SEARCH · PubMed Health

Results for “SSB”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1992

Properties of single-stranded DNA-binding proteins (SSB-proteins) from chromatin and nonchromatin fractions of Ehrlich ascites tumour: phosphorylation enhances the affinity of SSB-proteins for single-stranded DNA.

To assess the possible functional role of single-strand DNA-binding (SSB) proteins in eucaryotic cell, a comparative study was made of SSB-proteins isolated from chromatin and the nonchromatin fractions of Ehrlich ascites tumour (EAT) cells. No appreciable differences between the two groups could be found either in SDS-gel electrophoretic patterns or in the ssDNA-binding capacity and stimulation of DNA replication in permeable EAT cells. However, the chromatin SSB-proteins incorporated 1.4-times more labelled phosphate in vivo; phosphate assays in the isolated chromatin and nonchromatin SSB-proteins yielded ca. 3 and 2 moles Pi/mole protein, respectively. Both preparations could be further phosphorylated in vitro with Ca-phospholipid-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase, but the non-chromatin proteins were phosphorylated to a greater degree. In parallel with phosphorylation, the SSB-proteins displayed stronger binding to ssDNA cellulose. Phosphorylation may thus be a means of regulating the functions of SSB-proteins, in particular their interaction with chromatin.

Animals↗

PubMed1991

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA.

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

Bacterial Proteins↗

PubMed1992

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46.

Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1. A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113. It has very little or no effect on ssb-1 or ssb-3. All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels. The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda. The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411. The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain.

Bacterial Proteins↗

PubMed1991

Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein.

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

DNA-Binding Proteins↗

PubMed2026

SSB deficiency-induced R-loop accumulation triggers podocyte inflammation in DKD.

INTRODUCTION: Diabetic kidney disease (DKD) is fundamentally a podocytopathy in which sterile inflammation plays a central pathogenic role, yet the upstream triggers that initiate inflammatory cascades in podocytes remain elusive. R-loops are critical regulators of genomic stability, and their pathological accumulation triggers DNA damage and innate immune activation. Whether R-loop dysregulation contributes to podocyte-driven inflammation in DKD is unknown. METHODS: We integrated single-cell transcriptomic profiling, dual machine learning algorithms, and functional experiments to dissect the R-loop regulatory network in the diabetic kidney. RESULTS: Integrated analysis of human diabetic kidney single-cell RNA-seq data revealed a globally compromised R-loop regulatory network selectively within podocytes. Intersection of podocyte-specific transcriptomic shifts with validated R-loop regulators identified 93 candidate genes, from which dual machine learning algorithms pinpointed SSB (Sjögren syndrome antigen B) as the principal podocyte-selective R-loop resolver and a superior diagnostic biomarker (AUC = 0.983). SSB expression was selectively downregulated in diabetic podocytes and showed the strongest positive correlation with the R-loop resolution module. Mechanistically, SSB loss impaired RNA splicing and stability pathways, leading to aberrant R-loop accumulation that activated the cGAS-dependent inflammatory signaling in podocytes. In two murine DKD models and high glucose-challenged podocytes, SSB was markedly reduced. Remarkably, SSB knockdown in podocytes alone sufficed to trigger R-loop accumulation and pro-inflammatory cytokine expression, whereas both RNase H1-mediated R-loop removal and cGAS co-depletion blunted this response. DISCUSSION: These findings suggest that an SSB-governed R-loop -cGAS -inflammatory signaling axis may link genomic instability to podocyte inflammation and contribute to DKD progression, nominating R-loop homeostasis as a previously unrecognized potential therapeutic target.

Podocytes↗

PubMed1991

Requirement for the replication protein SSB in human DNA excision repair.

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.

Antibodies, Monoclonal↗

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↗

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↗

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↗

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↗

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↗

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↗

PubMedDate not supplied

Formation of ssb, dsb, and uracil in monofilarly and bifilarly bromouracil-substituted DNA molecules.

Bifilarly BU-substituted ColE 1 plasmid and monofilarly BU-substituted M 13 phage DNA were irradiated with UV light of 313 nm. Using agarose gel electrophoresis and "reversed phase" HPLC technique ssb, dsb induction and uracil formation, respectively, could be detected in the irradiated DNA in dependence on the UV fluence. The analysis of the strandbreaks in bifilar ColE 1 DNA shows a significant part of directly induced dsb. Cross sections of ssb induction from 4.1 m2/J x 10(7) in 28%, 3.9 m2/J x 10(7) in 55% and 3.1 m2/J x 10(7) in 85-90% BU-substituted DNA were calculated. The cross section for dsb induction was found to be 0.04 m2/J x 10(7), estimated from the linear part of the fluence effect curve. In monofilar M 13 DNA a linear fluence effect curve for dsb induction was obtained. Excluding other than the direct production of dsb by using an in vitro approach for M 13 DNA, the results strongly support the hypothesis that dsb can be induced by one photochemical absorption event. The cross section for ssb was 3.8 m2/J x 10(7) and for dsb 0.05 m2/J x 10(7) in 41.5% monofilarly BU-substituted M 13 DNA. The comparison of ssb, dsb, and uracil production in bifilar and monofilar DNA with similar BU substitution showed no significant difference between the two DNA systems (ColE 1, M 13), indicating that the location of BU molecules in one or in both DNA strands will not lead to a different number of lesions after UV313 exposure.

Bromouracil↗

PubMed1991

[Phosphorylation and other properties of proteins binding single-stranded DNA (SSB-proteins) from chromatin and extrachromatin fractions of Ehrlich ascites carcinoma].

A comparative study of single-stranded DNA-binding proteins (SSB-proteins) isolated from chromatin and the extrachromatin fraction of Ehrlich ascites tumour cells was carried out. No differences were found either in SDS-gel electrophoretic mobility or in the single-stranded DNA-binding capacity and stimulation of the replicative synthesis of DNA. However, chromatin SSB-proteins contained 1.4-1.5 times more phosphate than extrachromatin proteins. Both preparations could be phosphorylated in vitro by protein kinase C and cAMP-dependent protein kinase, but the chromatin proteins were phosphorylated in a lesser degree. In parallel with phosphorylation the SSB-proteins displayed a higher binding affinity for ssDNA-cellulose. Phosphorylation can thus be regarded as a means of regulation of the SSB-protein function, in particular, their interaction with chromatin DNA.

Animals↗

PubMed1992

SSB, an antigen that selectively labels morphologically distinct synaptic boutons at the Drosophila larval neuromuscular junction.

In this report we describe the expression of Small Synaptic Bouton (SSB), an antigen that is selectively expressed in a specific subset of neuromuscular junction terminals in the body wall of Drosophila larva. The expression of SSB was studied with a polyclonal antibody raised against the cAMP phosphodiesterase of the Drosophila learning mutant dunce (Nighorn et al., 1991, Neuron 6:455-467); however, immunoreactivity was not abolished by the dunce (dnc) alleles dncM14 and dncM11 or deficiencies of the dnc gene, indicating that the antigen labelled could not be the dnc gene product, but another antigen that we termed SSB. Immunoreactivity was localized in the body wall muscles to a specific subset of neuromuscular junction terminals that have been implicated in activity-dependent plasticity. This demonstrates that these morphologically distinct terminals can be immunocytochemically distinguished and that they probably represent innervation by a distinct neuronal population. Confocal and electron microscopic examination demonstrated that staining was restricted to the synaptic boutons themselves, not to neurites or motor axons. Ultrastructural analysis showed label close to synaptic vesicles in the presynaptic terminal and in the surrounding subsynaptic reticulum. Central nervous system (CNS) staining was restricted to a segmentally repeated pattern of cell bodies in the ventral ganglion and to a few small groups of cells in the brain lobes.

Animals↗

PubMed1992

A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.

Adenosine Triphosphate↗

PubMed1992

Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Incl1 plasmid Collb-P9.

The Incl1 conjugative plasmid Collb-P9 carries a psiB gene that prevents induction of the SOS response in host bacteria. This locus is located 2.5 kb downstream of the ssb (single-stranded DNA-binding protein) gene in the leading region. This portion of Collb is strikingly similar to part of the leading region of the otherwise distinct F plasmid. Expression of psiB and ssb is increased when the host cell is exposed to an SOS-inducing treatment or the Collb transfer system is derepressed. Moreover, expression of both genes on a derepressed plasmid is strongly enhanced in conjugatively infected recipient cells. Carriage of the psiB gene by Collb is shown to prevent a low level of SOS induction following conjugation. Plasmid ssb and psiB genes may function to promote installation of the replicon in the new cell.

Amino Acid Sequence↗

PubMed1990

Use of the Escherichia coli SSB gene to prevent bioreactor takeover by plasmidless cells.

Reactor takeover by plasmidless cells is a major problem encountered when producing proteins from plasmid-borne genes in genetically engineered bacteria. We have approached this problem by deleting the essential ssb gene from the Escherichia coli chromosome and placing it on a plasmid. Plasmidless cells do not accumulate even after growing such strains under non-selective continuous culture conditions for extended periods of time. Other ssb-containing plasmids can be readily introduced into this E. coli strain by a plasmid-displacement technique. Using this system, we have achieved very high levels of beta-lactamase production in continuous culture without selective pressure.

Bacterial Proteins↗