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PubMed1985

Analysis of the regulatory region of the ssb gene of Escherichia coli.

The regulation of the ssb gene of E. coli has been studied. We reported earlier that the SOS box of the neighbouring uvrA gene also controls the transcription of the ssb gene. Detailed analysis of the upstream region of ssb by S1 mapping reveals the existence of three in vivo functional promoters of which the most upstream one (PI) is inducible by DNA damage. Measurement of galactokinase synthesis using galK fusion plasmids indicates that the uninduced level of transcription from the PI promoter is low. Ssb multicopy plasmids lacking the PI promoter still complement the UV sensitivity of an Ssb mutant. The role of the three promoters in the regulation of the level of Ssb protein in the cell, is discussed.

Chromosome Mapping↗

PubMed1995

Analysis of a suppressor mutation ssb (kinC) of sur0B20 (spo0A) mutation in Bacillus subtilis reveals that kinC encodes a histidine protein kinase.

sur0B20 is a mutation that suppresses the effects of spo0B delta B or spo0F221 mutations in Bacillus subtilis, sur0B20 is an allele of the spo0A gene (Glu-14 to Val-14 conversion) and restores the sporulation of spo0B or spo0F mutants to the wild-type level. Here, we report the isolation of suppressor mutations of sur0B20 (ssb). One of these mutations, ssb-12, severely impairs the suppressor activity of sur0B20. A 2.5-kbp MboI fragment which complements the ssb-12 mutation was cloned by the prophage transformation method using phi CM as a vector. Nucleotide sequencing of the fragment revealed two open reading frames (orf1 and orf2). Gene disruption and complementation experiments showed that orf2 is the ssb gene. ssb was shown to encode a protein with a molecular weight of 48,846 (428 amino acid residues) showing strong similarity to transmitter kinases, especially KinA, of two-component regulatory systems. Therefore, ssb was renamed kinC. Deletion of kinC had no observable effect on sporulation. kinC transcription was induced at the onset of sporulation, probably from a sigma A-dependent promoter, and its expression was shut off at T3. DNase I protection experiments showed that the Spo0A protein binds to two adjoining sites in the kinC promoter region with different affinities. These results suggest that kinC expression might be regulated by Spo0A.

Amino Acid Sequence↗

PubMed2002

Diagnostic associations in a large and consecutively identified population positive for anti-SSA and/or anti-SSB: the range of associated diseases differs according to the detailed serotype.

OBJECTIVE: To determine the diagnostic distribution in a consecutive anti-SSA and/or anti-SSB positive population. METHODS: A total of 15 937 serum samples from 10 550 consecutive patients were analysed for antinuclear antibodies (ANAs) on HEp-2 cells. Serum samples positive for ANAs were analysed by immunodiffusion and line immunoassay with recombinant SSA-Ro52, natural SSA-Ro60, and recombinant SSB. RESULTS: Among ANA positive patients in whom clinical information was available, 181 consecutive patients with anti-SSA and/or anti-SSB antibodies were identified, Disease associations were systemic lupus erythematosus (SLE) (45.3%), primary Sjögren's syndrome (pSS) (14.4%), scleroderma (8.8%), RA (7.7%), cutaneous lupus (7.7%), and dermatomyositis (2.2%). The ratio of diagnoses differed according to the anti-SSA/anti-SSB serotype. Scleroderma and dermatomyositis were enriched among mono-Ro52 reactive serum samples (34.2% and 10.5% respectively). Single reactivity towards Ro60 or anti-Ro60 with anti-Ro52 predisposed for SLE (80.0% and 52.2% respectively). Triple reactivity towards Ro52, Ro60, and SSB was primarily linked with SLE (55.8%) followed by pSS (20.9%). Anti-SSA on immunodiffusion increased the chance for SLE (62.8%), whereas isolated anti-SSB reactivity on immunodiffusion was less indicative for SLE (14.3%) and predisposed more for cutaneous lupus (23.8%) and pSS (33.3%). CONCLUSION: The diagnostic range associated with anti-SSA or anti-SSB reactivity differs significantly according to the detailed serotype defined by line immunoassay and immunodiffusion.

Antibodies, Antinuclear↗

PubMedDate not supplied

[Antigenic properties of heterogeneous nuclear ribonucleoprotein particles weakly binding nonhistone proteins and proteins binding single-stranded DNA (SSB proteins) from Ehrlich ascites tumor].

Antigenic properties of the proteins of heterogeneous nuclear ribonucleoprotein particles, (hnRNP), weakly bound nonhistone chromatin proteins (WB(N)P) and single-strand DNA-binding proteins (SSB proteins) from chromatin and extrachromatin fraction of the Ehrlich ascites tumor cells have been comparatively studied. The chromatin and extrachromatin SSB proteins displayed similar mobility in the tube and slab SDS/PAGE, had the same ssDNA-binding capacity and similarly stimulated the replicative synthesis in permeable cells. However, the chromatin SSB proteins contained 1.4 times higher phosphate amount than the extrachromatin ones (3.1 and 2. 2. moles phosphorus per 1 mole protein, respectively). The study of four protein groups with the use of a rabbit antiserum to/against extrachromatin SSB proteins (titer 1:13000 by enzyme immunoassay) showed that the chromatin and the extrachromatin SSB proteins have similar antigenic properties. One fraction of the hnRNP proteins was also reactive with the antiserum, whereas the WB(N)P displayed no cross-reactivity. The specificity of the ferm "SSB proteins" as applied to eukaryotic cells, their affinity with hnRNP proteins and differences from the HMG proteins are discussed.

Animals↗

PubMed1986

Complement C4 fixation by the anti-La(SSB) found in normal donors and in patients with systemic lupus erythematosus.

The discovery that a common systemic lupus erythematosus (SLE) autoantibody (anti-La/SSB) occurs in normal individuals has made it possible to compare the specific complement binding capacity of normal autoantibody with SLE patient autoantibody. Normal donors with elevated levels of anti-La(SSB) binding were compared to negative normal donors and to 3 patients with SLE with high levels of anti-La(SSB) binding in assays for anti-La(SSB) and for the capacity of anti-La(SSB) to bind C4. The anti-La(SSB) found in normal sera and in SLE patient sera had no identical specific capacity to fix C4. For the case of anti-La(SSB), the autoantibody structure in relation to its specific capacity to activate complement, at least to the extent of fixing C4, does not differentiate SLE patients from normal individuals with this autoantibody.

Antibodies, Antinuclear↗

PubMed2000

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein.

Many macromolecular interactions, including protein-nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature-dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, DeltaH(obs), and accounts for as much as a half of the observed heat capacity change, DeltaC(p). However, there is still a substantial DeltaC(p) associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)(34) to SSB over a broad pH range (pH 5. 0-10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25 degrees C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)(34) binding over this entire pH range, with contributions from at least three sets of protonation sites (pK(a1) = 5.9-6.6, pK(a2) = 8.2-8.4, and pK(a3) = 10.2-10.3) and these protonation equilibria contribute substantially to the observed DeltaH and DeltaC(p) for the SSB-dT(pT)(34) interaction. The contribution of this coupled protonation ( approximately -260 to -320 cal mol(-1) K(-1)) accounts for as much as half of the total DeltaC(p). The values of the "intrinsic" DeltaC(p,0) range from -210 +/- 33 cal mol(-1) degrees K(-1) to -237 +/- 36 cal mol(-1)K(-1), independent of [NaCl]. These results indicate that the coupling of a temperature-dependent protonation equilibria to a macromolecular interaction can result in a large negative DeltaC(p), and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand-macromolecular interactions, as well as protein folding.

DNA, Single-Stranded↗

PubMed2006

Microsecond dynamics of protein-DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer.

The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the approximately 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex.

DNA, Single-Stranded↗

PubMed1983

Direct observation of complexes of ssb and recA proteins with a fluorescent single-stranded deoxyribonucleic acid derivative.

Evidence is presented from fluorescence and kinetic experiments that ssb binds to a recA-ssDNA-ATP complex causing a major structural change in which some 40% of the bound recA is released. On addition of ssb to recA-epsilon DNA-ATP (containing the fluorescent analogue of ssDNA epsilon DNA), there is a slow first-order decrease in fluorescence (t1/2 approximately 3 min). This is accompanied by a loss in the ATPase activity of recA protein. The resultant complex does not exchange epsilon DNA for added ssDNA. Measurement of the DNA-stimulated ATPase activity on addition of excess ssDNA reveals that 40% of the previously bound recA has been released. The stoichiometry of recA bound to epsilon DNA thus changes from 1 mol per six nucleotides to 1 per 10 on addition of ssb. Formation of the ssb-recA-epsilon DNA complex is dependent on ATP, and the rate varies with the concentration of ssb.

Adenosine Triphosphatases↗

PubMed2006

Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein.

The Deinococcus radiodurans SSB protein has an occluded site size of 50 +/- 2 nucleotides on ssDNA but can form a stable complex with a 26-30-nucleotide oligodeoxynucleotide using a subset of its four ssDNA binding domains. Quantitative estimates of D. radiodurans SSB protein in the D. radiodurans cell indicate approximately 2500-3000 dimers/cell, independent of the level of irradiation. At biologically relevant concentrations, when bound at single-strand-double-strand DNA junctions in vitro, D. radiodurans SSB protein has a limited capacity to displace the shorter strand of the duplex, permitting it to bind to single-strand extensions shorter than 26-30 nucleotides. The capacity to displace the shorter strand of the duplex shows a pronounced bias for extensions with a free 3' end. The Escherichia coli SSB protein has a similar but somewhat less robust capacity to displace a DNA strand annealed adjacent to a single-strand extension. These activities are likely to be relevant to the action of bacterial SSB proteins in double-strand break repair, acting at the frayed ends created by ionizing radiation.

DNA↗

PubMed1981

Sequences of the ssb gene and protein.

We have determined the sequences of the ssb gene and protein of Escherichia coli. The coding region of ssb is 534 base pairs and is preceeded and followed by dyad symmetries of 39 base pairs and 27 base pairs, respectively. The promoter for ssb is close to that for uvrA and these two genes are transcribed in opposite directions: ssb clockwise and uvrA counterclockwise on the standard E. coli genetic map. The DNA helix-destabilizing protein encoded by the ssb gene (single-strand binding protein) contains 177 amino acids and has a calculated molecular weight of 18,873. Although there is no extensive sequence homology among the three helix-destabilizing proteins whose sequences are now known, both the E. coli and bacteriophage T4 DNA helix-destabilizing proteins do contain an acidic, alpha-helical region at their carboxy termini that may be functionally homologous. The remainder of the E. coli helix-destabilizing protein can be divided into two apparent domains on the basis of its amino acid sequence. The amino-terminal region (residues 1-105) contains 79% of the charged residues (27 out of 34 total) in the protein and is predicted to have a high degree of secondary structure (alpha helix and beta pleated sheet). In contrast, the region including residues 106-165 contains only two charged amino acids and is devoid of alpha helix or beta pleated sheet.

Amino Acid Sequence↗

PubMed2001

Yields of SSB and DSB induced in DNA by Al(K) ultrasoft X-rays and alpha-particles: comparison of experimental and simulated yields.

PURPOSE: To compare experimental yields of single strand breaks (SSB) and double strand breaks (DSB) induced in plasmid DNA in aqueous solution by alpha-particles and Al(K) ultrasoft X-rays (USX) with the corresponding yields, generated via computer simulations, for a range of mean diffusion distances of the hydroxyl radical (*OH). MATERIALS AND METHODS: Aerobic, aqueous solutions of plasmid DNA were irradiated at 277K with 238Pu alpha-particles or USX in the presence of 10(-4) to 0.33 mol dm(-3) Tris and the yields of SSB and DSB determined by gel electrophoresis. Computer simulations, using Monte Carlo track-structure codes for 1.5keV electrons (CPA100) and 3.2MeV alpha-particle track segments (PITS), were used to obtain yields of DNA SSB and DSB at different *OH scavenger conditions. RESULTS: The experimental yield of SSB and DSB induced by AlK USX and SSB induced by alpha-particles and the dependences on the mean diffusion distance of the *OH are in reasonable agreement with the corresponding simulated yields and their corresponding dependences. However, for DSB induced by alpha-particles, a significant systematic difference exists between the simulated and experimental yields over the full *OH scavenging range, with the simulated yields being a factor of two to three greater than the experimental values. CONCLUSION: That the simulated yields of strand breaks are generally in reasonable agreement with those determined experimentally over a wide range of *OH scavenging capacities, increases confidence in the use of these simulations as a valuable source of quantitative, mechanistic information on DNA damage induced at very low radiation doses.

Alpha Particles↗

PubMed1997

Deletion formation between the two Salmonella typhimurium flagellin genes encoded on the mini F plasmid: Escherichia coli ssb alleles enhance deletion rates and change hot-spot preference for deletion endpoints.

Deletion formation between the 5'-mostly homologous sequences and between the 3'-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5' sequences, but not between the 3' sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the delta recA allele. Rates of the deletion between the 3' sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA+ background and 88% in delta recA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background > 50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3' sequences. The deletion formation between the homeologous sequences in RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.

Alleles↗

PubMed2004

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase.

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Archaeal Proteins↗

PubMed1997

Isolation and characterization of the gene encoding single-stranded-DNA-binding protein (SSB) from four marine Shewanella strains that differ in their temperature and pressure optima for growth.

The ssb gene, coding for single-stranded-DNA-binding protein (SSB), was cloned from four marine Shewanella strains that differed in their temperature and pressure optima and ranges of growth. All four Shewanella ssb genes complemented Escherichia coli ssb point and deletion mutants, with efficiencies that varied with temperature and ssb gene source. The Shewanella SSBs are the largest bacterial SSBs identified to date (24.9-26.3 kDa) and may be divided into conserved amino- and carboy-terminal regions and a highly variable central region. Greater amino acid sequence homology was observed between the Shewanella SSBs as a group (72-87%) than with other bacterial SSBs (52-69%). Analysis of the amino acid composition of the Shewanella SSBs revealed several features that could correlate with pressure or temperature adaptation. SSBs from the three low-temperature-adapted Shewanella strains were an order of magnitude more hydrophilic than that from the mesophilic strain, and differences in the distribution of eight amino acids were identified which could contribute to either the temperature or pressure adaptation of the proteins. The SSBs from all four Shewanella strains were overproduced and partially purified based upon their ability to bind single-stranded DNA. The differences found among the Shewanella SSBs suggest that these proteins will provide a useful system for exploring the adaptation of protein-protein and protein-DNA interactions at low temperature and high pressure.

Adaptation, Physiological↗

PubMed1996

A hierarchy of SSB protomers in replication protein A.

Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.

Amino Acid Sequence↗

PubMed1991

The single-stranded-DNA-binding protein encoded by the Escherichia coli F factor can complement a deletion of the chromosomal ssb gene.

Genes encoding single-stranded-DNA-binding proteins (SSBs) are carried by a variety of large self-transmissible plasmids, and it previously has been shown that these plasmid-borne genes can complement conditional lethal alleles of the ssb gene on the Escherichia coli chromosome for cellular viability. We have tested one of the plasmid-borne ssb genes, the ssf gene from the E. coli F factor, for its ability to complement total deletion of the chromosomal ssb gene for viability. We have found that ssf can complement the ssb deletion, but only when it is present on a high-copy-number plasmid. Cells that are totally dependent on the F-factor-encoded SSB for viability manifest growth properties indicative of problems in DNA replication.

Amino Acid Sequence↗

PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗

PubMed1992

[The binding of SSB-proteins with DNA in chromatin of Ehrlich ascites carcinoma cells].

Using UV-induced cross-linking between proteins and DNA, the contacts between single-stranded DNA-binding proteins (SSB proteins) and chromatin DNA have been demonstrated. Ehrlich ascites tumour DNA was labeled in vivo by inoculation of tumour-bearing mice with 3H-thymidine. The cells were irradiated with the UV light dose of 3000 J/m2, destroyed in a Triton X-100-containing hypotonic medium, and separated by centrifugation into the extrachromatin fraction and chromatin. Chromatin DNA was digested with DNAase 1, and the chromatin proteins were extracted with 2 M NaCl-polyethyleneglycol. SSB proteins from the extrachromatin fraction and chromatin were purified. Only SSB proteins from UV-irradiated cell chromatin appeared to possess a high specific radioactivity which exceeded 7.5-fold that of non-irradiated cells. There were no differences between chromatin SSB proteins in control and irradiated cells as could be evidenced from SDS electrophoresis data. It is assumed that in irradiated cells SSB proteins of DNA-digested chromatin are covalently cross-linked with DNA fragments.

Animals↗