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1.  Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins 
Nucleic Acids Research  2012;40(12):5546-5559.
We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.
doi:10.1093/nar/gks173
PMCID: PMC3384303  PMID: 22373918
2.  Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans 
FEBS Open Bio  2015;5:378-387.
Highlights
•Deinococcal Ssb variants with only N or C-terminal OB fold were constructed.•The ssDNA binding affinity decreased in the order SsbFL > SsbC > SsbNC > SsbN.•SsbNC or SsbN formed a binary complex with SsbC to enhance ssDNA binding.•Individual Ssb variants modulated DNA relaxation by topoisomerase I.•Only SsbFL facilitated strand exchange with cognate RecA.
Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo-dimers while SsbNC/SsbN formed different oligomeric forms. In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization.
doi:10.1016/j.fob.2015.04.009
PMCID: PMC4427625  PMID: 25973364
Deinococcus radiodurans; Ssb protein; OB folds; EMSA; RecA; Strand exchange; Topoisomerase activity; ESDSA, extended synthesis-dependent strand annealing; OB fold, oligonucleotide/oligosaccharide binding fold; RPA, Replication protein A; Ssb, single-stranded DNA binding protein; SsbC, C-terminal Ssb; SsbN, N-terminal Ssb without connector; SsbNC, N-terminal Ssb with connector; ssDNA, single-stranded DNA
3.  Plasmodium falciparum SSB Tetramer Binds Single Stranded DNA only in a Fully Wrapped Mode 
Journal of molecular biology  2012;420(0):284-295.
The tetrameric E. coli single stranded (ss)DNA binding protein (Ec-SSB) functions in DNA metabolism by binding to single stranded (ss) DNA and interacting directly with numerous DNA repair and replication proteins. Ec-SSB tetramers can bind ssDNA in multiple DNA binding modes that differ in the extent of ssDNA wrapping. Here, we show that the structurally similar SSB protein from the malarial parasite Plasmodium falciparum (Pf-SSB) also binds tightly to ssDNA, but does not display the same number of ssDNA binding modes as Ec-SSB, binding ssDNA exclusively in fully wrapped complexes with site sizes of 52 – 65 nucleotides/tetramer. Pf-SSB does not transition to the more cooperative (SSB)35 DNA binding mode observed for Ec-SSB. Consistent with this, Pf-SSB tetramers also do not display the dramatic intra-tetramer negative cooperativity for binding of a second (dT)35 molecule that is evident in Ec-SSB. These findings highlight variations in the DNA binding properties of these two highly conserved homotetrameric SSB proteins and these differences might be tailored to suit their specific functions in the cell.
doi:10.1016/j.jmb.2012.04.022
PMCID: PMC3894689  PMID: 22543238
DNA repair; recombination; replication; structure; malaria
4.  Binding Specificity of E. coli SSB protein for the χ subunit of DNA pol III Holoenzyme and PriA helicase.† 
Biochemistry  2010;49(17):3555-3566.
The E. coli single stranded DNA binding (SSB) protein plays a central role in DNA metabolism through its high affinity interactions with ssDNA, as well as its interactions with numerous other proteins via its unstructured C-termini. Although SSB interacts with at least 14 other proteins, it is not understood how SSB might recruit one protein over another for a particular metabolic role. To probe the specificity of these interactions we have used isothermal titration calorimetry to examine the thermodynamics of binding of SSB to two E. coli proteins important for DNA replication, the χ subunit of DNA polymerase III holoenzyme and the PriA helicase. We find that an SSB tetramer can bind up to four molecules of either protein primarily via interactions with the last ~ 9 amino acids in the conserved SSB C-terminal tails (SSB-Ct). We observe intrinsic specificity for the binding of an isolated SSB-Ct peptide to PriA over χ due primarily to a more favorable enthalpic component. PriA and χ also bind with weaker affinity to SSB (in the absence of ssDNA) than to isolated SSB-Ct peptides, indicating an inhibitory effect of the SSB protein core. Although the binding affinity of SSB for both χ and PriA is enhanced if SSB is prebound to ssDNA, this effect is larger with PriA indicating a further enhancement of SSB specificity for PriA. These results also suggest that DNA binding proteins such as PriA, which also interact with SSB, could use this interaction to gain access to ssDNA by first interacting with the SSB C-termini.
doi:10.1021/bi100069s
PMCID: PMC2861366  PMID: 20329707
5.  Plasmodium falciparum SSB Tetramer Wraps Single Stranded DNA with Similar Topology but Opposite Polarity to E. coli SSB 
Journal of molecular biology  2012;420(0):269-283.
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. Pf-SSB shares a high degree of sequence homology with bacterial SSB proteins, but differs in the composition of its C-terminus, which in E. coli SSB interacts with more than a dozen other proteins. Using sedimentation methods we show that Pf-SSB forms a stable homo-tetramer alone and when bound to single stranded DNA. 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 E. coli SSB tetramer and ssDNA wraps completely around the tetramer with a “baseball seam” topology that is similar to E. coli SSB in its “65 binding mode”. However, the polarity of the ssDNA wrapping around Pf-SSB is opposite to that observed for E. coli SSB. The interactions between the bases in the DNA and the amino acids side chains also differ from those observed in the E. coli SSB-DNA structure suggesting that other differences may exist in the DNA binding properties of these structurally similar proteins.
doi:10.1016/j.jmb.2012.04.021
PMCID: PMC4017622  PMID: 22543099
DNA repair; recombination; replication; structure; Plasmodium; malaria
6.  Binding of the Dimeric Deinococcus radiodurans SSB Protein to Single-stranded DNA† 
Biochemistry  2010;49(38):8266-8275.
D. radiodurans single stranded (ss) DNA binding protein (DrSSB) originates from a radiation-resistant bacterium and participates in DNA recombination, replication and repair. Although it functions as a homodimer, it contains four DNA binding domains (OB folds) and thus is structurally similar to the E. coli SSB (EcoSSB) homotetramer. We examined the equilibrium binding of DrSSB to ssDNA to compare with EcoSSB. We find that the occluded site size of DrSSB on poly(dT) is ~45 nucleotides in low salt (<0.02M NaCl) but increases to 50–55 nucleotides at [NaCl] ≥ 0.2M. This suggests that DrSSB undergoes a transition between ssDNA binding modes as is observed for EcoSSB, although the site size difference between modes is not as large as for EcoSSB, suggesting that the pathways of ssDNA wrapping differ for these two proteins. The occluded site size corresponds well to the contact site size (52 nucleotides) determined by Isothermal Titration Calorimetry (ITC). Electrophoretic studies of complexes of DrSSB with phage M13ssDNA indicate the formation of stable, highly cooperative complexes at low salt conditions. Using ITC we find that DrSSB binding to oligo(dT)s with lengths close to the determined site size (50–55 nts) is stoichiometric with ΔHobs ≈-94±4 kcal/mole, somewhat smaller than for EcoSSB (≈-130 kcal/mole) under the same conditions. The observed binding enthalpy shows a large sensitivity to NaCl concentration, similar to that observed for EcoSSB. With the exception of the less dramatic change in occluded site size, the behavior of DrSSB is similar to that of EcoSSB protein (although, clear quantitative differences exist). These common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.
doi:10.1021/bi100920w
PMCID: PMC2963097  PMID: 20795631
7.  Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA 
Nucleic Acids Research  2013;42(6):3821-3832.
Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus–Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20–500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.
doi:10.1093/nar/gkt1316
PMCID: PMC3973332  PMID: 24371279
8.  Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity 
PLoS Genetics  2011;7(6):e1002156.
Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB−) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.
Author Summary
Natural genetic transformation can compensate for the absence of sexual reproduction in bacteria, allowing genetic diversification by frequent recombination. In many species, transformability is a transient property relying on a specialized membrane-associated machinery for binding exogenous double-stranded DNA and internalization of single-stranded DNA (ssDNA) fragments extracted from exogenous DNA. Subsequent physical integration of internalized ssDNA into the recipient chromosome by homologous recombination requires dedicated cytosolic ssDNA–processing proteins. Here, we document the roles in the model transformable species Streptococcus pneumoniae of one of these processing proteins, SsbB, a paralogue of SsbA the ssDNA–binding protein essential for genome maintenance in bacteria, which is expressed uniquely in cells competent for genetic transformation. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; that it protects and stabilizes internalized ssDNA; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. We conclude that SsbB creates a reservoir of ssDNA, presumably allowing multiple transformations in the same cell, and that S. pneumoniae has evolved SsbB to optimize chromosomal transformation, thereby contributing to its remarkable genetic plasticity.
doi:10.1371/journal.pgen.1002156
PMCID: PMC3128108  PMID: 21738490
9.  SSB diffusion on single stranded DNA stimulates RecA filament formation 
Nature  2009;461(7267):1092-1097.
Single stranded (ss)DNA generated in the cell during DNA metabolism is stabilized and protected by binding of single stranded DNA binding (SSB) proteins. E. coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high affinity protein-DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here, we show, using single molecule two and three-color FRET, that tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This first observation of diffusional movement of a protein on ssDNA introduces a new paradigm for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.
doi:10.1038/nature08442
PMCID: PMC2782680  PMID: 19820696
10.  Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island 
PLoS ONE  2012;7(4):e35285.
Background
Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins.
Methodology/Principal Findings
In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity.
Conclusions/Significance
We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.
doi:10.1371/journal.pone.0035285
PMCID: PMC3334931  PMID: 22536367
11.  In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein. 
Nucleic Acids Research  1996;24(14):2706-2711.
We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.
PMCID: PMC145992  PMID: 8759000
12.  The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks 
PLoS Genetics  2010;6(12):e1001238.
We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.
Author Summary
Cell multiplication relies primarily on the complete and accurate duplication of the genome. Thus, all organisms have evolved multiple mechanisms to protect, repair, and re-activate the DNA replication forks. A large body of research is currently aimed at deciphering the mechanisms that precisely direct the proteins involved in these rescue pathways towards the chromosome replication forks. Here, we have used the model bacterium Bacillus subtilis to demonstrate that the active chromosomal DNA replication forks are pre-equipped with many such rescue effectors via their direct physical interaction with the carboxy-terminal end (Cter) of the Single-Stranded DNA Binding protein (SSB). A detailed analysis of the multiple defects of viable B. subtilis mutants deleted for the Cter of SSB (SSBCter) revealed the vital role of this domain for the maintenance of genome integrity and fork propagation. The inability to grow at high temperature is a major defect of the ssbΔCter mutant. We show that this lethality can be specifically suppressed by overexpression of RecO, one of the numerous partners of SSB, apparently by mediating the loading of the RecA recombinase on ssDNA.
doi:10.1371/journal.pgen.1001238
PMCID: PMC3000357  PMID: 21170359
13.  Differential functional behavior of viral φ29, Nf and GA-1 SSB proteins 
Nucleic Acids Research  2000;28(10):2034-2042.
DNA replication of φ29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage φ29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of φ29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of φ29 DNA polymerase to ssDNA and their stimulatory effect on replication by φ29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during φ29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by φ29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the φ29 and Nf SSBs do not show any effect. On the other hand, the φ29 SSB is the only one of the three SSBs able to increase the replication rate of φ29 DNA polymerase in primed M13 ssDNA replication. From the fact that the φ29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of φ29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on φ29-like DNA replication has been proposed.
PMCID: PMC105360  PMID: 10773070
14.  Peptide inhibitors identify roles for SSB C-terminal residues in SSB/Exonuclease I complex formation† 
Biochemistry  2009;48(29):6764-6771.
Bacterial single-stranded (ss) DNA-binding proteins (SSBs) facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C-terminus of SSB (SSB-Ct) as a common binding site for SSB’s protein partners. The SSB-Ct is a highly conserved, amphipathic sequence, comprising acidic and hydrophobic elements. A crystal structure of E. coli Exonuclease I (ExoI) bound to a peptide comprising the E. coli SSB-Ct sequence shows that the C-terminal-most SSB-Ct Phe anchors the peptide to a binding pocket on ExoI and implicates electrostatic binding roles for the acidic SSB-Ct residues. Here, we use SSB-Ct peptide variants in competition experiments to examine the roles of individual SSB-Ct residues in binding ExoI in solution. Altering the C-terminal-most Pro or Phe residues in the SSB-Ct strongly impairs SSB-Ct binding to ExoI, confirming a major role for the hydrophobic SSB-Ct residues in binding ExoI. Alteration of N-terminal SSB-Ct residues leads to changes that reflect cumulative electrostatic binding roles for the Asp residues in SSB-Ct. The SSB-Ct peptides also abrogate SSB stimulation of ExoI activity through a competitive inhibition mechanism, indicating that the peptides can disrupt ExoI/SSB/ssDNA ternary complexes. Differences in the potency of the SSB-Ct peptide variants in the binding and nuclease inhibition studies indicate that the acidic SSB-Ct residues play a more prominent role in the context of the ternary complex than in the minimal ExoI/SSB-Ct interaction. Together, these data identify roles for residues in the SSB-Ct that are important for SSB complex formation with its protein partners.
doi:10.1021/bi900361r
PMCID: PMC2746433  PMID: 19527069
15.  ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants 
PLoS Genetics  2014;10(10):e1004719.
The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.
Author Summary
Both replication polymerases and single-stranded DNA binding proteins (SSB, which associate with single-stranded DNA exposed transiently during replication) are ubiquitous and show high levels of functional and structural conservation across all species. Among the nine different polypeptides that compose the bacterial replicative polymerase, the HolC-HolD (χψ) complex interacts with SSB, and is crucial for normal growth in the model bacteria Escherichia coli. Interestingly, many bacterial species lack this complex, where its function is presumably carried out by other polymerase components. With the aim of better understanding HolC-HolD (χψ) complex function in E. coli, we isolated growth defect suppressor mutations of the holD mutant. We found that ssb gene duplication and the consequent doubling of SSB protein expression, renders the entire χψ complex dispensable for growth. We also show that growth-defect suppression requires the presence of the SSB C-terminal amino acids in both ssb gene copies. This C-terminal tail promotes interaction between SSB and its partner proteins. Thus, our results indicate that in vivo SSB concentration plays a key role in maintaining polymerase stability and replication efficiency, in a reaction that involves SSB interactions with protein partner(s) other than χψ.
doi:10.1371/journal.pgen.1004719
PMCID: PMC4199511  PMID: 25329071
16.  Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity 
PLoS ONE  2015;10(5):e0126563.
Background
SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.
Results
This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis.
Conclusion
NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.
doi:10.1371/journal.pone.0126563
PMCID: PMC4431734  PMID: 25973760
17.  Mechanism of Exonuclease I stimulation by the single-stranded DNA-binding protein 
Nucleic Acids Research  2011;39(15):6536-6545.
Bacterial single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during cellular DNA replication, recombination and repair reactions. SSBs also form complexes with an array of genome maintenance enzymes via their conserved C-terminal tail (SSB-Ct) elements. In many cases, complex formation with SSB stimulates the biochemical activities of its protein partners. Here, we investigate the mechanism by which Escherichia coli SSB stimulates hydrolysis of ssDNA by Exonuclease I (ExoI). Steady-state kinetic experiments show that SSB stimulates ExoI activity through effects on both apparent kcat and Km. SSB variant proteins with altered SSB-Ct sequences either stimulate more modestly or inhibit ExoI hydrolysis of ssDNA due to increases in the apparent Michaelis constant, highlighting a role for protein complex formation in ExoI substrate binding. Consistent with a model in which SSB stabilizes ExoI substrate binding and melts secondary structures that could impede ExoI processivity, the specific activity of a fusion protein in which ExoI is tethered to SSB is nearly equivalent to that of SSB-stimulated ExoI. Taken together, these studies delineate stimulatory roles for SSB in which protein interactions and ssDNA binding are both important for maximal activity of its protein partners.
doi:10.1093/nar/gkr315
PMCID: PMC3159472  PMID: 21572106
18.  Mechanism of Interaction between Single-Stranded DNA Binding Protein and DNA† 
Biochemistry  2009;49(5):843-852.
A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where ∼70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB·ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB2·dT70 to be 1.1 μM at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.
doi:10.1021/bi901743k
PMCID: PMC2827191  PMID: 20028139
19.  Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein 
Nucleic Acids Research  2011;39(14):6305-6314.
RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.
doi:10.1093/nar/gkr199
PMCID: PMC3152348  PMID: 21504984
20.  DNA Binding Compatibility of the Streptococcus pneumoniae SsbA and SsbB Proteins 
PLoS ONE  2011;6(9):e24305.
Background
Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multiple SsbAs and SsbBs onto single-stranded DNA was investigated.
Methodology/Principal Findings
The complexes that were formed by the SsbA and SsbB proteins on dTn oligomers of defined lengths were examined by polyacrylamide gel electrophoresis. Complexes containing either two SsbAs or two SsbBs, or mixed complexes containing one SsbA and one SsbB, could be formed readily, provided the dTn oligomer was long enough to satisfy the full binding mode capacities of each of the bound proteins under the particular solution conditions. Complexes containing two SsbAs or two SsbBs could also be formed on shorter dTn oligomers via a “shared-strand binding” mechanism in which one or both proteins were bound using only a portion of their potential binding capacity. Mixed complexes were not formed on these shorter oligomers, however, indicating that SsbA and SsbB were incompatible for shared-strand binding. Additional experiments suggested that this shared-strand binding incompatibility may be due in part to differences in the structure of a loop region on the outer surface of the subunits of the SsbA and SsbB proteins.
Conclusion/Significance
These results indicate that the SsbA and SsbB proteins may co-assemble on longer DNA segments where independent binding is possible, but not on shorter DNA segments where coordinated interactions between adjacent SSBs are required. The apparent compatibility requirement for shared-strand binding could conceivably serve as a self-recognition mechanism that regulates the manner in which SsbA and SsbB interact in S. pneumoniae.
doi:10.1371/journal.pone.0024305
PMCID: PMC3168475  PMID: 21915308
21.  Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA 
Nucleic Acids Research  2012;40(13):6039-6048.
DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.
doi:10.1093/nar/gks264
PMCID: PMC3401449  PMID: 22447448
22.  DYNAMIC STRUCTURAL REARRANGEMENTS BETWEEN DNA BINDING MODES of E. coli SSB PROTEIN 
Journal of molecular biology  2007;369(5):1244-1257.
Summary
Escherichia coli (E. coli) single stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)35 and (SSB)65 formed on (dT)70, with rates of interconversion on time scales that vary as much as 200-fold for a mere 4-fold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C-terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)35 mode, suggesting that interactions of proteins with the C-terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.
doi:10.1016/j.jmb.2007.03.079
PMCID: PMC2041828  PMID: 17490681
DNA-Protein Interactions; Single-stranded DNA Binding Protein; Binding Modes; Replication; FRET; single molecule; kinetics
23.  RecO-mediated DNA homology search and annealing is facilitated by SsbA 
Nucleic Acids Research  2010;38(20):6920-6929.
Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation.
doi:10.1093/nar/gkq533
PMCID: PMC2978338  PMID: 20581116
24.  Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization 
Nucleic Acids Research  2005;33(5):1662-1670.
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.
doi:10.1093/nar/gki310
PMCID: PMC1069009  PMID: 15781492
25.  The Mycoplasma pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange 
BMC Microbiology  2008;8:167.
Background
Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms.
Results
The MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae.
Conclusion
The M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.
doi:10.1186/1471-2180-8-167
PMCID: PMC2572620  PMID: 18831760

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