Bacterial Single-stranded DNA-binding Proteins Research Articles

High-Throughput Screening to Identify Inhibitors of SSB-Protein Interactions.

The bacterial single-stranded DNA-binding protein (SSB) uses an acidic C-terminal tail to interact with over a dozen proteins, acting as a genome maintenance hub. These SSB-protein interactions are essential, as mutations to the C-terminal tail that disrupt these interactions are lethal in Escherichia coli. While the roles of individual SSB-protein interactions have been dissected with mutational studies, small-molecule inhibitors of these interactions could serve as valuable research tools and have potential as novel antimicrobial agents. This chapter describes a high-throughput screening campaign used to identify inhibitors of SSB-protein interactions. A screen targeting the PriA-SSB interface from Klebsiella pneumoniae is presented as an example, but the methods may be adapted to target nearly any SSB interaction.

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.

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

Harnessing single-stranded DNA binding protein to explore protein-protein and protein-DNA interactions.

Proteins must interact with a variety of other cellular components to properly perform their functions. We have developed a series of five experiments based on the robust bacterial single-stranded DNA binding protein (SSB) to characterize both known and unknown protein-protein and protein-DNA interactions. Students work in groups to generate and process data from electrophoretic mobility shift assays (EMSA), yeast two-hybrid, far Western, chromatin immunoprecipitation (ChIP), and fluorescence microscopy experiments, including choosing a novel condition for each. A gamification approach was used to encourage student participation and laboratory safety. Student learning was assessed using pre- and post-surveys and course grade data. The results indicate a clear increase in both content knowledge and confidence in the topics presented. Ranking of course activities indicated that performing the hands-on laboratory exercises was the most valuable course component, and over half of the students would choose to take another course with a similar gamification component. Each of the five laboratory experiments can be performed in combination with each other or integrated separately into a related course, and the gamification structure can be applied to any course.

Structural Mechanisms of Cooperative DNA Binding by Bacterial Single-Stranded DNA-Binding Proteins

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

A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein–Protein Interactions in an Academic Screening Facility

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

Distinct properties of a hypoxia specific paralog of single stranded DNA binding (SSB) protein in mycobacteria

In addition to the canonical Single Stranded DNA Binding (SSBa) protein, many bacterial species, including mycobacteria, have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation during sporulation. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most bacterial SSB proteins, M. smegmatis SSBb (MsSSBb) forms a stable tetramer. However, solution studies indicate that MsSSBb is less stable to thermal and chemical denaturation than MsSSBa. Also, in contrast to the 5–20 fold differences in DNA binding affinity between paralogous SSBs in other organisms, MsSSBb is only about two-fold poorer in its DNA binding affinity than MsSSBa. The expression levels of ssbB gene increased during UV and hypoxic stresses, while the levels of ssbA expression declined. A direct physical interaction of MsSSBb and RecA, mediated by the C-terminal tail of MsSSBb, was also established. The results obtained in this study indicate a role of MsSSBb in recombination repair during stress.

Variations in amino acid composition in bacterial single stranded DNA–binding proteins correlate with GC content

Background and purposeSSB proteins are essential for the maintenance of the genome in all domains of life. Most bacterial SSBs are active as homotetramers. Each monomer comprises N-terminal domain (OB-fold) which is responsible for ssDNA binding and a disordered C-terminal domain (Ct) with a conserved acidic tail responsible for protein interactions.The variations in these essential proteins prompted us to conduct in silico analyses of the aa composition and properties of two distinct SSB domains in relation to bacterial GC content.Materials and methodsSSB sequences were collected from genomes covering a wide range of GC content from 14 bacterial phyla. The maximum-likelihood (ML) trees were constructed for SSB sequences and corresponding 16S rRNA genes. The aa contents of OB folds and Ct domains were subsequently analysed. ResultsWe showed that SSB proteins followed predicted amino acid (aa) composition as a function of genomic GC content. However, two distinct domains of SSB exhibit significant differences to the expected aa composition. Variations in aa proportion were more prominent in Ct domains. Elevated accumulation of Gly (up to 60 %) and Pro (up to 24 %), significant drop in the proportion of basic Lys and reduction in hydrophobic Leu, Ile and Val were identified in Ct domains of SSBs from high GC genomes. Consequently, this influences the biochemical properties of Ct domains.ConclusionsBased on this comparative study of SSBs we conclude that genomic GC content and two distinct domains with different functional roles participate in shaping aa composition of SSB proteins.

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

The bacterial single-stranded DNA binding protein (SSB) acts as an organizer of DNA repair complexes. The radD gene was recently identified as having an unspecified role in repair of radiation damage and, more specifically, DNA double-strand breaks. Purified RadD protein displays a DNA-independent ATPase activity. However, ATP hydrolytic rates are stimulated by SSB through its C terminus. The RadD and SSB proteins also directly interact in vivo in a yeast two-hybrid assay and in vitro through ammonium sulfate co-precipitation. Therefore, it is likely that the repair function of RadD is mediated through interaction with SSB at the site of damage.

Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

Plasmodium falciparum SSB Tetramer Wraps Single-Stranded DNA with Similar Topology but Opposite Polarity to E. coli SSB

Single-stranded DNA binding (SSB) proteins play central roles in genome maintenance in all organisms. Plasmodium falciparum, the causative agent of malaria, encodes an SSB protein that localizes to the apicoplast and likely functions in the replication and maintenance of its genome. P. falciparum SSB (Pf-SSB) shares a high degree of sequence homology with bacterial SSB proteins but differs in the composition of its C-terminus, which interacts with more than a dozen other proteins in Escherichia coli SSB (Ec-SSB). Using sedimentation methods, we show that Pf-SSB forms a stable homo-tetramer alone and when bound to single-stranded DNA (ssDNA). We also present a crystal structure at 2.1 Å resolution of the Pf-SSB tetramer bound to two (dT)35 molecules. The Pf-SSB tetramer is structurally similar to the Ec-SSB tetramer, and ssDNA wraps completely around the tetramer with a “baseball seam” topology that is similar to Ec-SSB in its “65 binding mode”. However, the polarity of the ssDNA wrapping around Pf-SSB is opposite to that observed for Ec-SSB. The interactions between the bases in the DNA and the amino acid side chains also differ from those observed in the Ec-SSB–DNA structure, suggesting that other differences may exist in the DNA binding properties of these structurally similar proteins.

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance.

Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.

DdrB Protein, an Alternative Deinococcus radiodurans SSB Induced by Ionizing Radiation

Deinococcus radiodurans exhibits an extraordinary resistance to the effects of exposure to ionizing radiation (IR). DdrB is one of five proteins induced to high levels in Deinococcus following extreme IR exposure and that play a demonstrable role in genome reconstitution. Although homology is limited, DdrB is a bacterial single-stranded DNA-binding protein. DdrB features a stable core with a putative OB-fold, and a C-terminal segment with properties consistent with other bacterial SSBs. In solution, the protein functions as a pentamer. The protein binds single-stranded DNA but not duplex DNA. Electron microscopy and assays with two RecA proteins provide further structural and functional identification with bacterial SSB. Overall, the results establish DdrB as the prototype of a new bacterial SSB family. Given the role of SSB as a mobilization scaffold for many processes in DNA metabolism, the induction of an alternative and quite novel SSB following irradiation has potentially broad significance for the organization of genome reconstitution functions.

Engineering of Functional Replication Protein A Homologs Based on Insights into the Evolution of Oligonucleotide/ Oligosaccharide-Binding Folds

The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains Bacteria and Eukarya, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon Methanosarcina acetivorans harbors multiple functional RPAs named MacRPA1 (for M. acetivorans RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in Methanococcoides burtonii and Methanosaeta thermophila. We further created two RPAs that mimicked the RPAs in Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus through fusions of modules from MacRPA1 and M. thermautotrophicus RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.

Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis

The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.

Biophysical Analysis of Thermus aquaticus Single-Stranded DNA Binding Protein

Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the χ-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria.

Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization

The highly conserved bacterial single-stranded DNA-binding (SSB) proteins play an important role in DNA replication, repair and recombination and are essential for the survival of the cell. They are functional as tetramers, in which four OB(oligonucleotide/oligosaccharide binding)-folds act as DNA-binding domains. The protomer of the SSB protein from the extremely radiation-resistant organism Deinococcus radiodurans (DraSSB) has twice the size of the other bacterial SSB proteins and contains two OB-folds. Using analytical ultracentrifugation, we could show that DraSSB forms globular dimers with some protrusions. These DraSSB dimers can interact with two molecules of E.coli DNA polymerase III χ subunit. In fluorescence titrations with poly(dT) DraSSB bound 47–54 nt depending on the salt concentration, and fluorescence was quenched by more than 75%. A distinct low salt binding mode as for EcoSSB was not observed for DraSSB. Nucleic acid binding affinity, rate constant and association mechanism are quite similar for EcoSSB and DraSSB. In a complementation assay in E.coli, DraSSB took over the in vivo function of EcoSSB. With DraSSB behaving almost identical to EcoSSB the question remains open as to why dimeric SSB proteins have evolved in the Thermus group of bacteria.

Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein.

Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.

Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein.

Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A. The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.