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PubMed1992

[Use of benzoyl-naphthyl-DEAE-cellulose chromatography for determining the ability of proteins from Ehrlich ascites carcinoma, binding with single-stranded DNA (SSB-proteins), to destabilize the DNA double helix].

Single-stranded DNA-binding proteins (SSB-proteins) isolated from Ehrlich ascites tumour (EAT) cells were incubated for 30 min at 5 mM NaCl with salmon sperm DNA or [3H]DNA from EAT at the SSB-protein/DNA ratio (w/w) of 0 to 4.5. After addition of sodium dodecyl sulfate up to a 0.05% concentration, the proteins were applied to columns with benzoylated naphthoylated DEAE-cellulose. Double-stranded DNA was eluted by 1 M NaCl; the DNA containing single-stranded regions was eluted by 50% dimethylformamide. There was a progressive lowering of the DNA content in the first eluate and a rise in the second eluate, as could be evidenced from the increase in the SSB-protein/DNA w/w ratio. This effect was more pronounced in the case of homologous DNA and was not coupled with the nuclease activity of SSB proteins. It was concluded that EAT SSB-proteins are "DNA-unwinding" proteins.

Animals↗
PubMedDate not supplied

[Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation].

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.

Animals↗
PubMed1988

[Isolation and characteristics of single-stranded DNA-binding protein (SSB-protein) from Ehrlich ascites carcinoma cells].

Single-stranded DNA-binding protein (SSB-protein) has been purified and characterized from Ehrlich ascite tumour (EAT) cells. The purification procedure was performed in analytical and preparative variants. It was shown that in the analytical variant of the purification procedure can be used to determine protein concentration in the cell. The molecular mass of the SSB-protein as determined by SDS polyacrylamide gel electrophoresis is 36 and 43 kD; that determined by gel filtration is 27, 28, 43 and 44 kD; pI is 7.4. The use of nitrocellulose filters showed that the SSB-protein binds preferentially to ss-DNA. The protein contains no admixtures of DNA-polymerases, endo- or exonucleases, DNA-dependent ATPase, lactate dehydrogenase and HMG-proteins. The SSB-protein stimulates 1.5-2-fold the activity of DNA-polymerase alpha from EAT, it does not activate DNA-polymerase beta from EAT and strongly inhibits the activity of exonuclease (snake venom phosphodiesterase). The specificity of the term "SSB-protein" which makes it different from other non-histone proteins of chromatin is discussed.

Animals↗
PubMed1988

[The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair].

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.

Animals↗
PubMed1994

Relationship between maternal and neonatal levels of antibodies to 48 kDa SSB(La), 52 kDa SSA(Ro), and 60 kDa SSA(Ro) in pregnancies complicated by congenital heart block.

OBJECTIVE: Since there is suggestive but, to date, indirect evidence that maternal anti-SSA(Ro) and SSB(La) antibodies are pathogenic in congenital heart block (CHB), we explored the hypothesis that binding to fetal tissues would result in selective depletion of autoantibodies from the neonatal circulation. METHODS: Maternal and umbilical cord levels of anti-48 kDa SSB(La), anti-52 kDa SSA(Ro) and anti-60 kDa SSA(Ro) antibodies were measured by ELISA and immunoprecipitation at parturition in 15 pregnancies complicated by CHB. A control group consisted of 13 pregnancies in which the mother was known to have antibodies to either SSA(Ro) and/or SSB(La) and the children did not have CHB. RESULTS: The ratios of maternal to cord serum levels of anti-48 SSB(La), anti-52 SSA(Ro) and anti-60 kDa SSA(Ro) antibodies ranged from 0.71 to 2.38 in both affected and unaffected pregnancies. The mean ratio obtained for each of the 3 autoantibodies was not significantly different between the 2 groups. Moreover these ratios did not significantly differ from the mean ratios obtained for total IgG levels in either group. CONCLUSION: These data demonstrate that maternal antibodies to all components of the SSA(Ro) SSB(La) system are efficiently transported across the placenta and are not selectively depleted in the circulation of neonates with CHB.

Antibodies, Antinuclear↗
PubMed1992

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.

Amino Acid Sequence↗
PubMed1995

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Adenosine Triphosphatases↗
PubMed1998

Calorimetric studies of E. coli SSB protein-single-stranded DNA interactions. Effects of monovalent salts on binding enthalpy.

Isothermal titration calorimetry (ITC) was used to examine the effects of monovalent salts (NaCl, NaBr, NaF and ChCl) on the binding enthalpy (DeltaHobs) for E. coli SSB tetramer binding to the single-stranded oligodeoxythymidylates, dT(pT)69 and dT(pT)34 over a wide range of salt concentrations from 10 mM to 2.0 M (25 degrees C, pH 8.1), and when possible, the binding free energy and entropy (DeltaG degrees obs, DeltaS degrees obs). At low monovalent salt concentrations (<0.1 M), the total DeltaHobs for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (DeltaHobs=-150(+/-5) kcal/mol). This is much larger than any DeltaHobs previously reported for a protein-nucleic acid interaction. However, at salt concentrations above 0.1 M, DeltaHobs is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (DeltaHobs=-70(+/-1)-kcal/mol in 2 M NaBr). These salt effects on DeltaHobs were mainly a function of anion type and concentration, with the largest effects observed in NaBr, and then NaCl, with little effect of [NaF]. These large effects of salt on DeltaHobs appear to be coupled to a net release of weakly bound anions (Br- and Cl-) from the SSB protein upon DNA binding. However, at lower salt concentrations (</=0.1 M), specific cation effects on DeltaHobs also are observed. Under conditions where we can determine DeltaG degrees obs, DeltaS degrees obs, and DeltaHobs (25 degrees C, pH 8.1, 0.17 to 2 M NaBr), SSB binding to dT(pT)69 is enthalpically driven with a large unfavorable entropic contribution, both of which are dependent upon [NaBr]. These studies show that weak anion binding to a protein can result in large effects of salt concentration on DeltaHobs (as well as DeltaG degrees obs and DeltaS degrees obs) for a protein-ssDNA interaction. The possibility of such effects needs to be considered in any interpretation of the thermodynamics of this and other protein-nucleic acid interactions.

Anions↗
PubMed2000

Structural and functional comparative study of the complexes formed by viral ø29, Nf and GA-1 SSB proteins with DNA.

Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their single-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related bacteriophages, ø29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ø29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ø29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity for single-stranded DNA (GA-1 SSB, K(eff)=18.6 x 10(5) M(-1); o29 SSB, K(eff)=2.2 x 10(5) M(-1); Nf SSB, K(eff)=2.9 x 10(5) M(-1)). Altogether, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ø29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ø29 DNA amplification.

Bacillus↗
PubMed1991

Properties of RecA441 protein reveal a possible role for RecF and SSB proteins in Escherichia coli.

We examined the possibility that the recA441 mutation, which partially suppresses the UV sensitivity of uvr recF mutant bacteria, exerts its effect by coding for an altered RecA protein that competes more efficiently than the RecA+ protein with SSB for ssDNA in vivo. Using an assay measuring recombination between UV-damaged lambda DNA and intact homologous DNA, we found that the introduction of the recA441 mutation partially suppressed the defects in recombination in bacteria lacking RecF activity but not in bacteria with excess SSB, although recombination was affected more in recF mutants than in bacteria overproducing SSB. These results therefore do not support the hypothesis that RecA441 protein, or RecA protein with the help of RecF protein, is required during recombination of UV-damaged DNA to compete with SSB for ssDNA.

Bacterial Proteins↗
PubMed1989

The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro.

The formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA double-strand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and 3H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of 3H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl2 concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.

DNA Repair↗
PubMed1982

Role of SSB protein in RecA promoted branch migration reactions.

The RecA protein of Escherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of phi X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981 a0. In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.

Adenosine Triphosphatases↗
PubMed1982

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12.

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.

Bacterial Proteins↗
PubMed1993

[Development of an immunoenzyme assay technique of ELISA type for detecting anti-SSB antibodies. Comparative study of 2 kits].

The anti-SSB antibodies were measured using two enzyme immunoassays (ELISA). The difference between the both results from the preparation of the SSB antigenic extract. The first method, developed in our laboratory, uses a purified SSB antigen extracted from calf thymus, while the other uses an antigen cloned by genetic engineering. We have realized an analytic investigation about the repetability, the reproducibility and the detection limit of our ELISA method and we have refined its evaluation in using a clinic study carried out on 203 subjects (55 had a Sjögren syndrome, 47 had a systemic lupus erythematosus, 17 had a rheumatoid arthritis, 13 a progressive systemic sclerosis, 10 a polymyositis and 61 healthy subjects (blood donors)). These measures were worked out in order to compare them with the Ouchterlony method of reference. The results we have obtained are totally similar to the ELISA methods, with a global correlation factor of 0.96 in spite of the difference on the preparation of SSB antigenic extract. The enzyme immunoassay is a lot more sensitive than the Ouchterlony method since, for a Sjögren sample, we obtain a sensitivity of 0.69 while the sensitivity is only of 0.51 for the immunoprecipitation. In the lupus sample, the sensitivity is respectively of 0.42 and 0.25.

Antibodies, Antinuclear↗
PubMed1997

The ssb-113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli.

Mutations in the dnaQ gene, which encodes the proofreading epsilon-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the epsilon-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.

Alleles↗
PubMed1988

Suppression of the Escherichia coli ssb-1 mutation by an allele of groEL.

A series of spontaneous suppressors to the temperature-sensitive phenotype of the single-stranded DNA-binding protein mutation ssb-1 were isolated. A genomic library of EcoRI fragments from one of these suppressor strains was prepared by using pBR325 as the cloning vector. A 10.0-kilobase class of inserts was identified as carrying the ssb-1 gene itself. A second class of 8.3-kilobase inserts was shown to contain the groE region by (i) restriction analysis, (ii) Southern hybridization of the 8.3-kilobase insert to groE+ DNA, and (iii) identification of the gene products by similar migration on polyacrylamide gels. Subcloning demonstrated that an intact mutant groEL gene was necessary for suppression and that plasmids carrying the 8.3-kilobase insert could suppress mutants carrying groES- but not groEL- genes for phage lambda growth. The suppressor, designated as groEL411, was specific for the ssb-1 allele. In ssb-1 groEL411 cells, DNA synthesis stopped after a shift to 42.5 degrees C but rapidly recovered within minutes. The data suggest a direct interaction between the single-stranded DNA-binding protein and GroEL proteins in DNA replication.

Bacterial Proteins↗
PubMed2006

3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins.

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis-trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the chi subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli.

Amino Acid Sequence↗