The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

Abstract

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.

1. INTRODUCTION

The SSB interactome plays an important role in maintaining the integrity of the genome.1, 2 The central player regulating interactome function is the essential single‐strand DNA binding protein (SSB). How the activities of the interactome proteins are regulated and directed to the DNA at the correct time when needed is essential to cell viability. This regulation involves the binding of SSB to either itself or, to other interactome partners. The former activity is required for rapid and cooperative single‐stranded DNA (ssDNA) binding while the latter is necessary for dynamically mediating partner‐DNA interactions essential to maintaining genome integrity. The linker/oligonucleotide binding folds (OB‐fold) model was proposed to explain how these two disparate SSB activities could be linked. ³

The central player in the linker/OB‐fold model is the SSB protein which exists as a stable homo‐tetramer. ⁴ Each 178‐residue monomer is divided into two fragments defined by proteolytic cleavage: an N‐terminal portion comprising the first 115 residues and a C‐terminal tail comprising residues 116–178.5, 6 The N‐terminal domain is well conserved and contains elements critical to tetramer formation and the oligonucleotide‐oligosaccharide binding fold (OB‐fold) required for ssDNA binding. ⁷ The flexible C‐terminal tail is required for protein–protein interactions, is essential in vivo and is further divided into two regions: a sequence of approximately 54 amino acids known as the intrinsically disordered linker or linker, and the last eight residues known as the acidic tip or C‐peptide.4, 5, 8, 9, 10 While the acidic tip is very well conserved among eubacterial proteins, the overall sequence of the linker domain is not.1, 9 Consequently, this has resulted in the misconception that the linker is unimportant for biological function while the acidic tip is essential and mediates all protein–protein interactions.

The importance of the tip comes from several biochemical and biophysical studies. First, studies show that when the tip is deleted, protein–protein interactions are lost.3, 11, 12 Second, cocrystals of the acidic tip peptide and a partner protein show that tip residues can bind to partners.10, 13, 14, 15 Third, binding studies using short peptides comprising the acidic tip demonstrated binding, but in these studies, the binding sites on the partner proteins were not identified.16, 17, 18, 19, 20 Collectively these studies have been interpreted to mean that the acidic tip is solely responsible for mediating protein–protein interactions between an SSB tetramer and a partner.

However, these studies can be interpreted differently. First, the deletion of the tip could impair the overall structure of the SSB C‐terminal domain so that the partner interaction domain is secluded and/or otherwise inactivated, thereby eliminating partner interactions. Second, both the crystallographic and binding studies used short peptides corresponding to the acidic tip and, a full‐length partner protein. While peptides can bind to targets, there are a large number of potential binding sites on intact partner proteins. In studies with Exonuclease I, cocrystals show that the peptide bound to two distinct sites, with only one of these proposed to have a biological function. ¹⁷ If intact SSB and full‐length partners were used instead, it is conceivable that the protein–protein interaction domains identified could be very different. Third, even though there are several cocrystals of tip peptides and partners, no highly conserved binding pocket has been revealed. This is surprising given that the acidic tip is almost invariant among several hundred SSB sequences aligned.1, 21 One would have expected that the binding site on the partner proteins would also be well conserved either in sequence, structure or both, as seen in many other cases.22, 23 Intuitively, this makes sense because if interfaces are functionally important, one would expect them to be conserved on both sides. Instead, results show that the tip binding sites do share electrostatic similarity and that the terminal phenylalanine residue of the tip is inserted into the base of the partner binding pocket. ¹⁵ Surprisingly, no binding site has been identified on the SSB protein itself, even though the C‐terminus can bind to the OB‐fold in an SSB monomer (not the intact tetramer), albeit transiently at pH 3.4 and, a model has been proposed invoking a role for the tip in SSB–SSB interactions.10, 18, 20, 24, 25 Therefore, while it is clear that peptides comprising the acidic tip can bind to partner proteins, it is unclear what the biological significance of these interactions is.

Recently, an alternative proposal was presented which suggested that instead of being the primary domain mediating protein–protein interactions, the tip has a regulatory function, employing long‐range electrostatic effects to regulate the C‐terminal tail of SSB. ²⁶ This regulatory function is afforded by the four aspartic acid residues in the tip (N‐DFDDDIPF‐C). These residues confer an isoelectric point of 3.7 on the C‐terminus, which contains only two other charged residues in this 62‐residue region of the protein.3, 4, 8

If the tip is not the primary protein–protein interaction domain, then another region of SSB must be responsible for mediating protein–protein interactions essential to interactome function. We proposed that this is the linker domain of SSB which binds to an OB‐fold in an adjacent partner. ³ The data showed that when the linker domain was deleted or replaced with that from another bacterial species, partner binding was lost. This was surprising given that a functional acidic tip was present in each SSB protein.

We proposed that the mechanism employed for binding in the linker/OB‐fold model is similar to that used by eukaryotic Src homology 3 (SH3) domains that bind peptide ligands containing one or more, PXXP motifs to mediate cellular processes.3, 23, 27, 28 This model makes sense for three reasons. First, OB‐folds and SH3 domains are structurally almost identical, with the folds aligning very well with an average root mean square deviation of less than 2.0 Å for the β‐strands. ²⁹ Second, SSB and many of its interactome partners each contain an OB‐fold.3, 9, 30, 31, 32, 33, 34 Third, the SSB linker contains three, well‐conserved conserved PXXP motifs. ²¹ The conservation of the PXXP motifs originally went unnoticed because of the presence of 1–4 sequence insertions in the linker domains that resulted in poor overall sequence conservation in this region among eubacterial SSB proteins. However, when sequence alignments are adjusted, the PXXP motifs are well conserved. ²¹

In the linker/OB‐fold model, one or more of the conserved PXXP motifs in the SSB linker bind to the structurally conserved OB‐fold in the adjacent partner. The partner is either another SSB tetramer or, an interactome member. OB‐fold binding could potentially stabilize the intrinsically disordered linker, with the OB‐fold functioning as a scaffold, similar to what has been observed for the binding of the intrinsically disordered PXXP‐domain of the c‐Src protein to its SH3 partner.35, 36 In support of the linker/OB‐fold model, deletion or mutation of the linker or replacement of Escherichia coli SSB linkers with that from other bacterial species eliminates SSB function.3, 37, 38, 39 Consequently, the linker/OB‐fold network of interactions could be responsible for regulating the SSB interactome as suggested previously. ³

To determine if the linker/OB‐fold model is correct, we focused on the C‐terminus of SSB and the mechanism of partner binding. First, the OB‐folds in three interactome partners were deleted and their ability to bind SSB in vivo assessed. Next, using RecG as a prototypical binding partner, 10 carefully selected residues in the DNA helicase OB‐fold were mutated and binding of these mutant proteins to SSB assessed in vivo. This was followed by an evaluation of the effects of mutation of the PXXP motifs in the linker on SSB function, by assessing both RecG binding in vivo and cooperative ssDNA‐binding in vitro. Circular dichroism of both peptides and intact proteins was then used to assess the proposed role of the acidic tip in affecting the C‐terminal domain structure. Finally, a combination of in vivo and in vitro binding assays was used to determine the role of the acidic tip in linker function as it pertains to SSB–SSB and SSB–RecG interactions.

Collectively, the results show that the intrinsically disordered linker is the primary domain in SSB that mediates protein–protein interactions. Single point mutations in linker PXXP motifs reduce SSB–SSB interactions and separately, eliminate RecG binding. On the other side of the interface, the deletion of partner OB‐folds in RecG, PriA, or RecO eliminates SSB interactions. This was confirmed using single point mutations in the RecG OB‐fold, which abolished SSB binding. Further support for the linker/OB‐fold model comes from the unexpected result showing that the acidic tip is not required for RecG binding. Finally, the data demonstrate that the acidic tip imparts significant structure to the C‐terminal domain that prevents the linker from binding to SSB OB‐folds while simultaneously enabling partner binding. When the tip is mutated, the structure of at least the C‐terminal 30 residues is impaired and the linker collapses back onto SSB where it binds to the OB‐fold. This prevents both ssDNA and interactome partner binding, thereby inactivating SSB and consequently, interactome function.

Consequently, the results herein support the linker/OB‐fold network of interactions as being responsible for regulating SSB interactome function. The findings are consistent with our previous work and that of others, demonstrating that the linker is the primary protein–protein interacting domain of SSB.3, 37, 40 Consequently, we propose that the SSB interactome should be designated as the first OB‐fold family of genome guardians in prokaryotes, similar to what is found in higher eukaryotes. ⁴¹

2. RESULTS

2.1. SSB binding requires a functional OB‐fold in the partner

To assess SSB–protein interactions, we used a well‐established in vivo binding assay.3, 11 Here, the two proteins being tested for binding are coexpressed to comparable levels and one partner is histidine‐tagged to facilitate complex isolation. Cells are lysed and the cleared cell lysate applied to a nickel column, which is then extensively washed and proteins eluted with an imidazole gradient. Using this assay system, coelution of SSB and a co‐expressed partner is observed in both high (600 mM) and low (100 mM) NaCl conditions and requires that proteins had bound in vivo, prior to cells being lysed. ¹¹ As the SSB–partner complexes do not contain DNA, coelution demonstrates that the two proteins directly bound to one another. Finally, the presence of the histidine tag on either SSB or the binding partner does not influence binding, producing results that are consistent with those obtained from our group and of others using different approaches.3, 12, 18, 33, 42 Using this assay system, in vivo complex formation between SSB and RecG; SSB and PriA; and SSB and RecO was demonstrated.3, 11 Complex formation between SSB and the above‐mentioned partners was eliminated when the linker domain of SSB was deleted or replaced with the corresponding Mycobacterial SSB sequence. ³

The linker/OB‐fold model requires a functional SSB linker and an OB‐fold be present in the binding partner. To test if partner OB‐folds are required for SSB binding, OB‐fold deletion mutants of RecG, PriA, and RecO were constructed (Figure S1a,b,d; OB‐folds highlighted in orange). OB‐fold deletions remove the N‐terminus of each protein. For RecG the remaining helicase domains express well and are active in vitro while for PriA, the C‐terminal domain of the protein is active in vivo.43, 44 Following construction, the OB‐fold deletion mutants were separately coexpressed with his‐SSB. Cleared cell lysates from each, dual‐expression culture were separately applied to nickel columns and proteins eluted with an imidazole gradient. Control experiments utilized full‐length partners and show that as before, full‐length RecG, PriA, and RecO coelute with SSB (Figure 1a 3, 11). In sharp contrast, the deletion of the OB‐fold results in a 25‐fold reduction in binding of RecG to SSB, whereas for PriA and RecO, binding was undetectable.

FIGURE 1.

Interactome partner oligonucleotide binding folds (OB‐fold) are critical for SSB binding. (a) The deletion of partner OB‐folds eliminates SSB binding. Analysis of sodium dodecyl sulfate‐polyacrylamide (SDS‐PAGE) gels of nickel‐column elution profiles of cleared cell lysates from hisSSB/ΔOB‐fold‐RecG; hisSSB/ΔOB‐fold‐PriA; hisSSB/ΔOB‐fold‐RecO. The ratio of hisSSB:partner was calculated as described in Section 4.2. (b) The structure of the RecG OB‐fold (blue) is shown with residues colored according to their importance in SSB binding. Red (high importance), mutation severely impairs binding; orange, moderate effects, and green (unimportant), the mutation has no effect on binding. Yellow oval, location of the DNA. The complete RecG structure bound to a model fork is presented in the Figure S1 to orient the reader. (c) A typical SDS‐PAGE gel showing the purification of WtRecG and his‐SSB. W, whole‐cell lysate; C, cleared cell lysate; F, flow‐through, M, marker and representative peak fractions from the elution profile. The red boxed region indicates the apex fractions used in analyses to calculate RecG to SSB ratios. (d) Key residues in the RecG OB‐fold are essential for SSB binding and these overlap the fork DNA binding site. Analysis of SDS‐PAGE gels of nickel column elution profiles of cleared cell lysates from hisSSB/RecG cultures is shown. For each mutant, expression was comparable to that of wild type (typical expression elution profiles for four mutants are shown in Figure S2). The ratio of SSB:RecG was determined as described in Section 4.2. In these experiments, only the three central peak fractions of the elution profile were used in the analysis. The ratio for wild type RecG was assigned a value of 1 with other ratios normalized to this value. Bars are colored to match the residues highlighted in (b). *, residues that make contact with the ssDNA ⁴⁷

To further refine the location of the SSB binding site on a partner, RecG was selected for further study as SSB binds to RecG both in vivo and in vitro and the interaction results in remodeling of the helicase.11, 12, 42 First, the location of 10 residues predicted to be involved in linker binding as determined from analysis of the published structure of Thermotoga maritima RecG and the molecular model of E. coli RecG were identified and mutated independently (Figures 1b and S1a,b9, 21, 45). Then, each mutant was separately coexpressed with his‐SSB, cleared cell lysates applied to nickel columns and proteins eluted with imidazole gradients.

As before, wild type RecG and his‐SSB co‐eluted (Figure 1c 3, 11, 46). In contrast, analysis of gels like those in Figure 1c revealed that the mutation of R95A reduces SSB binding 24‐fold; F75A and M80A reduce binding 12–14‐fold, respectively, and F97D has a ninefold effect (Figures 1d and S2). Notably, analysis of the gels in Figure S2 reveals that each mutant RecG expressed as well or better than his‐SSB. Further, the ratio of RecG to SSB in the cleared cell lysates is comparable to that of wild type (Figure 1c). Therefore, the failure to coelute is not due to differences in expression between wild type and mutant RecG proteins but is instead due to the inability of the mutant proteins to bind to SSB.

Importantly, the location in the helicase OB‐fold of the residues defective for SSB binding overlaps that of the binding site for the leading strand arm of the fork in RecG (Figure 1b, yellow oval; Figure S1a,b ³² ). Previous work has shown that F97A RecG is defective for DNA binding and fork unwinding. ⁴⁷ Thus, this residue plays a key role in binding to both DNA and SSB and this finding is consistent with previous work showing that SSB and fork DNA binding are mutually exclusive. ⁴²

Additional residues identified by modeling, Q84, S86, and I91 are also affected by mutation as SSB binding to these mutants is reduced threefold to sixfold (Figures 1d and S2). This suggests that this region of the OB‐fold, located almost on the opposite face of RecG is less important for linker binding (Figure 1b,d). In contrast, mutation to alanine of residues R78, K123, and M128, which are positioned above and below the predicted linker binding site, respectively, has no effect on binding (Figures 1b,d and S2). Collectively, these results show that the SSB binding site on RecG is the helicase OB‐fold and overlaps that of the nascent, leading strand arm of the fork as proposed previously (Figure S1b ⁹ ).

2.2. PXXP motifs are essential for RecG binding

A critical component of the OB‐fold linker model is the presence of three, well‐conserved PXXP motifs that are key to mediating protein–protein interactions (Figure 2a 3, 9, 21). This made sense because, in eukaryotes, these motifs are known to bind to SH3 domains, and SH3 domains and OB‐folds are structurally almost identical.29, 48

FIGURE 2.

The PXXP motifs are required for RecG binding. (a) The location of mutants in the primary amino acid sequence of SSB are shown. Amino acid numbering is from position 1, so that the Asn insertions are N142, 159, and 164, whereas for the proline to alanine substitutions, the residue position number remains unchanged. (b) Mutations in motifs I and II impair RecG binding. SDS‐PAGE gels comparing the apex fractions from each RecG/SSB purification. Wild type lanes are from the red boxed region in Figure 1c. The remaining apex fractions were subjected to electrophoresis in a single Criterion gel. This gel was performed after all protein purifications were complete. For N164, these lanes are not as clear due to the aggregation/precipitation that occurred during short term storage. (c) Analysis of the gel panels shown in Figure 1b. The ratio of SSB:RecG was determined as described in Section 4.2. Normalized SSB:RecG ratios are shown above each bar

To test whether these motifs in SSB are critical for function, mutants were again constructed (Figure 2a). First, each motif in SSB was interrupted by the insertion of an asparagine residue as this has been shown to impair PXXP–SH3 binding. ⁴⁹ Second, the proline residues at each end of motifs I and III were mutated to alanine either individually, or as pairs. The proline mutants are predicted to perturb the structure of the PXXP motif so that it cannot dock onto the OB‐fold of the partner.28, 50, 51 Each mutant was then tested for RecG binding in vivo as before.

Results show that interruption of the PXXP spacing of motif I by asparagine reduced binding to RecG 34‐fold and interruption of motif II reduced binding 24‐fold (Figure 2b,c). In contrast, the asparagine insertion into motif III did not affect binding. Mutation of the first proline residue in motif I reduced binding 27‐fold (P140A), whereas the corresponding mutation in motif III, P162A, showed a 2.5‐fold reduction in binding. Similarly, P143A reduced binding whereas P165A had only a small effect (not shown). Therefore, PXXP motifs I and II are critical for RecG binding and that motif III appears to be less important. As motifs II and III are near, it is conceivable that the reduction in binding observed for P162A is due to a distortion induced in motif II instead.

2.3. PXXP motifs are critical for ssDNA binding by SSB

To assess the role of the PXXP motifs in cooperative ssDNA binding where SSB–SSB interactions are important, each mutant protein was purified to near homogeneity. The purified SSB proteins all contain an N‐terminal histidine tag. The presence of the tag does not influence tetramer stability, even in chimeric tetramers where one or more subunits are fused to GFP. ⁵² Furthermore, the presence of the N‐terminal tag has no detectable effect on partner‐protein binding, as identical results are observed when the tag was present on the binding partner instead of SSB. ¹¹ Finally, the presence of the tag does not affect the intrinsic site‐size of SSB.3, 46, 52 Consequently, for the data presented below and in subsequent sections, the differences between wild type his‐SSB, and mutant his‐SSB proteins can be attributed to the presence of the mutation only.

First, the ability of each protein to bind ssDNA was assessed by monitoring the quenching of the intrinsic fluorescence of each protein on binding to ssDNA. ⁵² The controls for these assays are his‐SSB and his‐SSBΔC8 which have site sizes of 10 nt/monomer (Table 1 and Figure S3). Overall, the data show that each mutant retains the ability to bind ssDNA. However, the size site is affected by mutations in the linker domain in unexpected ways (Table 1). Interruption of motif I by asparagine reduced the site size slightly. Interruption of motifs II and III (N159 and N164 SSB proteins, respectively) increased site size to 13–14 nt/monomer (Table 1 and Figure S3c).

TABLE 1.

Site size of SSB proteins ^a

His‐SSB protein present b	Site size (nt)
Wild type	10 ± 0.2
SSBΔC8	10 ± 0.6
N142	8 ± 0.2
N159	13 ± 0.6
N164	14 ± 0.4
P140A	14 ± 0.5
P140A, P143A	8 ± 0.1
P162A	10 ± 0.1
P162A, P165A	6 ± 0.5
D4‐N4 c	15 ± 1.1
D4‐A4	35 ± 0.3
A179	10 ± 0.4
S179	9 ± 0.1

^aAssays were done as described in Section 4.2. Each site size value is the average of multiple separate experiments done in duplicate on separate days. Representative titrations are presented in Figure S2.

^bThe location of each mutation is shown in Figure 2a.

^cThe SSB D4K4 mutant was constructed but the protein is insoluble (Ding and Bianco, unpublished).

When the proline substitution mutants were analyzed, the single substitution in motif I (P140A) showed an increase in site size and this effect was reduced to 8 ± 0.1 by the double substitution. In contrast, for motif III, the single substitution did not affect site size whereas the double mutant (P162A and P165A), has a twofold lower site size. The effects of PXXP mutations on site size are consistent with the effects of mutations of the glycine‐rich region within the linker domain on site‐size. ³⁷ Collectively these data demonstrate that each mutant retains the ability to bind ssDNA and that the PXXP motifs play a role in ssDNA binding, possibly at the level of cooperativity between SSB tetramers, as explained below.

To test the effects of PXXP motif mutations on cooperative ssDNA binding, an agarose gel‐based assay was used. ¹⁰ Here, under conditions of low salt, M13 ssDNA is titrated with increasing amounts of SSB and the resulting protein‐DNA complexes subjected to electrophoresis. The binding of SSB to ssDNA retards the mobility of the DNA in the gel. For wild type SSB, a characteristic sigmoid‐shaped set of complexes are observed, reaching a maximum shift at R values between 0.8 and 1.0 (Figure 3a). This behavior is consistent with cooperative ssDNA binding by wild type. In contrast, for SSB N142, the shift in migration appears linear as protein concentration increases, indicating cooperativity has been compromised (Figure 3b). Analysis of the resulting gels shows that for Wt‐ and SSB N164, curves are sigmoid‐shaped, consistent with cooperative ssDNA binding (Figure 3c ¹⁰ ). In contrast, for SSB N142, the curve is almost linear, indicating that cooperative ssDNA binding is impaired. For N159, the ssDNA migration pattern in the gels was found to be intermediate between wild type and N142, consistent with a mild defect in cooperative binding (data not shown). The remaining PXXP mutants were characterized using the agarose gel analysis and show behavior comparable to wild type (data not shown). As mutations in motif I affect cooperative ssDNA binding, we conclude this motif is critical for this aspect of SSB function, whereas motif II is involved in binding but appears to be less important. Therefore, these data are consistent with a model involving interactions between the linker domain(s) of one SSB tetramer with the OB‐folds of another tetramer nearby as being critical for SSB–SSB interactions.

FIGURE 3.

The PXXP motifs are required for cooperative SSB–ssDNA binding. (a and b) Representative agarose gels showing the alteration in migration of M13 ssDNA as a function of increasing SSB concentration. Complexes were formed at different protein/DNA ratios: R ₃₅ = 35[SSB]_tot/[M13nts]_tot as described previously. ¹⁰ Assays, gel electrophoresis and imaging were done as described in Section 4.2. (c) Analysis of agarose gels quantifying the effects of PXXP mutations on SSB–ssDNA binding. Only wild type and two mutants are shown for clarity. Data were well approximated by the Hill equation (wild type and N164; Hill coefficients of 3 and 2, respectively) whereas N142 was not well approximated due to the lack of cooperative binding

2.4. Mutations in the SSB acidic tip have a profound effect on the C‐terminal tail

In each of the mutant SSB proteins studied above, a wild type acidic tip was present and single amino acid changes in the PXXP motifs impaired both SSB–SSB and SSB–RecG binding. Therefore, the results are consistent with the model that the linker mediates protein interactions between SSB and a partner.3, 9, 21 What then is the role of the acidic tip? The Dixon group proposed that instead of being involved in direct binding, long‐range electrostatic effects from the tip regulated the tail of SSB. ²⁶ It follows that mutations in the tip might alter the structure of the C‐terminal tail possibly rendering it non‐functional.

To test this, C‐terminal peptides were designed starting at amino acid 148, corresponding to the middle of the linker and, ending at the C‐terminal residue (Figure S4). We synthesized wild type, SSB113 and SSBΔC8 peptides. SSBΔC8 lacks the terminal eight residues comprising the acidic tip and this mutant does not bind partner proteins.11, 12, 53 SSB113 was selected as the in vitro phenotype of this mutant is in‐between that of wild type and ΔC8. ¹² The synthesized peptides were analyzed using circular dichroism spectroscopy (CD) as described. ⁵⁴

The CD spectrum for the wild type peptide exhibits negative peaks at 200 and 230 nm (Figure 4a). These peaks are consistent with the presence of either a partially unfolded poly‐l‐proline type II (PPII) helix or random coil, and α‐helical structure, respectively. ⁵⁵ Surprisingly, the introduction of a single P to S mutation at the penultimate position in SSB113 eliminates α‐helical content and causes a significant reduction in the negative peak at 200 nm. The deletion of the acidic tip further exacerbates these effects and eliminates virtually all secondary structure in the 23‐residue peptide. Therefore, tip mutations have a significant impact on the overall structure of at least the C‐terminal 31 aa, and could conceivably impact the structure of the intact protein as well.

FIGURE 4.

The acidic tip regulates the structure of the SSB C‐terminus. (a) CD spectra of peptides comprising residues 148 to the end of the protein. For clarity, the sequence of only the last 13 residues are shown (Wt and SSB113) or last 5 (SSBΔC8). Arrow, small negative peak at 230 nm. The location and the full sequence of peptides are shown in Figure S3. (b) CD spectra of wild type and SSBΔC8 proteins. (c) Analysis of the CD spectra in (b) by Bestsel ⁵⁶

To determine if this might be occurring, wild type and SSBΔC8 proteins were analyzed using CD in the same buffer as for peptides and the resulting spectra analyzed using Bestsel. ⁵⁶ Results show that for wild type there is a negative peak at 206 nm and a second region with negative ellipticity between 230 and 240 nm in the CD spectrum (Figure 4b). SSBΔC8 exhibits a similar spectrum although the minimum at 206 nm is reduced compared to wild type and ellipticity is greater than zero above 224 nm.

Secondary structure analysis of these spectra reveals that wild type is comprised of 11.7% turns, 40.4% β‐sheet, 44.6% other, and 3.4% α‐helical secondary structure (Figure 4c). The low α‐helical content is consistent with the available crystal structures showing the presence of a lone α‐helix in the N‐terminal domain and a small amount of α‐helical character that we detected in the wild type 31‐residue peptide (Figure 4a ⁷ ). In contrast, the analysis of the SSBΔC8 spectrum reveals no α‐helical content and a 3% increase in β‐sheet (Figure 4c). Collectively, the CD data show that the acidic tip imparts secondary structural features to the C‐terminal domain of SSB. The data also show that when the tip is mutated, there are significant changes to the overall structure of the C‐terminal 31 residues and these are imparted to the intact protein.

2.5. The acidic tip is not required for RecG binding

Previously, we demonstrated using modeling that an SH3 domain bound to a PXXP‐ligand, superimposed well with one OB‐fold of SSB. ²¹ In this model, the PXXP ligand bound to the OB‐fold in the same location as ssDNA. If PXXP/OB‐fold binding resulted in stable complex formation, SSB would be inactivated and this would be lethal to the cell. Therefore, a mechanism must be in place to prevent this from happening. This could be the long‐range electrostatic effects afforded by a functional acidic tip as proposed by Dixon. ²⁶ It follows then, that mutations in the tip might alter the structure of the C‐terminal tail so that it can bind to the OB‐fold via its PXXP motifs, thereby inactivating SSB.

To test this, additional acidic tip mutants were constructed (Figure 2a). First, the four invariant aspartic acid residues were changed to either alanine (D4A4) or asparagine (D4N4). This eliminates the net negative charge from the C‐terminus and increases the pI of the protein from 5.44 to 9.05, and that of the C‐terminal domain from 3.71 to 6.0. ⁵⁷ Second, crystallographic studies using short peptides show that the terminal residue of SSB, F178, fits into a binding pocket in the target protein. ¹⁵ To test this directly using intact proteins, serine and alanine were added separately after the terminal phenylalanine residue at position 178 creating the A‐ and S179 mutant proteins. These four mutants were purified to near homogeneity and tested for their ability to bind ssDNA

For the A‐ and S179 SSB mutants, site size was unaffected by the additional residue (Table 1). In contrast, the site size for the D4N4 protein was 15 nt/monomer, while that of the D4A4 mutant was 35 ± 0.3 nt/monomer (Table 1 and Figure S3d). The fourfold increase in site size for D4A4 relative to wild type indicates that when the aspartates are mutated to alanine, the absence of a net negative charge in the C‐terminus enables the linker to bind to the OB‐fold where it competes directly with ssDNA for binding. Consequently, a fourfold higher concentration of poly d(T) is required to fully saturate ssDNA‐binding for this protein.

Previous work has shown that binding of ssDNA to SSB results in a conformational change in the protein that increases the exposure of the C‐termini to proteolysis and enables them to bind interactome partners with greater affinity.6, 18 For the acidic tip mutants, the increase in site size comes from the competition between the mutant C‐termini and ssDNA for binding to SSB OB‐folds. Consequently, we predict that partner binding for acidic tip mutants would be inhibited in the absence of DNA as the mutant C‐termini are sequestered by the OB‐fold. It follows then, that when ssDNA is bound, a conformational change is induced in SSB so that the previously sequestered linkers become available for partner binding. To determine whether this might be occurring, the ability of the acidic tip mutants to bind RecG in the presence and absence of ssDNA was tested.

To do this, the histidine‐tagged versions of each SSB were bound to magnetic, nickel‐coated beads, washed to remove excess SSB and RecG bound (Figure 5a). Proteins were subsequently eluted by the addition of buffer containing 500 mM imidazole, aliquots subjected to electrophoresis in SDS‐PAGE gels and bands analyzed. As expected, and consistent with previous work, binding was observed for wild type SSB (Figure 5b ¹² ). In contrast, binding was reduced 35‐fold for SSBΔC8 and SSB D4A4, and 10‐fold for the A‐ and S179 mutants. Next, a sub‐stoichiometric amount of poly d(T) was added to SSB‐coated beads, washed, and RecG bound (Figure 5a). For wild type, RecG binding increased slightly by 5%. In contrast, for SSBΔC8, RecG binding increased fourfold to 7% and for the S‐ and A179 mutants, binding increased to 24% (Figure 5b). Finally, when stoichiometric poly d(T) was added, the amount of RecG bound by the ΔC8, S‐, and A179 SSB mutants was indistinguishable from that of wild type. For SSB D4A4, the addition of 150 μM poly d(T) did not affect RecG binding while the addition of 200 μM increased binding to only 60%. This follows because this concentration of ssDNA is sub‐stoichiometric for this mutant which has a site size of 35 nt/monomer and thus the wild type level of binding could only be achieved at 750 μM nt.

FIGURE 5.

The acidic tip of SSB is not required for RecG binding. (a) A schematic of the bead‐binding assay. Top, SSB proteins are bound to magnetic nickel beads, washed to remove unbound SSB and RecG added. Bottom, SSB proteins are bound to magnetic nickel beads, washed to remove unbound SSB and poly d(T) added. Beads were washed to remove unbound ssDNA and RecG bound. In both assays, the [NaCl] was 600 mM and elution of bound species achieved by the addition of buffer containing 500 mM imidazole. Eluted proteins were subjected to SDS‐PAGE. (b) Mutated and sequestered SSB C‐termini become available for partner binding in the presence of ssDNA. Analysis of magnetic bead binding experiments is shown. Assays and analyses are described in Section 4.2. Site sizes for ssDNA binding are shown below each protein

The increase in RecG binding observed in the presence of ssDNA is not due to the helicase binding to poly d(T) as the assay buffers contain 600 mM NaCl. At this high concentration of salt, RecG does not bind to ssDNA or poly d(T). ⁵⁸ This follows because the salt‐titration midpoint for poly d(T) is 38 mM and the salt concentration used in these binding and elution buffers is 16‐fold higher. Therefore, the only way RecG can be retained on the beads is by direct binding to SSB.

Furthermore, we interpret the increase in RecG binding in the presence of poly d(T) for the mutants is due to the availability of the linker domains brought about by ssDNA binding inducing a conformational change in SSB. When ssDNA is present, it binds to the OB‐folds with a greater affinity (K _a = 5.1 × 10⁷ M⁻¹), out‐competing the mutant C‐termini making them available for RecG binding. ⁶ As RecG binding is observed for each acidic tip mutant when linker domains are made available, these results show that in the intact SSB protein, the acidic tip is not essential for RecG binding.

2.6. In the absence of ssDNA, the tip minimizes the linker–SSB core interactions

To further assess the role of the acidic tip in linker function, fusions of SSB C‐termini to the green fluorescent protein (GFP) were used. The rationale behind this fusion design was to assess the binding ability of SSB C‐termini without the complications afforded by competition by the OB‐folds present in the tetramer which they emanate. GFP was selected as it is almost exclusively β‐strand and it was anticipated to have minimal interaction with the additional SSB components. ⁵⁹

Two his‐GFP fusions were constructed. The first had the wild type SSB C‐terminus (aa117 to 178) fused to the C‐terminus of GFP while the second had the ΔC8 C‐terminus attached (Figure 6a). As the binding partner, an untagged SSB core construct was made that expressed only residues 1–116; that is, the OB‐fold (MW = 12,115 Da). The wild type and ΔC8 GFP‐tail constructs were separately coexpressed with the SSB core and cleared cell lysates applied to nickel columns as before. We reasoned that if the Dixon model is correct then the tip would prevent the GFP‐Wt fusion from binding to OB‐fold of SSB protein. Instead, the SSB OB‐folds will only co‐elute with the GFP‐ΔC8 fusion protein.

FIGURE 6.

The acidic tip prevents the linker from binding to the SSB OB‐folds. (a) A schematic of the assay showing each GFP‐fusion and the SSB core tetramer. Top, his‐GFP‐Wt linker with the tip represented by the red rectangle; bottom, his‐GFP‐ΔC8 linker. (b) The acidic tip prevents the SSB C‐terminus from binding to the core. An SDS‐PAGE gel showing three different amounts of the apex fractions from nickel column elution profiles of cleared cell lysates coexpressing his‐GFP‐Wt tail/SSB core and separately, his‐GFP‐ΔC8 tail/SSB core. In these gels, the GFP‐fusions migrate at 36 kDa and the SSB core migrates at 13 kDa (the predicted MW is 12.1 kDa). In lanes 2–4, the ratio of fusion to the core, when corrected for Coomassie staining, is 3

SDS‐PAGE analysis shows that the SSB core co‐eluted with the ΔC8 protein fusion only and the ratio of fusion to the core in the resulting SDS‐PAGE gel is 3 (Figure 6b). Coelution was not detectable for the wild type fusion protein. Control experiments show that the SSB core tetramer does not co‐elute with the his‐GFP‐his protein (data not shown). Therefore, the acidic tip prevents the SSB C‐terminus from binding to the SSB OB‐fold in the absence of ssDNA.

3. DISCUSSION

The primary conclusion of this study is that the primary protein–protein interaction domain of SSB is the intrinsically disordered linker. One or more of the conserved PXXP motifs in the linker bind to the OB‐fold present in the interactome partners. This conclusion has important implications for SSB interactome function as discussed below. The secondary conclusion is that the acidic tip is not the primary protein–protein interaction domain of the intact SSB protein as previously thought. Surprisingly it is not required for RecG binding at all. Instead, the tip has two roles. First, and as proposed by Dixon, the acidic tip uses long‐range electrostatic effects to regulate the structure of the C‐terminus maintaining it in a configuration that is competent for function. ²⁶ Second, we propose that the tip is a secondary binding site interacting with partner proteins at a site distant from the OB‐fold.

The role of the intrinsically disordered linker or linker, in SSB function, has become increasingly clear over the past few years. First, when the linker is partially or completely deleted, binding to RecG and RecO is lost even when a wild type acidic tip is present. ³ Second, when the linker of E. coli SSB is swapped out with that from another bacterial species, physical and functional interactions between SSB and RecG, and separately between SSB and PriA are lost.3, 37 Furthermore, when the E. coli SSB liker is replaced with that of Mycobacterium tuberculosis SSB, this produces non‐functional proteins in vivo.38, 39 These studies show the importance of the linker and this is not limited to E. coli as linker‐partner interactions were demonstrated using proteins from M. tuberculosis, Klebsiella pneumoniae, Salmonella enterica, and Pseudomonas aeruginosa.

Additional support for the role of the linker in partner binding comes from a recent study using Alkylation protein B (AlkB). ⁴⁰ Here, the AlkB binding site on SSB was mapped to linker residues 152–169, a region that spans PXXP motifs II and III. Finally, the work in this study demonstrates that binding between SSB and a partner requires one, or more, of the three conserved PXXP motifs in the SSB linker and a functional OB‐fold in the partner (Figures 1, 2, 3). Consistent, when the PXXP motifs are mutated, SSB function is significantly impaired. This is observed as diminished cooperative ssDNA binding or a failure to bind to RecG (Figures 2 and 3). On the opposite side of the interface, when partner OB‐folds are deleted or, mutated at key locations, binding to SSB is abolished (Figures 1 and 2). Results show that a single amino acid change in the OB‐fold in the RecG helicase reduces SSB binding 25‐fold (Figure 1d). The final piece of the puzzle demonstrating the linker domain binds to the OB‐fold in a partner comes from assays using GFP‐fusions. Binding between the linker fusion and the core of SSB required the linker only and was inhibited by the acidic tip (Figure 6). Therefore, the linker/OB‐fold model accurately describes how SSB binds to both itself and partner proteins.

Consequently, we propose that the acidic tip of SSB is not the primary domain of the intact protein responsible for mediating protein–protein interactions as we and others had previously thought.1, 11, 12 This was demonstrated using nickel‐bead‐binding assays and the GFP‐fusions. In the fusion assay, binding between GFP‐ΔC8 and the SSB core was observed. Surprisingly, binding was not detected using the GFP‐WT SSBCt construct. In the nickel‐bead assays, SSB proteins with mutant acidic tips bound to RecG in the presence of ssDNA (Figure 5). If the acidic tip was required for binding, these mutant SSB proteins would not bind RecG, regardless of the presence of nucleotide. Enhanced binding observed in the presence of poly d(T) is the result of a conformational change in SSB brought about by ssDNA binding.6, 18 Work from the Lohman group demonstrated that the affinity of SSB for the chi subunit of DNA polymerase and separately for PriA, increased when SSB was pre‐bound to ssDNA. ¹⁸ In the work described herein, ssDNA‐binding to the acidic tip mutants likely displaces the mutant C‐termini from the OB‐fold enabling the linker domains to bind RecG. This is evident for SSBD4A4, which requires four‐fold higher concentrations of poly d(T) to saturate binding; is defective for RecG binding in the absence of DNA and binding is partially rescued by the addition of sub‐stoichiometric amounts of poly d(T).

At present, there is a large volume of data from studies using acidic tip peptides and intact partner proteins.13, 15, 16, 17, 18, 20, 33, 60 These studies concluded that the tip is the primary protein–protein interaction domain where it mediates binding to a pocket in the partner, with the terminal phenylalanine residue inserted into the base of the pocket. If the acidic tip‐pocket binding is essential to mediating protein–protein interactions then the addition of an extra residue should impair partner binding both in the absence, and presence, of ssDNA. This was directly tested in bead‐binding assays using the SSB A‐ and S179 mutants. The results demonstrate wild type levels of helicase binding for each mutant in the presence of stoichiometric ssDNA where SSB C‐termini are available for partner binding (Figure 5b). The inability of the mutants to bind RecG in the absence of ssDNA likely comes from structural changes in the C‐termini brought about by the additional alanine or serine residues. This is currently being tested.

Similar to the tip mutants, single amino acid changes in the PXXP motifs reduce RecG binding in the absence of ssDNA by 24–34‐fold (Figure 2). However, for each of these mutants, a wild type acidic tip was present. Collectively these data are consistent with a model that the acidic tip is not required for partner binding. If the tip is essential, then in each of the PXXP mutants RecG binding should be unaffected. Consequently, the protein–protein interface that mediates SSB interactome function involves the PXXP motifs in the SSB linker on one side and a functional OB‐fold in the partner on the other side. This makes sense because, for critical interfaces, both sides of the interface should be conserved.22, 23, 61

Accordingly, we propose that instead of being the primary protein–protein interaction domain, the acidic tip has two different but important roles in SSB function. First, the tip regulates the structure of the SSB C‐terminus as proposed previously and supported by our CD data (Figure 4 ²⁶ ). One important consequence of the structure regulation afforded by the tip is to prevent the C‐terminus from binding the OB‐folds of SSB. It was not surprising then, that when the aspartic acid residues are changed to alanine, the effects on SSBD4A4 were dramatic. First, electrostatic charges are lost and the pI of the protein increases from 5.44 to 9.05, while that of the C‐terminal domain increases from 3.71 to 6.0. As a result, the PXXP motifs in the mutant C‐termini are now able to bind to the SSB OB‐fold where they compete with ssDNA for binding as predicted by modeling. ²¹ This is observed as a requirement for a fourfold higher ssDNA concentration to fully occlude the OB‐folds of the SSB D4A4 protein (Table 1; site size is 35 nt/monomer vs. 10 for wild type). Furthermore, the sequestered C‐termini cannot bind RecG as they are already bound to an OB‐fold. This was observed as a 35‐fold reduction in RecG binding (Figure 5b). Inhibition was partially alleviated in the presence of sub‐stoichiometric poly d(T) which binds to OB‐folds with greater affinity than the mutant C‐termini. The effects observed in these assays are not due to the acidic tip competing with ssDNA for binding as this likely does not occur. ⁶² Therefore, the data support the long‐range electrostatic effect model of Dixon wherein a functional acidic tip regulates the structure and function of the C‐terminus essentially keeping it away from the SSB OB‐folds thereby making it available for partner binding. ²⁶

The regulatory role for the tip is critical because if the linkers were to bind the SSB OB‐folds forming a stable complex, the interactome would be inactivated. The reason for this is two‐fold. First, the SSB OB‐folds would be occluded so ssDNA‐binding is impossible. Second, the C‐termini would be sequestered so that partner binding is not feasible. Therefore, a wild type acidic tip ensures the linker is maintained in a functional configuration that is competent to bind to interactome partners or, to other SSB tetramers at the appropriate time. Furthermore, a functional acidic tip also ensures that the OB‐folds are clear for ssDNA binding, guaranteeing that exposed single strands of DNA are rapidly protected.

A second role for the acidic tip of SSB may be as a secondary partner binding site. The primary interface is formed first when the linker of one SSB subunit binds to the partner OB‐fold. Then, the tip from another subunit of the same tetramer binds to the secondary binding site in the partner, which is distant from the OB‐fold binding site. Consistent, in the available crystal structures, the tip binding sites are distal to the OB‐fold (Figure S1c–e). If this model is correct, then the stoichiometry of binding of an SSB tetramer to a partner binding should be 2. This was demonstrated for RecG. ¹²

The linker/OB‐fold model of interactome control requires that functional OB‐folds be present in SSB and its partners. To date, the clear designation of OB‐folds in interactome members has only been made in PriB, RecO, and SSB.33, 63, 64 For RecJ, it was not initially identified but in a subsequent study Cheng et al., correctly identified the presence of an OB‐fold.30, 65 For Exonuclease I, a region encompassing the OB‐fold was identified as an “extended SH3 domain” of unknown function. ⁶⁶ In RecG this region was initially termed a Greek Key motif located in the wedge domain of the helicase. ³² In the first structure of the N‐terminal domain of PriA, the OB‐fold was not identified and was instead listed as a unique fold. ⁶⁷ In a subsequent study of the apo‐form of the enzyme, the OB‐fold was also not identified. ¹³ However, in the most recent structure of PriA bound to DNA, a well‐formed OB‐fold is visible and interacts with the DNA. ⁶⁸ Therefore, OB‐folds are discerned in seven interactome partners.

For other partners, such as the chi‐psi complex of the clamp loader, DnaG, and DNA Polymerase β, a clearly defined OB‐fold was not highlighted in the available structures, even though upon closer examination the fold is present (Bianco, unpublished69, 70, 71). It is conceivable that a more readily discernable OB‐fold in this latter group of partners proteins could occur once the linker of SSB binds. At present, OB‐folds are present in as many as 12 of 18 interactome partners, with this number likely to increase as more proteins are analyzed. The OB‐fold plays a key role in SSB–partner binding as when it is deleted or mutated at key residues, SSB binding is significantly impaired (Figure 1).

How does a single SSB interact with distinct binding partners? The data demonstrate that PXXP motifs I and II are both important for RecG binding and motif I (and to a lesser extent motif II) are important for SSB–SSB interactions (Figures 2 and 3). In contrast, for AlkB the region spanning motifs II and III is required for binding. ⁴⁰ This suggests that each motif has a distinct binding role and this may be a result of the sequence context for each motif. The unique context enables classification of the SSB PXXP motifs into different classes similar to that observed for those that bind SH3 domains.28, 72 SSB motifs II and III more closely resemble Class I (+xxPXXP; for SSB—QxRPxxP and QxxPxxP, respectively), whereas SSB motif I represents a unique class (WGQPxxPQG). As each motif is unique, this enables the single SSB protein to differentially bind to both itself and the functionally distinct members of the interactome. Further specificity could be provided by the unique sequence composition of each structurally conserved OB‐fold. This is analogous to what is observed for PXXP–SH3 domain interactions.29, 73, 74, 75, 76

In summary, the results herein are consistent with the linker/OB‐fold model being the primary protein–protein interface of the SSB interactome, which functions in a manner analogous to the SH3 domain family of proteins.28, 51, 75, 77 Here, the PXXP motifs within the SSB linker domain and, OB‐folds present in both SSB and interactome partners, form dynamic protein–protein interfaces at critical times. These linker/OB‐fold interactions regulate SSB interactome function, ensuring that unwound ssDNA is protected, DNA replication and repair proteins are loaded onto the DNA at the right time and genome stability is maintained.1, 2

It is known that the maintenance of genomic stability relies on the coordinated actions of the OB‐fold family of genome guardians. In eukaryotic cells, many of the proteins functioning in DNA replication, telomere homeostasis, activation of the DNA‐damage checkpoint and DNA repair contain OB‐folds.78, 79 Two key examples are Replication protein A and BRCA2. ⁸⁰ Not surprisingly, these OB‐folds play critical roles in DNA binding, protein complex assembly, and are involved in regulating complex protein–protein interactions. ⁴¹ A similar set of functions occurs in prokaryotes making the SSB interactome the first prokaryotic OB‐fold family of genome guardians to be identified. However, it is to our knowledge, the first OB‐fold family to demonstrate competitive protein and nucleic acid binding as a means of regulating function. Recently, an additional level of interactome regulation has been shown. Here, SSB binding to phospholipids of the inner membrane of E. coli sequesters free protein and which is released following DNA damage. ⁸¹ Phospholipid binding involves the OB‐fold as well, further contributing to the importance of the regulation of this domain and its interactions in SSB interactome function.

4. MATERIALS AND METHODS

4.1. Chemicals and reagents

All chemicals were reagent grade and were made up in Nanopure water and passed through 0.2 μm pore size filters. Isopropyl β‐d‐1‐thiogalactopyranoside (IPTG), sodium chloride (NaCl), calcium chloride (CaCl₂) and sodium phosphate dibasic (Na₂HPO₄) were from Fisher Scientific (NJ). Coomassie Brilliant Blue R‐250, Tris‐base, potassium chloride (KCl), sodium dodecyl sulfate (SDS), and acetic acid were from Amresco (OH). Ethylenediaminetetraacetic acid (EDTA), potassium acetate, magnesium chloride (MgCl₂), glycerol, potassium phosphate monobasic (KH₂PO₄), sodium phosphate monobasic (NaH₂PO₄), and nickel sulfate (NiSO₄[H₂O]₆) were from J.T. Baker (NJ). Imidazole, adenosine diphosphate (ADP), dithiothreitol (DTT) and magnesium acetate (MgOAc) were from Acros Organics (NJ). The infusion kit was from Clontech (CA) and the Quicksite Mutagenesis kit was from Agilent (CA). Phenylmethylsulfonyl fluoride (PMSF) was from Omnipur (NJ). HisTrap FF crude columns and His Mag Sepharose Ni Ni‐NTA Magnetic Agarose Beads were from GE Healthcare Life Sciences (NJ). Proteinase K was purchased from Roche (IN). Hydrochloric acid (HCl) was from Macron Chemical (PA). Potassium phosphate dibasic (K₂HPO₄) was from BDH Chemicals (PA). Ammonium sulfate was purchased from MP Biomedicals, LLC (OH). Poly d(T) was purchased from the Midland Certified Reagent Company (TX).

Oligonucleotides were purchased from Integrated DNA Technologies (IA). RecG point mutants were constructed by Genscript (NJ). Deoxyribonucleic acid−cellulose single‐stranded from calf thymus DNA (ssDNA cellulose resin) was from Sigma Aldrich (MO). Φx174 Virion DNA and restriction enzymes were purchased from New England BioLabs (MA).

Peptides were purchased from either Genscript (NJ) or Hangzhou Peptide Biochem Co. Ltd. (Hangzhou, China). The purity of peptides was >90%. Peptide sequences are presented in Figure S3. They were stored at −20°C in dried form. On the same day that CD measurements were done, 1 mg amounts were measured out and dissolved in CD buffer (100 mM NaHCO₃ containing 100 mM NaSO₄), and concentration determined spectrophotometrically at 257.5 nm using ε = 195 M⁻¹ cm⁻¹ for each phenylalanine present. ⁸²

4.2. Methods

4.2.1. Construction of OB‐fold deletion mutants of RecG, PriA, and RecO

The plasmids pGS772 (RecG), pet15b‐hisPriA, pet15b‐hisRecO were separately mutated to remove the oligonucleotide‐oligosaccharide binding folds.3, 11, 83 Deletion reactions were done using the Infusion kit with positive clones verified by DNA sequencing performed by Genscript (NJ).

4.2.2. Construction of GFP fusions

The GFP gene from pQBI25 (Q‐Biogene) was sub‐cloned into pET28 using the Infusion kit to create his‐GFP‐his. Positive clones were identified by restriction enzyme mapping, followed by DNA sequencing. Next, the C‐terminal domains of wild type (residues 114–178) and separately, SSBΔC8 (residues 114–170) were cloned using the Infusion Kit into the XhoI site placed between the C‐terminus of GFP and the second histidine tag. This strategy produced his‐GFP‐Wt‐linker and separately, his‐GFP‐ΔC8‐linker. Clones were verified by DNA sequencing.

4.2.3. Construction of SSB mutants

A pET28a⁺ plasmid containing the E. coli ssb gene was used as the initial clone. As the TAA stop codon functioned inefficiently, it was converted to TAG using the Quicksite Mutagenesis Kit (Agilent). Resulting clones were confirmed by DNA sequencing. Next, the modified ssb gene was cloned into pET28 in‐frame with the N‐terminal histidine tag to create his‐SSB. The wild type and his‐SSB plasmids were then used in separate mutagenesis reactions to (a) insert an asparagine residue into each of the PXXP motifs to produce PXNXP; (b) mutate the proline residues of each motif to alanine; (c) construct the double proline to alanine mutants (PXXP to AXXA); (d) mutate the acidic tip, or (e) add single residues to the C‐terminus. Mutagenesis was done using the Quicksite kit with positive clones verified by DNA sequencing.

SSB core constructs were created using the Infusion kit. The core is comprised of amino acids 1–116. This region was cloned into pET28 to create his‐core or pET15 to create an untagged SSB core. Clones were verified by restriction mapping and confirmed by DNA sequencing.

4.2.4. Wild type SSB protein

It was purified from strain K12ΔH1Δtrp as described. ⁸⁴ Histidine tagged SSBΔC8 was purified as described previously. ⁵² The concentration of the purified proteins was determined at 280 nm using ε = 30,000 M⁻¹ cm⁻¹. Protein preparations do not contain contaminating DNA or nuclease activity.

4.2.5. Histidine‐tagged mutant SSB proteins

Cultures were lysed and the cleared cell lysate was subjected to affinity chromatography using HisTrap FF crude columns, equilibrated in binding buffer (20 mM sodium phosphate, pH 7.4, 600 mM NaCl, 30 mM imidazole). The column was sequentially washed with 400 mL binding buffer, 350 mL binding buffer with 0.2% NP40 and 250 mL binding buffer only. A linear gradient of 30–500 mM of Imidazole was used to elute bound proteins from the column. Proteins were assessed by SDS‐PAGE. Following electrophoresis, gels were stained with Coomassie Brilliant Blue, de‐stained and photographed. Protein fractions were pooled, precipitated in 70% ammonium sulfate, resuspended and dialyzed overnight against storage buffer (20 mM Tris–HCl pH 8.0, 1 mM EDTA, 500 mM NaCl and 50% (vol/vol) glycerol). The concentration of the purified mutant proteins was determined at 280 nm using ε = 30,000 M⁻¹ cm⁻¹. Protein preparations do not contain contaminating DNA or nuclease activity.

4.2.6. Purification of wild type RecG protein

It was done as described previously.12, 58 The purified protein is free of contaminating DNA and nuclease activity.

4.2.7. Dual plasmid expression and purification of protein complexes

These were done as described previously.3, 11

4.2.8. Site size determination of SSB proteins

The binding of SSB to poly d(T) was determined by monitoring the quenching of the intrinsic fluorescence of SSB that occurs on binding to ssDNA, as described. ⁵² Here, reactions were done at 25°C in a 500 μL volume. Reactions contained 10 mM Tris–HCl (pH 8.0), 1 mM DTT and 10 mM NaCl and 1 μM SSB, in monomer. In these assays, SSB was titrated with increasing amounts of poly d(T) until the maximum amount of intrinsic fluorescence was quenched. On each day, assays were done in parallel in separate cuvettes. Repeats were performed on separate days, also in parallel.

4.2.9. Circular dichroism measurements

These were done in a Jasco J‐710 CD spectrometer at room temperature in a 0.1 cm cuvette and a volume of 500 μL under nitrogen. Sample scanning (continuous) was done using the following parameters: standard sensitivity, 0.5 nm data pitch, at a speed of 20 nm/min, with a response time of 1 s, and a bandwidth of 1 nm. Twenty scans per sample were accumulated. Using these scan parameters, the high‐tension voltage at 190 nm was ≤600 V.

Peptides were prepared for CD as described above. Proteins are typically stored in buffers containing 50% glycerol and chloride ions which interfere with CD measurements. Therefore, the day before measurements were taken, protein samples were thawed and dialyzed overnight against a 100–200‐fold excess of CD buffer at 4°C with stirring. The following day, samples were subjected to centrifugation at 10,000g for 10 min to remove aggregates and protein concentration determined spectrophotometrically using the extinction coefficient for each protein. The resulting “stock” protein concentrations were diluted to the working concentration of 0.05 mg/mL immediately before scanning. Diluted samples were used only once and discarded. When measurements were repeated, fresh protein samples were thawed, dialyzed overnight and concentration determined.

4.2.10. CD data analysis

The resulting data sets were converted from millidegrees (machine units) into Mean residue ellipticity using the formulas below:

$${\left[\theta \right]}_{\mathrm{mrw},\lambda }=\frac{\mathrm{MRW}\bullet {\theta }_{\lambda }}{10\bullet d\bullet c}$$

and

$$\mathrm{MRW}=\frac{\mathrm{MW}}{n-1}$$

where θ_λ is the observed ellipticity (in degrees), d is the pathlength (0.1 cm), c is the concentration in g/mL (0.00005 g/mL) and n is the number of amino acids in the protein or peptide being scanned. Further analysis of spectra for structure determination was done using Bestsel. ⁵⁶

4.2.11. Cooperative ssDNA binding by SSB

Agarose gels were used to assess the cooperative binding of wild type and mutant SSB proteins to M13 ssDNA. Binding assays (30 μL) contained TE buffer, 20 mM NaCl (final), 30 μM nt of ssDNA and SSB protein. SSB proteins were diluted with TE buffer immediately before use and added to the reaction mix on ice. The binding reactions were initiated by the addition of ssDNA, with reaction mixes incubated at 4°C overnight. The next day, reactions mixed with loading buffer (for the recipe see Reference 10) and subjected to electrophoresis in 0.7% agarose gels in TAE buffer at 350 Vhr. Following electrophoresis, gels were rinsed in water and stained with ethidium bromide in water for 60 min. Then, stained gels were rinsed in water to remove the free dye and imaged. Assays were done in duplicate on the same day and repeated the next day to verify results.

4.2.12. Nickel magnetic bead binding

Reactions were done at 4°C and contained 10 μL of beads that had been washed three times with 500 μL of equilibration buffer (20 mM sodium phosphate; 600 mM NaCl; 30 mM Imidazole; pH 7.4). After each wash, the tube containing the beads was placed on a magnetic rack and buffer removed by aspiration with a pipettor. Immediately, a 30 μL solution of his‐SSB (20 μM final concentration) was added and the solution mixed by inverting the tube with slow end‐over‐end mixing for 30 min at 4°C. The tube was then placed on the magnetic rack and the protein solution aspirated as before and stored at 4°C (Wash I). Beads were then washed three times using equilibration buffer. Once the last wash solution had been removed, a 30 μL solution of RecG (20 μM final concentration) was added and the solution mixed to permit binding by inverting the tube with slow end‐over‐end mixing for 30 min at 4°C. The tube was again placed on the magnetic rack, the solution aspirated (flow‐through) and beads washed three times using equilibration buffer. Once the last wash was removed, 30 μL of elution buffer (20 mM sodium phosphate; 600 mM NaCl; 500 mM imidazole; pH 7.4) was added, the beads resuspended and mixed as before for 10 min at 4°C. The tube was placed on the magnetic rack and all of the solution containing eluted proteins removed and stored at 4°C (eluate). In reactions where poly d(T) was present, it was added to SSB‐coated beads after the first three wash steps to remove free SSB had been completed. Once assays were complete, aliquots from Wash I, flow‐through and eluate were mixed with SDS‐PAGE sample buffer and subjected to electrophoresis in 12% SDS‐PAGE gels. Following electrophoresis, gels were stained with Coomassie and photographed.

4.2.13. Gel photography and analysis

All gels were photographed using a Bio‐Rad ChemiDoc XRS+ imaging system. SDS‐PAGE and ethidium bromide‐stained gels were analyzed using Image Lab, version 6.0.0 build 25, standard edition (Bio‐Rad). For SDS‐PAGE, the area under each peak was determined from the trace of each lane. This value was then divided by the molecular weight of each protein to correct for the different amounts of Coomassie dye being present in bands, assuming each protein stained equally well with Coomassie. The ratio of protein A (RecG, PriA, or RecO): protein B (SSB) was then determined. Each coelution or bead‐binding experiment was repeated 2–5 times on separate days, with two to three lanes per gel analyzed and combined to yield the resulting graphs. For images of stained, agarose gels showing SSB–ssDNA binding, the position of migration of each band was measured relative to the well in each lane using Image Lab. Then, the position of migration was expressed relative to the migration of the DNA only, control lane, and graphed as a function of R = n × [SSB monomer]/([ssDNA] in nt), where n = site size (Table 1).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Wenfei Ding: Methodology. Hui Yin Tan: Formal analysis; methodology. Jia Xiang Zhang: Formal analysis; investigation. Luke Wilczek: Formal analysis; investigation. Karin Hsieh: Investigation. Jeffrey Mulkin: Investigation. PIERO Bianco: Conceptualization; formal analysis; methodology; writing‐review and editing.

Supporting information

Data S1. Supporting Information.

Ding W, Tan HY, Zhang JX, et al. The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function. Protein Science. 2020;29:1211–1227. 10.1002/pro.3855