).

Interestingly, we find that nearest-neighbor cooperativity is not affected by replacing chloride with glutamate at moderate salt concentrations, although the affinity of SSB for DNA increases as expected (38). This suggests that the significant effects of KGlu versus NaCl on SSB cooperativity on ssM13DNA (22) are due to non-nearest-neighbor SSB-SSB interactions that involve the linker, possibly via direct linker-linker interactions. The formation of such non-nearest-neighbor SSB interactions occurs only on longer ssDNA and is accompanied by additional ssDNA compaction (collapse) beyond that expected from formation of the (SSB)₆₅ binding mode (37).

Functional implications of the multiple SSB-ssDNA cooperativities

The functional role of cooperative binding of SSB proteins to ssDNA has been considered since Alberts and Frey (59) first showed that the phage T4 gene 32 protein binds ssDNA with high cooperativity. This T4 SSB is simple in that it is a monomer and appears to show only one type of nearest-neighbor cooperativity (ω ∼ 10³) (41,60,61). However, the EcSSB protein is a tetramer that can bind ssDNA in multiple binding modes (14,15) and with multiple cooperativities (18,22,33) involving both nearest-neighbor (32,38) and non-nearest-neighbor interactions (22,37).

High “unlimited” nearest-neighbor cooperative binding of an SSB to ssDNA enables formation of protein clusters along the ssDNA, which would be needed to protect the ssDNA from damage and to remove unwanted DNA secondary structures that would inhibit DNA replication. As such, we have suggested (18,32,62) that the (SSB)₃₅ mode would most likely be used during DNA replication because of its very high nearest-neighbor cooperativity (ω₃₅ = 5 × 10⁴–1 × 10⁶). Recent studies indeed implicate the use of the (SSB)₃₅ mode during DNA replication (36,63). In addition, the (SSB)₃₅ mode also is needed for an SSB tetramer to undergo a direct transfer from one DNA segment to another (34), a reaction that we suggested might be used to recycle SSB from one lagging strand to another during replication (34) and that has recently been demonstrated both in vitro and in vivo (36). In this study, we also show that high nearest-neighbor cooperativity (ω_m ∼ 10⁴) also occurs between the (SSB)₃₅ and (SSB)₆₅ binding modes. Hence, SSB would not need to be bound exclusively in the (SSB)₃₅ mode to exhibit high nearest-neighbor cooperativity.

We find a much lower nearest-neighbor cooperativity for the (SSB)₆₅ mode, consistent with previous studies (28,38) showing that the (SSB)₆₅ mode displays only “limited” cooperativity, forming dimers of tetramers (octamers) rather than long clusters (16,28). The experiments reported here with (dT)₁₄₀ are unable to differentiate between “limited” and “unlimited” cooperativity because (dT)₁₄₀ is too short to observe an “unlimited” cooperativity in the (SSB)₆₅ mode. However, the consequences of a low cooperativity in the (SSB)₆₅ mode, regardless of which type, are that long clusters of SSB protein on the DNA are much less probable. Because there are many processes, e.g., recombination and repair of DNA, that occur during genome maintenance that require other proteins to access the ssDNA even when it is bound with SSB, this mode might be used under such circumstances (62). Also, the transient unwrapping of short segments of ssDNA from the SSB, which is more likely in the (SSB)₆₅ mode (20), would also enable other proteins to access the DNA, as has been shown recently (64,65).

A fourth level of cooperativity involving non-nearest-neighbor interactions has recently been identified for EcSSB binding to long polymeric ssDNA (22,33,37). This was first observed in single-molecule experiments that showed an additional compaction of a single DNA molecule bound with SSB in the (SSB)₆₅ mode that is induced at high Na acetate concentrations. Bell et al. (37) suggested that this compaction, beyond what is expected because of the wrapping of ssDNA around SSB in the (SSB)₆₅ mode, is due to interactions between non-nearest-neighbor SSB tetramers on the ssDNA. Binding of a second protein to SSB, either RecO or RecOR, led to even further compaction. Interestingly, the experiments of Bell et al. (37) were performed in acetate salts. The ability of an SSB tetramer to interact simultaneously, although transiently, with distant regions of the same ssDNA molecule has also been suggested based on single-molecule experiments (35). Related to these findings, the high-cooperativity, bimodal SSB-ssM13 DNA populations that we observe in sedimentation velocity experiments at low [NaCl], are also promoted at higher salt concentration when chloride is replaced with acetate or glutamate, and these interactions require the SSB IDL (22). Hence, these observations likely reflect the same non-nearest-neighbor interactions involving the IDL. However, we show here that glutamate has no effect on any of the nearest-neighbor cooperative interactions on (dT)₁₄₀. One possible role of non-nearest-neighbor SSB-ssDNA interactions might be to facilitate homology-dependent annealing of DNA that is important for recombination (37).

Author Contributions

A.G.K. and T.M.L. designed the research. A.G.K. and M.K.S. performed the research. A.G.K. analyzed the data. A.G.K. and T.M.L. wrote the manuscript.

Editor: James Cole.