Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein
© Biswas-Fiss et al.; licensee BioMed Central Ltd. 2012
Received: 19 January 2012
Accepted: 21 May 2012
Published: 14 June 2012
Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.
We have cloned and purified SSB from Bacillus anthracis (SSBBA). In the absence of DNA, at concentrations ≤100 μg/ml, SSBBA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSBBA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)70 binding suggested that SSBBA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.
Unlike other prokaryotic SSB proteins, SSBBA from Bacillus anthracis appeared to be monomeric at concentrations ≤100 μg/ml as determined by SE-HPLC. SSBBA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSBBA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSBBA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.
KeywordsSingle-stranded DNA binding protein (SSB) DNA replication Fluorescence anisotropy ssDNA binding Protein-DNA complex
Nearly all cellular nucleic acid transactions, including DNA replication, repair and recombination require the activity of a single stranded DNA binding protein (SSB) [1–7]. SSB proteins and are found throughout nature and their functional importance is underscored by their presence in prokaryotes, archaea, and eukaryotes including mammals . Among its multifaceted roles, upon binding to ssDNA, SSB prevents the reformation of duplex DNA making it possible for other enzymes such as DNA polymerase to use ssDNA as substrate. In addition, the binding of SSB-type proteins protects the ssDNA molecules from attack by intracellular nucleases. Although not possessing intrinsic enzymatic activity in and of themselves, SSB proteins are known to influence the activities of many enzymes as well as to organize the multi-protein complexes required for processes such as DNA replication, recombination and DNA repair [8–11].
The function of SSB during DNA replication has been extensively studied in E. coli, which serves as the prototypical model system for prokaryotes and eukaryotes alike. In E. coli, the large nucleoprotein replication initiation complex is stabilized by single stranded DNA binding protein, following which DNA is unwound by the DnaB helicase protein. Efficient DNA unwinding activity of DnaB protein in progression of the replication fork in E. coli is strongly dependent on the continued action of a cognate SSB [12, 13]. SSB works in concert with DnaB helicase, DNA primase, and DNA polymerase III holoenzyme during E. coli DNA replication [5, 9, 12, 14, 15]. Phage λ DNA replication requires the participation of host E. coli SSB as well [16, 17]. In archaea and eukaryotes, its functional homolog, Replication Protein A (RPA), carries out the role of organizing and stabilizing the replisome during DNA replication [1, 3, 10, 18–21].
Vital to its function in DNA metabolism is the structure of SSB. In the Gram-negative bacteria, SSB is homotetrameric, with each monomer contributing a single ssDNA-binding domain to the functional form. The eukaryotic RPA is composed of three subunits (RPA70, RPA32, and RPA14) and functions as a heterotrimer through the use of four ssDNA-binding domains [2, 3, 18].
Unlike E. coli SSB, single stranded DNA binding protein from bacteriophage T4, the gene 32 protein, is a monomer. T4 gene 32 protein can form multimers at high concentration induced by high salt and high pH . Kim and Richardson demonstrated that the bacteriophage T7 SSB, the gene 2.5 protein, is a dimer . The T7 gene 2.5 SSB appears to bind DNA as a dimer. The ssDNA binding affinities of both T4 and T7 SSBs are lower than that observed with E. coli SSB. Despite these differences, ssDNA binding of SSB proteins using OB fold-domains (oligosaccharide/oligonucleotide binding domains) appears to be universal throughout all systems described to date .
The E. coli SSB is highly cooperative in ssDNA binding that is influenced by salt concentration [24, 25]. Recent studies indicate that SSB has at least two distinct modes of ssDNA binding . The binding is modulated by monovalent salts. At very low salt concentration (<10 mM NaCl), SSB binds ssDNA using two of its four subunits in a highly cooperative manner and occludes only 35 nucleotides [(SSB)35 mode]. On the other hand, at higher salt concentrations (>200 mM NaCl), it binds to ssDNA using all four subunits and protects ~65 nucleotides [(SSB)65 mode]. It is not clear how the ssDNA binding is altered between 10 and 200 mM NaCl. Higher-order forms of SSB in ssDNA bound states, based on high resolution electron microscopic studies of SSB-ssDNA complex, have also been reported . Chrysogelos and Griffith discovered that repeated freezing-thawing of E. coli SSB leads to the formation of unique strings of tetramers .
Gram-positive bacterial protein sequences do not form a monophyletic group, but are intermixed with plasmid and phage sequences [29, 30]. Gene organization in these organisms can differ from that observed in Gram-negative E. coli and these organisms may contain multiple paralogues [31, 32]. Sequence analysis indicated that Gram-positive SSBs have a highly conserved nearly-identical (>90% identity) N-terminal ssDNA binding as well as monomer-monomer interaction domains but they differ to some extent from the Gram-negative SSBs. We have investigated the structure and ssDNA binding of a Gram-positive bacillus SSB (SSBBA) in order to understand its mechanism of action of SSBs in these organisms. We present here a report of a Gram-positive SSB that utilizes a novel structural mechanism for protein-DNA interaction using a transient tetramer formation.
The single-stranded DNA binding protein ORF of B. anthracis (BAS5326) was identified by BLAST search of the annotated sequenced genome of B. anthracis Stern strain [33, 34]. The ORF encodes a polypeptide of 172 amino acid residues with a predicted molecular weight of 19.2 kDa.
Sequence analysis of SSBBA
Purification of SSBBA
To test the biological activity of SSBBA we measured its ability to stimulate its cognate DnaB, DnaBBA, using a FRET based DNA helicase assay. It was based on the ability of SSBBA to stimulate DNA unwinding activity of its cognate DNA helicase, DnaBBA (Figure 2). In the absence of SSBBA, DNA unwinding by DnaBBA was limited (Figure 2B) and was greatly stimulated in the presence of SSBBA (Figure 2C). The stimulation of the DNA helicase activity of DnaBBA in the presence of the purified SSBBA was >10 fold which was as expected for the cognate SSB .
Mechanism of ssDNA binding by SSBBA
Thermodynamics of ssDNA binding
The dissociation constants obtained at varying temperatures were used to evaluate the thermodynamic properties of DNA binding. We have plotted the dissociation constants using the Van’t Hoff equation, lnKD = −ΔH°/RT, where ΔH° is the enthalpy change and T and R are the temperature and gas constant respectively, with the dissociation constants derived from 20, 25, 30, and 37°C (Figure 5B). The plot is linear for temperatures 20°C to 37°C and it diverges from linearity below 20°C. The slope of the Van’t Hoff plot was used to derive the change in enthalpy (ΔH°) at 25°C (33.9 kJ mol−1). The change in entropy (ΔS°) was calculated to be 56.9 J mol−1 K−1. Thus, it appears that the formation of the SSBBA·ssDNA complex has a strong entropic or hydrophobic component to the overall protein-DNA interaction.
The analysis suggests that upon SSBBA·ssDNA complex formation, only one ion was released from the protein-DNA interface. These results appeared to indicate a small but significant contribution of ionic interaction in the ssDNA binding.
Structural analysis of SSBBA by homology modeling
The SSBBA sequence was further analyzed for secondary structure using Rosetta software (http://robetta.org/fragmentsubmit.jsp). Rosetta analysis indicated that there are at least five significant β strand structures and a single α-helix in the N-terminal half of the molecule (data not shown). The structure between the residues 101–170 appeared to be a random coil. These secondary structures are consistent with known features of SSBEC monomer, as determined earlier by X-ray crystallography [37, 39, 42].
SSB proteins are known to bind ssDNA through their oligonucleotide/oligosaccharide binding fold (OB fold) as described by Murzin . The OB fold is characterized by a β-barrel consisting of five β-strands capped by an α-helix. Despite sequence differences between SSBBA and SSBEC, the OB fold observed in SSBEC remained intact in SSBBA including the β-turn regions, particularly L45 between β sheets 4 and 5 (Figure 7). It has been shown that the β sheet 1 of SSBEC with the sequence VNKVILV is in the monomer-interface of the SSBEC dimer . In SSBBA, this β sheet remains partially intact (NKVILV) with the loss of the Val5 residue. However, the His56 of one monomer in SSBEC forms a hydrogen bond with Asn6 and the carbonyl oxygen of Leu83 of another monomer, which is essential for a stable dimer/tetramer formation. Although Asn6 (Asn2 in SSBBA) and Leu83 (Leu76 in SSBBA) remained conserved, one of the most important residues, His56 in SSBEC was altered to Ile (Ile47) in SSBBA (Figure 1). It should be noted that in the temperature-sensitive E. coli mutant, ssb-1, His56 was mutated to Tyr56 leading to the ts-phenotype. E. coli ssb-1 ts- mutant does not form a stable tetramer at non-permissive temperature [5, 45]. Thus, the lack of this His residue in SSBBA will likely hinder a stable dimer formation.
The SSBEC tetramer is formed by the interaction of two dimers [37, 39]. The dimer-dimer interface involves two six-stranded surfaces, each comprised of β1, β4, and β5 from two monomers. The structure of SSBBA, as shown in Figure 7, could form such a tetramer interface, had it not been for the difficulty associated with the dimer formation. It has been shown with a number of SSB crystal structures that a network of hydrogen bonds among the side chains in this six-stranded interface is necessary for a stable tetramer formation. The residues that were shown to be important in SSBEC for this network of hydrogen bond formation are Lys8, Tyr79, Gln77, Glu81, and Gln111. Sequence comparison (Figure 1) between SSBEC and SSBBA indicated that all of these residues in SSBBA underwent changes and are as follows: Lys8→Arg2, Tyr79→Gly71, Gln77→Leu69, Gln83→Arg75, and Gln111→Phe104. Although all of the changes may not be significant, three of these five changes are significant in terms of hydrogen bond formation. Therefore, these amino acid changes in SSBBA are likely to impede tetramer formation further. Taken together, inhibition of both monomer-monomer interaction leading to dimer formation as well as dimer-dimer interaction leading to tetramer formation, the amino acid sequence of SSBBA does not support formation of stable dimer or tetramer.
Analysis of the structure of ssDNA binding pocket in SSBBA
Single-stranded DNA binding by prokaryotic SSBs has been shown by several groups to be carried out exclusively by tetrameric forms of SSBs containing four OB folds or dimeric forms with each monomer containing two OB folds [37, 39, 46]. Thus, the presence of four OB folds in SSBs appears essential for high affinity ssDNA binding. Our studies indicated that SSBBA bound ssDNA with very high affinity (1.0 ± 0.1 x 10−9 M) even though it did not appear to form a stable tetramer in the absence of DNA at the concentration range examined (Figures 3 & 7).
Subunit structure of SSBBA in the SSBBA·ssDNA complex
SSB protein is required for a variety of processes such as DNA replication, recombination and DNA repair despite its lack of any enzymatic activity [5, 9, 49]. Among its multifaceted cellular activities, a common feature of all of these processes is to bind ssDNA with high affinity and protect it from reannealing and/or degradation. Of emerging importance is SSB’s role in protein-protein interaction during various DNA transactions. Most studies involving SSB proteins demonstrated that ssDNA wraps around a tetrameric form of SSB.
SSBBA does not form a stable tetramer
E. coli SSBEC is a stable tetramer with high solubility and tremendous thermal stability . The majority of prokaryotic cellular SSBs are homotetramers, where each monomer harbors an OB fold. However there are exceptions. SSBDR from Deinococcus radiodurans is a homodimer, where each monomer is quite large and contains two OB folds [37–39, 46]. Each OB fold is capable of binding ssDNA independently. In both cases, a stable SSB protein complex with four OB folds is required for ssDNA binding. SSBBA was found to be not tetrameric at or above ambient temperature by size exclusion HPLC (Figure 3). This physicochemical property of SSBBA is closely comparable to the T4 bacteriophage SSB, which is monomeric.
Molecular basis of SSBBA structure
Sequence alignment and three dimensional structure of SSBBA, generated by homology-based modeling were utilized to probe the molecular basis of ssDNA binding (Figures 1). Sequence alignment and secondary structure prediction (data not shown) clearly indicated the presence of an OB fold in SSBBA, which is a characteristic of SSBs and required for high affinity ssDNA binding (Figure 4).
Both sequence alignment and homology modeling (Figures 1 & 10) indicated lack of several residues that are important for monomer-monomer and dimer-dimer interactions leading to the formation of a stable tetramer in SSBEC. His56 (as well as Glu54) of β sheet 3 (Figure 1) in SSBEC plays an important role by forming a hydrogen bond with Asn7 in β sheet 1, the carbonyl group in Leu84 and Thr100 at the base of loop L45. Notably, in E. coli temperature-sensitive mutant, ssb-1, His56 is mutated to Tyr56 . This mutant does not form a stable tetramer with respect to monomers at non-permissive temperatures [5, 45, 50]. Thus, a lack of the corresponding His residue (His→Ile change) in SSBBA may be one of the important contributors to its structure. Moreover, in B. anthracis the sequence Glu54. Trp55.His56 in the E. coli β sheet 3 is altered to Asp46.Phe47.Ile48 (Figure 1). This change did not alter the β sheet structure but may have altered the contributions of these residues in the monomer-monomer interaction in the stable dimer and tetramer formation. It appears that although β sheet 1 remained conserved in SSBBA, this region lacks the valine residue which may have attenuated the interaction of the β sheet 1 with β sheet 1′ in the monomer-monomer interface of the dimer. The shortening of the N-terminus in SSBBA may also have deleterious effect in the interactions involving H-bonds in this region and can contribute to the lack of tetramer formation. Taken together, the amino acid residue substitutions in SSBBA, as described above, are likely contributed to the disruption of monomer-monomer interaction leading to dimer formation.
The dimer-dimer interface in the SSBEC tetramer is primarily a six-stranded β sheet-mediated. Residues that are important in SSBEC for the network of hydrogen bond formation at the dimer-dimer interface are Lys8, Tyr71, Gln77, Glu81, and Gln111 . Sequence comparison (Figure 1) between SSBEC and SSBBA indicated that all of these residues in SSBBA underwent alteration and are as follows: Lys8→Arg3, Tyr79→Gly71, Gln77→Leu69, Gln83→Arg75, and Gln111→Phe104. Some of these changes are chemically significant leading to possible disruption of the network of hydrogen bond formation that is required for a stable tetramer formation. In addition, Gln77 and Gln111 are located in the dimer-dimer interface and have been implicated in the tetramer formation. As described earlier, Gln111 is altered to Phe104 in SSBBA. An equally significant change is observed with Gln77 which is changed to Leu69 in SSBBA. Taken together, these changes in amino acid sequence may disrupt both monomer-monomer and dimer-dimer interactions leading to a monomeric SSBBA at a physiological temperature.
Energetics of SSBBA·ssDNA binding
Protein-DNA recognition and binding involve complex interactions. Earlier, we have used fluorescence anisotropy analysis of DNA binding by E. coli DNA primase and determined the thermodynamic parameters of protein-DNA interaction (38). We have used a similar analysis to probe the ssDNA binding by SSBBA. In addition, we have analyzed contribution of electrostatic and ionic interactions in the binding by analyzing the dependence of binding on the ionic strength of the environment. Together, these two analyses provided a detailed picture of the forces in SSBBA·ssDNA binding.
The KD values were determined at different temperatures (20–37°C) (Figure 5). Our data showed that although SSBBA was able to bind DNA at a wide range of temperatures, it bound with the highest affinity at 20–25°C. The free energy change for SSB·ssDNA association was −23 kJ mol−1 at 25°C. Using the two equations: ΔG° = −RT lnKD and ΔG° = ΔH°−TΔS° and the slope of the plot, we determined that ΔS° was ~188 J mol−1 K−1 in this temperature range.
We determined the KD value of SSBBA binding to ssDNA at different salt concentrations (0–200 mM NaCl) (Figure 6A). The binding is progressively weakened with an increase in ionic strength. The KD values were then analyzed using a linkage plot to determined ionic interactions in the binding. The negative slope of the plot in Figure 5B indicated a release of ions from. Our analysis determined the release of one Na+ and one Cl- ion during the binding process. In addition, our results also pointed out that SSBBA likely formed a tetrameric species at 0 mM NaCl and became monomeric at higher NaCl concentration. It is perhaps possible that the tetramer formation could be dependent on the ionic strength.
Thus our results suggest that ionic interaction or salt bridge formation between the protein and the DNA made specific contribution to the overall free energy change. In order to determine the contribution we first extrapolated KD value of the complex at infinite salt concentration (KD∞) by nonlinear regression of KD versus log[NaCl] plot (data not shown). The value of ΔG°ionic was −8 kJ mol−1.
Mechanisms of ssDNA binding by SSBBA
Despite differences between its Gram-negative counterpart, SSBBA bound to ssDNA with high affinity (Figure 4). The ssDNA binding affinity (KD) for a SSBBA monomer binding to a small oligonucleotide was 1.0 ± 0.1 x 10−9 M at 25°C. Even though many changes in amino acid sequence of SSBBA directly relate to ssDNA binding, such as Trp55→Phe47, Trp90→Tyr81, Phe61→Trp53, the changes are not drastic enough to alter ssDNA binding (Figure 8). Three dimensional structure as well as electrostatic surface potential in Figure 8 indicates that ssDNA binding remained unperturbed. A temperature-sensitive mutant of E. coli SSB, ssb-1, is unable to form a stable tetramer at a non-permissive temperature [5, 45, 50]. This mutant is also defective in supporting DNA replication at non-permissive temperature. Thus, it appears a SSB tetramer formation is a prerequisite for DNA replication. Consequently, we sought to explore whether ssDNA template could influence the ability of SSBBA to form tetramers upon DNA binding. A likely possibility is that SSBBA is capable of forming a normal tetrameric structure containing four OB-folds, as seen in other SSBs, upon sufficiently long ssDNA. This possibility was examined using a recently developed FRET assay for SSB·ssDNA interaction .
Previous studies with SSBEC have shown that its SSB35 and SSB65 binding modes can be distinguished by a FRET assay . Both of these ssDNA binding modes require a tetrameric (or di-tetrameric) structure of bound SSB. A similar FRET assay was used to probe the structure of SSBBA in ssDNA bound state. As the ssDNA binding constant of SSBBA is very high, the possibility that it may form a tetramer only in the ssDNA bound state was investigated. As shown in Figure 9A, SSBEC formed both SSB35 and SSB65 structures with the 5′-Cy5(dT)70Cy3-3′ oligonucleotide as evidenced by FRET analysis. As described earlier, SSB35 represented the intermediate-FRET complex and SSB65 represented the high-FRET SSB-ssDNA complex. At a low SSB to dT70 ratio, it formed the high FRET complex and at a high SSB to dT70 ratio, it formed the intermediate FRET complex. In the FRET analysis, SSBBA formed only a high-FRET complex but not the intermediate-FRET complex (Figure 9B). In addition, the slope of the plot with SSBBA is different from that of SSBEC. As SSBEC is a stable tetramer, the high FRET complex formed rapidly with increasing SSB concentration and it formed much slowly with SSBBA because of the lack of a stable tetramer formation. Our results appeared to indicate (i) a tetrameric structure of SSBBA in the SSBBA-ssDNA complex, and (ii) the SSBBA-ssDNA complex was formed only in the SSB65 mode. Due to high affinity of ssDNA binding, perhaps four monomers can bind the oligo(dT)70 prior to the tetramer formation. Once this multi-SSBBA complex is formed, the bound SSBBA monomers undergo conformational transition and form tetrameric structure in SSB65 mode. A hypothetical model is proposed in Figure 10. In this proposed model, all four monomers in the tetramer would first bind to the ssDNA, which would likely lead to the formation of the SSB65 complex and prevent the formation of a SSB35 complex. In the case of SSBBA, a two-fold higher concentration of protein was needed to observe the high-FRET complex. We believe this is due to the following reasons. First, the SSBBA is in essence a mutant form of SSBEC and as a result its ssDNA binding mechanism is likely somewhat different. Second, higher concentration of SSBBA might have favored the binding of all four monomers to the ssDNA. Initial slope of the plot in Figure 9B appeared to support this pathway.
SSBBA appears to undergo structural transformations which may support its high affinity binding to ssDNA. Its structure is due to a cumulative effect of multiple changes in key amino acid residues in its sequence which resulted in the loss of stable tetramer formation. Nonetheless, the SSBBA bound oligo(dT)20 with high affinity as shown in Figures 4, 5, 6. Therefore, multiple monomers will bind to oligo(dT)70 due to its long size. Thus, it is reasonable to assume that four monomers are binding to a long ssDNA (≥70 nucleotides). FRET data presented in Figure 9 established that SSB65-like structure is being formed upon oligo(dT)70 binding. Therefore, the ssDNA binding is leading to the formation of an SSB65 complex in which ssDNA is bound to a SSBEC-like tetrameric structure. We have proposed a hypothetical model, presented in Figure 10, which may explain the mechanism of formation of a SSBBA tetramer assembly upon ssDNA binding which require further studies of such complex formation. The proposed model represents a stepwise process by which SSBBA can achieve high affinity DNA binding through a tetramer formation. This mechanism of SSB-ssDNA complex formation and its reversal may aid in the rapid removal of SSB, a necessary step, by enzymes such as a DNA polymerase during DNA replication as well as in other processes. In essence, SSBBA could actually be more effective than its tetrameric orthologs in executing its multifaceted functions in cellular DNA transactions.
Our studies suggest that the structural properties of SSBBA differ from that of its Gram-negative counterpart, SSBEC, and that furthermore its structure is modulated in the presence an ssDNA template. It is noteworthy that despite complexities in structure and oligomerization, SSBBA retains high-affinity ssDNA binding, which is its primary function. Its unique structure may be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to alteration of stable dimer and tetramer formation. In the presence of a long ssDNA (≥70 nucleotides) appears to form with SSBBA a SSB65 complex in which ssDNA is bound to all four SSB monomers in a tetrameric structure. A proposed model may explain the mechanism of such SSBBA-ssDNA complex formation through a transient tetramer formation. This model indicates that SSBBA may be more efficient in assembly and disassembly of the protein-DNA complex particularly during DNA replication. The physiological consequence(s) of the unusual structural dynamics of SSBBA, could be significant. Further studies are required to fully elucidate the role of protein·DNA and protein·protein interactions on SSBBA protein structure.
Nucleic acids and other reagents
Ultra pure nucleotides were obtained from GE Biosciences (Piscataway, NJ) and were used without further purification. All other chemicals used to prepare buffers and solutions were reagent grade and were purchased from the Fisher Chemical Company (Pittsburgh, PA). HPLC ion exchange columns, ion exchange chromatography matrix, and the Bio-Cad 20 HPLC instrument were from Applied Biosystems Inc., Woburn, MA. The gel filtration column, TSK gel 3000SW, was from Tosoh Bioscience, King of Prussia, PA. Custom oligonucleotides for PCR and fluorescently labeled oligonucleotides were from Sigma-Aldrich (St. Louis, MO).
Lysis buffer contained 25 mM Tris–HCl, (pH 7.9), 10% sucrose, 250 mM NaCl, and 0.001% NP40. Buffer A contained 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 10% glycerol, 5 mM DTT, and NaCl in mM as indicated in the subscript. Buffer B, used for all fluorescence studies, contained 20 mM Hepes-NaOH (pH 7.5), 5 mM MgCl2 and 1 mM DTT and 25 mM (unless otherwise indicated) ultrapure NaCl. In temperature and salt titration experiments, buffer B containing 5% ultrapure glycerol was used. Buffers for fluorescence measurements were prepared with HPLC-grade water (with minimal background fluorescence), fluorescence grade reagents, and filtered through a 0.2 μm nylon filter, examined for background fluorescence and Raman spectrum before use in anisotropy measurements. Background fluorescence was subtracted where necessary.
Cloning and expression of SSBBA
The SSBBA gene was amplified by PCR using B. anthracis genomic DNA, obtained as a gift from Dr. Theresa M. Koehler of the University of Texas Houston Health Science Center, Houston (33, 34). This ORF codes for a 172 amino acid polypeptide with a predicted molecular weight of 19.2 kDa. The amplified gene was cloned into a pET29b vector (Novagen, Inc., Madison, WI) under the control of a T7 promoter (pET29b-SSBBA recombinant plasmid). The presence of the correct insert was confirmed by DNA sequencing. The SSBBA protein was over-expressed in E. coli strain BL21(DE3)RIL (Agilent Technologies Inc., Santa Clara, CA) harboring pET29b-SSBBA plasmid. Cells harboring the recombinant plasmid were grown in 2X-YT media containing 50 μg/ml of kanamycin, 20 μg/ml of tetracycline and 12 μg/ml of chloramphenicol with shaking at 37°C to an optical density at 600 nm of 0.4. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.25 mM. The cells were shaken for an additional two hours at 25°C, then harvested by centrifugation for 10 min at 5,000 x g. The cells were resuspended in 2.5% of the original culture volume of lysis buffer at 4°C and stored at −80°C until further use.
Purification of SSBBA
Cells were thawed, adjusted to pH 8.0 with 1 M Tris base, and lysed using 0.25 mg/ml lysozyme, 5 mM MgCl2, 5 mM spermidine·HCl, and 2.5 mM DTT via incubation at ambient temperature for 60 min. The mixture was Dounce homogenized followed by centrifugation. The lysate was centrifuged at 43,000 x g for 30 min at 23°C. The supernatant was precipitated overnight using 0.25 g/ml ammonium sulfate at 4°C. This precipitate was collected by centrifugation at 43,000 x g for 30 min at 4°C, and dissolved in buffer A0 (Fraction II). Fraction II was clarified by centrifugation at 43000 x g for 30 min. All steps were carried out at ambient temperature unless otherwise indicated.
The salt concentration of Fraction II was adjusted to the conductivity of buffer A50 by diluting with buffer A0. The protein fraction was then passed through a 5 ml POROS-Q column equilibrated with buffer A50. SSBBA protein was eluted with a 150 ml gradient from A100 to A500. The SSBBA fractions, identified by SDSPAGE, were pooled (fraction III). The salt concentration of Fraction III was adjusted to the conductivity of buffer A50 by diluting with buffer A0. Diluted Fraction III was bound to a 5 ml S-Sepharose column equilibrated with A50. SSBBA was eluted using a 150 ml gradient from A100 to A500. Fractions containing SSBBA were identified by SDS-PAGE and combined (Fraction IV). The Fraction IV, adjusted to 0.25 g/ml ammonium sulfate, was incubated on ice for two hours that resulted in the selective precipitation of SSBBA. The SSBBA precipitate was collected by centrifugation at 43000 x g for 60 min at 0–1°C. The pellet (Fraction V) was resuspended in 10 ml of buffer A100. Homogeneity was assessed by SDS-PAGE.
Assay of SSB biological activity
Italicized nucleotides denote non-complimentary bases that create the fork structure of the duplex. Fluorescence emission spectra of the samples, before and after reaction, were recorded between 550–750 nm with 519 nm excitation with 8 nm slit-width. Reaction was initiated by adding 0.5 μg/ml DnaBBA helicase to the reaction mixture and incubated for 15 min at 37°C and FRET was measured. SSBBA (3 μg/ml) was added to the reaction mixture where indicated. DnaBBA helicase unwinding of the duplex led to inhibition of the FRET between Cy3 and Cy5. SSBBA was required for efficient helicase action of DnaBBA which was the basis of the assay. By using native and heat denatured substrates, it was determined that 1% decrease in FRET is equivalent to 3 pmol DNA unwinding in terms of base pairs (bp).
Steady-state fluorescence measurements
Fluorescence anisotropy was measured to investigate DNA binding by SSBBA in solution [40, 52]. Fluorescence measurements were carried out using a steady-state-photon counting spectrofluorometer, PC1 with Vinci software, from ISS Instruments (Champaign, IL) and Fluoromax4-TCSPC with time-resolved fluorescence from Horiba Instruments Inc. (Edison, NJ). Excitation and emission slits were adjusted to 8 nm to maximize intensity counts . Temperature during measurements was maintained using a programmable Peltier-controlled cuvette holder from Quantum Northwest Inc. (Seattle, WA).
Fluorescence anisotropy analysis of equilibrium ssDNA binding
Ivv, Ivh, Ihv and Ihh represent the fluorescence signal for excitation and emission with the polarizers set at (00, 00), (00, 900), (900, 00) and (900, 900) respectively.
Where, R is the ligand i.e., labeled oligonucleotides and P is SSBBA.
where, YMIN and YMAX are the anisotropy values at the bottom and top plateaus respectively. X represents log[SSBBA] (where [SSBBA] represents total concentration of SSBBA) and X0 is the X value when the anisotropy is halfway between the top and the bottom of the plot and napp is the Hill coefficient.
FRET analysis of ssDNA binding by SSBEC and SSBBA
FRET analysis was used to monitor ssDNA binding by SSB as described (22). Reaction mixtures were assembled on ice and incubated at 25°C for 5 min before FRET analysis. Reaction mixtures contained 40 nM labeled Cy5-(dT)70-Cy3 oligonucleotide and the indicated amount of SSBEC or SSBBA in a total volume of 1 ml. SSBBA or SSBEC titrations were performed with PC1 spectrofluorometer with the monochromator set at 515 nm for excitation for the Cy3 donor and with the monochromator set at 665 nm emissions for the Cy5 acceptor. Slit width was 8 nm.
- B. anthracis:
Single stranded DNA
Sodium dodecyl sulfate
Polyacrylamide gel electrophoresis
Bovine serum albumin
Single stranded DNA binding protein
SSB of Bacillus anthracis
DnaB helicase of Bacillus anthracis
Fluorescence resonance energy transfer
Authors gratefully acknowledge support of this work by grants from the National Institute of Allergy & Infectious Diseases, NIH and UMDNJ Foundation. Authors wish to thank Ms. S. Rotoli and K-Y Luu for DNA helicase assay of SSBBA, Ms. J. Debski for critical review of the manuscript, Ms. Julia Crawford for F-test, Mr. Robert McBride of Educational Media, UMDNJ-SOM for illustration. We also thank the anonymous reviewers for helpful suggestions and comments.
- Pestryakov PE, Lavrik OI: Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism. Biochemistry (Mosc). 2008, 73 (13): 1388-1404. 10.1134/S0006297908130026.View ArticleGoogle Scholar
- Bochkarev A, Bochkareva E: From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. Curr Opin Struct Biol. 2004, 14 (1): 36-42. 10.1016/j.sbi.2004.01.001.PubMedView ArticleGoogle Scholar
- Iftode C, Daniely Y, Borowiec JA: Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol. 1999, 34 (3): 141-180. 10.1080/10409239991209255.PubMedView ArticleGoogle Scholar
- Salas M, Freire R, Soengas MS, Esteban JA, Mendez J, Bravo A, Serrano M, Blasco MA, Lazaro JM, Blanco L: Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication. FEMS Microbiol Rev. 1995, 17 (1–2): 73-82.PubMedGoogle Scholar
- Meyer RR, Laine PS: The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev. 1990, 54 (4): 342-380.PubMedPubMed CentralGoogle Scholar
- Sancar A, Williams KR, Chase JW, Rupp WD: Sequences of the ssb gene and protein. Proc Natl Acad Sci USA. 1981, 78 (7): 4274-4278. 10.1073/pnas.78.7.4274.PubMedPubMed CentralView ArticleGoogle Scholar
- Sancar A, Rupp WD: Cloning of uvrA, lexC and ssb genes of Escherichia coli. Biochem Biophys Res Commun. 1979, 90 (1): 123-129. 10.1016/0006-291X(79)91598-5.PubMedView ArticleGoogle Scholar
- Wold MS, Li JJ, Kelly TJ: Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci USA. 1987, 84 (11): 3643-3647. 10.1073/pnas.84.11.3643.PubMedPubMed CentralView ArticleGoogle Scholar
- Chase JW, Williams KR: Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986, 55: 103-136. 10.1146/annurev.bi.55.070186.000535.PubMedView ArticleGoogle Scholar
- Zou Y, Liu Y, Wu X, Shell SM: Functions of human replication protein A (RPA): from DNA replication to DNA damage and stress responses. J Cell Physiol. 2006, 208 (2): 267-273. 10.1002/jcp.20622.PubMedPubMed CentralView ArticleGoogle Scholar
- Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck JL: SSB as an organizer/mobilizer of genome maintenance complexes. Crit Rev Biochem Mol Biol. 2008, 43 (5): 289-318. 10.1080/10409230802341296.PubMedPubMed CentralView ArticleGoogle Scholar
- LeBowitz JH, McMacken R: The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986, 261 (10): 4738-4748.PubMedGoogle Scholar
- Biswas EE, Chen PH, Biswas SB: Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins. Nucleic Acids Res. 2002, 30 (13): 2809-2816. 10.1093/nar/gkf384.PubMedPubMed CentralView ArticleGoogle Scholar
- Stayton MM, Bertsch L, Biswas S, Burgers P, Dixon N, Flynn JE, Fuller R, Kaguni J, Kobori J, Kodaira M: Enzymatic recognition of DNA replication origins. Cold Spring Harb Symp Quant Biol. 1983, 47 Pt 2: 693-700.PubMedView ArticleGoogle Scholar
- Stayton MM, Kornberg A: Complexes of Escherichia coli primase with the replication origin of G4 phage DNA. J Biol Chem. 1983, 258 (21): 13205-13212.PubMedGoogle Scholar
- Learn BA, Um SJ, Huang L, McMacken R: Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA. Proc Natl Acad Sci USA. 1997, 94 (4): 1154-1159. 10.1073/pnas.94.4.1154.PubMedPubMed CentralView ArticleGoogle Scholar
- Biswas SB, Biswas EE: Regulation of dnaB function in DNA replication in Escherichia coli by dnaC and lambda P gene products. J Biol Chem. 1987, 262 (16): 7831-7838.PubMedGoogle Scholar
- Krejci L, Sung P: RPA not that different from SSB. Structure. 2002, 10 (5): 601-602. 10.1016/S0969-2126(02)00765-7.PubMedView ArticleGoogle Scholar
- Haseltine CA, Kowalczykowski SC: A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus. Mol Microbiol. 2002, 43 (6): 1505-1515. 10.1046/j.1365-2958.2002.02807.x.PubMedView ArticleGoogle Scholar
- Robbins JB, Murphy MC, White BA, Mackie RI, Ha T, Cann IK: Functional analysis of multiple single-stranded DNA-binding proteins from Methanosarcina acetivorans and their effects on DNA synthesis by DNA polymerase BI. J Biol Chem. 2004, 279 (8): 6315-6326.PubMedView ArticleGoogle Scholar
- Wadsworth RI, White MF: Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus. Nucleic Acids Res. 2001, 29 (4): 914-920. 10.1093/nar/29.4.914.PubMedPubMed CentralView ArticleGoogle Scholar
- Pant K, Karpel RL, Rouzina I, Williams MC: Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements. J Mol Biol. 2005, 349 (2): 317-330. 10.1016/j.jmb.2005.03.065.PubMedView ArticleGoogle Scholar
- Nakai H, Richardson CC: The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7. J Biol Chem. 1988, 263 (20): 9831-9839.PubMedGoogle Scholar
- Ferrari ME, Fang J, Lohman TM: A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding. Biophys Chem. 1997, 64 (1–3): 235-251.PubMedView ArticleGoogle Scholar
- Overman LB, Lohman TM: Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions. J Mol Biol. 1994, 236 (1): 165-178. 10.1006/jmbi.1994.1126.PubMedView ArticleGoogle Scholar
- Roy R, Kozlov AG, Lohman TM, Ha T: Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein. J Mol Biol. 2007, 369 (5): 1244-1257. 10.1016/j.jmb.2007.03.079.PubMedPubMed CentralView ArticleGoogle Scholar
- Griffith JD, Harris LD, Register J: Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments. Cold Spring Harb Symp Quant Biol. 1984, 49: 553-559. 10.1101/SQB.1984.049.01.062.PubMedView ArticleGoogle Scholar
- Chrysogelos S, Griffith J: Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proc Natl Acad Sci USA. 1982, 79 (19): 5803-5807. 10.1073/pnas.79.19.5803.PubMedPubMed CentralView ArticleGoogle Scholar
- Bendtsen JD, Nilsson AS, Lehnherr H: Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein. J Virol. 2002, 76 (19): 9695-9701. 10.1128/JVI.76.19.9695-9701.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Moreira D: Multiple independent horizontal transfers of informational genes from bacteria to plasmids and phages: implications for the origin of bacterial replication machinery. Mol Microbiol. 2000, 35 (1): 1-5. 10.1046/j.1365-2958.2000.01692.x.PubMedView ArticleGoogle Scholar
- Lindner C, Nijland R, van Hartskamp M, Bron S, Hamoen LW, Kuipers OP: Differential expression of two paralogous genes of Bacillus subtilis encoding single-stranded DNA binding protein. J Bacteriol. 2004, 186 (4): 1097-1105. 10.1128/JB.186.4.1097-1105.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Grove DE, Willcox S, Griffith JD, Bryant FR: Differential single-stranded DNA binding properties of the paralogous SsbA and SsbB proteins from Streptococcus pneumoniae. J Biol Chem. 2005, 280 (12): 11067-11073. 10.1074/jbc.M414057200.PubMedView ArticleGoogle Scholar
- Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Tettelin H, Fouts DE, Eisen JA, Gill SR: The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature. 2003, 423 (6935): 81-86. 10.1038/nature01586.PubMedView ArticleGoogle Scholar
- Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N: Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res. 2000, 28 (21): 4317-4331. 10.1093/nar/28.21.4317.PubMedPubMed CentralView ArticleGoogle Scholar
- Genschel J, Curth U, Urbanke C: Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease. Biol Chem. 2000, 381 (3): 183-192.PubMedView ArticleGoogle Scholar
- Curth U, Genschel J, Urbanke C, Greipel J: In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein. Nucleic Acids Res. 1996, 24 (14): 2706-2711. 10.1093/nar/24.14.2706.PubMedPubMed CentralView ArticleGoogle Scholar
- Matsumoto T, Morimoto Y, Shibata N, Kinebuchi T, Shimamoto N, Tsukihara T, Yasuoka N: Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-Ray structure analysis. J Biochem. 2000, 127 (2): 329-335. 10.1093/oxfordjournals.jbchem.a022611.PubMedView ArticleGoogle Scholar
- Murzin AG: OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J. 1993, 12 (3): 861-867.PubMedPubMed CentralGoogle Scholar
- Raghunathan S, Ricard CS, Lohman TM, Waksman G: Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-A resolution. Proc Natl Acad Sci USA. 1997, 94 (13): 6652-6657. 10.1073/pnas.94.13.6652.PubMedPubMed CentralView ArticleGoogle Scholar
- Khopde S, Biswas EE, Biswas SB: Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase. Biochemistry. 2002, 41 (50): 14820-14830. 10.1021/bi026711m.PubMedView ArticleGoogle Scholar
- Datta K, LiCata VJ: Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases. J Biol Chem. 2003, 278 (8): 5694-5701. 10.1074/jbc.M208133200.PubMedView ArticleGoogle Scholar
- Raghunathan S, Kozlov AG, Lohman TM, Waksman G: Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat Struct Biol. 2000, 7 (8): 648-652. 10.1038/77943.PubMedView ArticleGoogle Scholar
- Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T: Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc. 2009, 4 (1): 1-13.PubMedView ArticleGoogle Scholar
- Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T: The SWISS-MODEL Repository and associated resources. Nucleic Acids Res. 2009, 37 (Database issue): D387-D392.PubMedPubMed CentralView ArticleGoogle Scholar
- Williams KR, Murphy JB, Chase JW: Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation. J Biol Chem. 1984, 259 (19): 11804-11811.PubMedGoogle Scholar
- Bernstein DA, Eggington JM, Killoran MP, Misic AM, Cox MM, Keck JL: Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage. Proc Natl Acad Sci USA. 2004, 101 (23): 8575-8580. 10.1073/pnas.0401331101.PubMedPubMed CentralView ArticleGoogle Scholar
- Chan KW, Lee YJ, Wang CH, Huang H, Sun YJ: Single-stranded DNA-binding protein complex from Helicobacter pylori suggests an ssDNA-binding surface. J Mol Biol. 2009, 388 (3): 508-519. 10.1016/j.jmb.2009.03.022.PubMedView ArticleGoogle Scholar
- Lohman TM, Ferrari ME: Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem. 1994, 63: 527-570. 10.1146/annurev.bi.63.070194.002523.PubMedView ArticleGoogle Scholar
- Kornberg A, Baker TA: DNA Replication. 1992, Freeman, New York, NYGoogle Scholar
- Meyer RR, Glassberg J, Scott JV, Kornberg A: A temperature-sensitive single-stranded DNA-binding protein from Escherichia coli. J Biol Chem. 1980, 255 (7): 2897-2901.PubMedGoogle Scholar
- Aiello D, Barnes MH, Biswas EE, Biswas SB, Gu S, Williams JD, Bowlin TL, Moir DT: Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases. Bioorg Med Chem. 2009, 17: 4466-4476. 10.1016/j.bmc.2009.05.014.PubMedPubMed CentralView ArticleGoogle Scholar
- Gryczynski I, Gryczynski Z, Lakowicz JR: Fluorescence anisotropy controlled by light quenching. Photochem Photobiol. 1998, 67 (6): 641-646. 10.1111/j.1751-1097.1998.tb09467.x.PubMedView ArticleGoogle Scholar
- Lakowicz JR: Principles of Fluorescence Spectroscopy. 2006, Springer Sciences + Business Media, LLC, New YorkView ArticleGoogle Scholar
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