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PubMed1983

Study of the expression of UVRA and SSB proteins in vivo in lambda hybrid phages containing the uvrA and ssbA genes of Escherichia coli.

A 9.3-kb Eco RI fragment obtained by partial digestion of the plasmid pDR2000 and containing the uvrA and ssbA genes was subcloned in the insertion vector lambda gt4. Two hybrid bacteriophages carrying this fragment inserted in opposite orientations were isolated and used to lysogenize a uvrA and an ssbA mutant of Escherichia coli. Both phages conferred to these host bacteria the ultraviolet resistance of the wild-type parent indicating full complementation of the uvrA and of the ssbA defect. Two polypeptides corresponding to the molecular weights of the UVRA protein (115 000 dalton) and of the SSB protein (18 500 dalton) were synthesized and amplified after infection of a UV-irradiated lambda ind- lysogen with these 2 hybrid phages. The UVRA protein was not amplified after infection of a lex A3 host while SSB was still produced in large amount. These results establish that uvrA is repressed by lexA in vivo whereas ssbA is not.

Bacterial Proteins↗

PubMed1987

Complexes of the single-stranded DNA-binding protein from Escherichia coli (Eco SSB) with poly(dT). An investigation of their structure and internal dynamics by means of electron microscopy and NMR.

Based on electron microscopy and NMR spectroscopy it is deduced that Eco SSB binds with moderate cooperativity to polynucleotides. Evidence is provided that the protein binds in its tetrameric form to the nucleic acid forming a nucleosome-like structure. NMR-spectroscopic analysis of the complexes shows that the carboxy-terminal region of the Eco SSB maintains a high flexibility even when the protein is immobilized in large protein-protein clusters.

Escherichia coli↗

PubMed1980

Isolation of plasmids carrying either the uvrC or uvrC uvrA and ssb genes of Escherichia coli K-12.

A 3.4 kb PstI fragment containing the uvrC gene of Escherichia coli K-12 has been cloned into pBR322. Plasmids carrying this PstI fragment, in either orientation (pGY3233, pGY4211) relative to the cloning vehicle, complement uvrC mutants. A second plasmid (pGY3243) with a 21 kb HindIII fragment is shown to complement mutations in uvrA and ssb (single-strand binding protein). A composite plasmid (pGY4610) containing pBR322 and PstI fragments derived from pGY3233 (3.4 kb) and pGY3243 (11.05 kb) complements the uvrC, uvrA and ssb mutations.

Cloning, Molecular↗

PubMed1993

Cloning of human and rat cDNAs encoding the mitochondrial single-stranded DNA-binding protein (SSB).

We have retro-transcribed and amplified by PCR the full-length cDNAs specifying the rat and human precursors of the single-stranded mitochondrial DNA (mtDNA)-binding protein (mtSSB). Each deduced sequence is composed of a 16-amino-acid (aa) N-terminal basic pre-sequence and a mature protein (132 aa in humans and 135 aa in the rat). The mature proteins are highly conserved among themselves and with the mtSSB from Xenopus laevis (Xl). Moreover, three regions of the protein are similar to corresponding domains of the SSB of Escherichia coli and to the E. coli F-sex factor SSB, indicating the existence of a broad class of DNA-binding proteins with structural and functional similarities both in prokaryotes and in prokaryote-derived organelles of higher organisms.

Amino Acid Sequence↗

PubMed1994

RNA- and single-stranded DNA-binding (SSB) proteins expressed during Drosophila melanogaster oogenesis: a homolog of bacterial and eukaryotic mitochondrial SSBs.

Little is known about the identity and involvement of single-stranded (ss) DNA-binding (SSB) and RNA-binding proteins in developmental processes that occur during oogenesis in Drosophila melanogaster (Dm). Here, we describe a molecular approach designed to identify such proteins by virtue of their ssDNA-binding activity. We have constructed a directional ovarian cDNA library and conducted expression cloning screens which identified five unique cDNAs that encode proteins capable of binding ssDNA. All five represent previously unreported sequences. The remainder of this paper focuses on one of these cDNAs which encodes a Dm protein displaying significant sequence homology to Escherichia coli ssDNA-binding protein (SSB, involved in DNA replication, repair and recombination), as well as eukaryotic SSBs isolated from the mitochondria (mt) of rats, frogs, humans and yeast. The deduced amino acid (aa) sequence of this 15.6-kDa protein, which we will refer to as Dm mtSSB, displays average identities of 38.3% with eukaryotic mtSSBs and 23.4% with bacterial SSBs. Gel retardation analysis with an affinity-purified GST fusion protein confirms that Dm mtSSB specifically binds ss, but not double stranded DNA. Dm mtSSB is encoded by a nuclear gene whose expression appears to be developmentally regulated. It is expressed as a single 600-nucleotide (nt) transcript during oogenesis and embryogenesis. A larger transcript of 1500 nt is prevalent in some later stages of Dm development.

Amino Acid Sequence↗

PubMed2006

Identification, cloning, expression, and characterization of a highly thermostable single-stranded-DNA-binding protein (SSB) from Deinococcus murrayi.

We report identification and characterization of SSB-like protein from Deinococcus murrayi (DmuSSB). PCR-derived DNA fragment containing the complete structural gene for DmuSSB was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 826 nucleotides encoding a protein of 276 amino acid residues with a calculated molecular weight of 30.14 kDa. DmuSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT) DmuSSB bound 27-32 nt depending on the salt concentration, and fluorescence was quenched by about 62%. In a complementation assay in E. coli, DmuSSB took over the in vivo function of EcoSSB. DmuSSB maintained 100% activity after 120 min incubation at 80 degrees C, with half-lives of 50 min at 95 degrees C, 40 min at 100 degrees C and 35 min at 105 degrees C. DmuSSB is the most thermostable SSB-like protein identified to date, offering an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.

Amino Acid Sequence↗

PubMed2000

Structure of the DNA binding domain of E. coli SSB bound to ssDNA.

The structure of the homotetrameric DNA binding domain of the single stranded DNA binding protein from Escherichia coli (Eco SSB) bound to two 35-mer single stranded DNAs was determined to a resolution of 2.8 A. This structure describes the vast network of interactions that results in the extensive wrapping of single stranded DNA around the SSB tetramer and suggests a structural basis for its various binding modes.

Binding Sites↗

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↗

PubMed1991

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins.

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

Adenosine Triphosphate↗

PubMed1998

Evidence for a hypoxic fixation reaction leading to the induction of ssb and dsb in irradiated DNA.

PURPOSE: To measure hypoxic chemical fixation processes of radiation damage in both isolated plasmid DNA and in GSH-depleted E. coli cells. MATERIALS AND METHODS: Plasmid pBR322 DNA was irradiated with a single 5 ns pulse of 400 keV electrons under hypoxic conditions. At pre-set times, immediately before or after the electron pulse, the chamber containing the DNA was exposed to a high-pressure shot of hydrogen sulphide (H2S) gas. RESULTS: DNA irradiated before contact with the H2S pulse was more sensitive to the production of both single strand breaks (ssb) and double strand breaks (dsb) than DNA irradiated after the addition of H2S. The post-irradiation protection of DNA by H2S was time-dependent, having first-order rate constants of 21 s(-1) for ssb and 10 s(-1) for dsb. CONCLUSIONS: This is the first direct kinetic evidence for the involvement of a hypoxic fixation reaction in the production of DNA damage by ionizing radiation. It indicates that long-lived radical damage is induced in DNA which, even at times of 20-50ms after irradiation, can be chemically repaired, or rescued, by the addition of a thiol agent. This reaction may partially explain the predicted decrease in oxygen enhancement ratio (OER) with linear energy transfer (LET) on the basis of the increased clustering of radicals produced on the DNA by tracks of ionizing radiation. As radical multiplicity increases with LET there is a greater chance that some of the radicals will become fixed in the absence of oxygen leading to an increased probability of damage under hypoxia and a reduction in the OER.

DNA↗

PubMed2006

Structure of the single-stranded DNA-binding protein SSB from Thermus aquaticus.

The crystal structure of the single-stranded DNA-binding protein from Thermus aquaticus has been solved and refined at 1.85 A resolution. Two monomers, each encompassing two oligonucleotide/oligosaccharide-binding (OB) domains and a number of flexible beta-hairpin loops, form an oligomer of approximate D(2) symmetry typical of bacterial SSBs. Comparison with other SSB structures confirms considerable variability in the mode of oligomerization and aggregation of SSB oligomers.

Crystallography, X-Ray↗

PubMed2001

Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA.

Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.

Amino Acids↗

PubMed2005

Broad sensitivity of Saccharomyces cerevisiae lacking ribosome-associated chaperone ssb or zuo1 to cations, including aminoglycosides.

The Hsp70 Ssb and J protein Zuo1 of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, proposed to be involved in the folding of newly synthesized polypeptide chains. Cells lacking Ssb and/or Zuo1 have been reported to be hypersensitive to cationic aminoglycoside protein synthesis inhibitors that affect translational fidelity and to NaCl. Since we found that Deltassb1 Deltassb2 (Deltassb1,2), Deltazuo1, and wild-type cells have very similar levels of translational misreading in the absence of aminoglycosides, we asked whether the sensitivities to aminoglycosides and NaCl represent a general increase in sensitivity to cations. We found that Deltassb1,2 and Deltazuo1 cells are hypersensitive to a wide range of cations. This broad sensitivity is similar to that of cells having lowered activity of major plasma membrane transporters, such as the major K+ transporters Trk1 and Trk2 or their regulators Hal4 and Hal5. Like Deltahal4,5 cells, Deltassb1,2 and Deltazuo1 cells have increased intracellular levels of Na+ and Li+ upon challenge with higher-than-normal levels of these cations, due to an increased rate of influx. In the presence of aminoglycosides, Deltassb1,2, Deltazuo1, and Deltahal 4,5 cells have similarly increased levels of translational misreading. We conclude that, in vivo, the major cause of the aminoglycoside sensitivity of cells lacking ribosome-associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins.

Aminoglycosides↗

PubMed1994

Recurrent annular erythema with anti-SSA/Ro and anti-SSB/La antibodies localized on palms and fingers in an elderly man.

A 69-year-old Japanese man with recurrent annular erythema localized on palms and flexor surface of fingers with anti-SSA/Ro and anti-SSB/La antibodies, is reported. The present case indicates that even in an elderly man with atypical localization, annular erythema with autoimmunity may be considered and serological analysis should be performed. Systematic studies will be required to understand the relationship between 'recurrent annular erythema associated with anti-SSB/La antibodies' in Japanese, subacute cutaneous lupus erythematosus (SCLE) and 'annular erythema associated with Sjogren's syndrome'.

Aged↗

PubMed2004

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.

Adenosine Triphosphatases↗

PubMed1991

Anti-Ro(SSA) and anti-La(SSB) antibodies in lupus erythematosus and Sjögren's syndrome.

Anti-Ro(SSA) and La(SSB) antibody determinations have become important serologic tests in the evaluation of lupus erythematosus and Sjögren's syndrome patients. These antibodies appear to identify a group of lupus patients with prominent skin diseases. Although much of the initial investigations regarding the value of these antibodies has been performed in the United States and Europe, preliminary studies suggest that the frequency of these antibodies in Japanese and other Oriental patients may be double that seen in American patients. This article will briefly review our knowledge of the molecular, immunogenetic and clinical features of the anti-Ro(SSA) and anti-La(SSB) antibody responses.

Antibodies, Antinuclear↗

PubMedDate not supplied

[Binding of SSB-protein from Ehrlich ascites carcinoma cells with DNA and polyribonucleotides].

Binding of SSB-protein from Ehrlich ascites tumor to ssDNA from M13 phage leads to its compactization. The structure of the complex at the protein/DNA ratios far from the saturation level looks like "beads-on the string". DNA that was fully saturated with protein forms collapsed globular structure. Binding of the protein to the dsDNA from phage lambda increases its flexibility and decreases the coil dimensions; no "beads-on the string" structure are seen. The protein possess slight destabilizing effect on hairpin helices of M13DNA. Competition studies demonstrate that the binding properties of protein with polyribonucleotide lattices and DNA's decrease in ranking as follows: poly(rG) greater than or equal to poly(rI) greater than or equal to ssDNA greater than dsDNA greater than poly(rA) congruent to approximately poly(rU). Thus SSB-protein from Ehrlich ascites tumor differs significantly from its presumed prokaryotic analogs.

Animals↗

PubMed1987

[Functional properties of E. coli SSB-proteins in vivo].

An essential function of single-stranded DNA-binding (SSB) proteins--a defense from nuclease action, determined by their first and second domains, is realized in conditions of normal cell metabolism. With the inhibition of the replicative furcula growth and total protein synthesis (imbalance state), the SSB protein function associated with the third domain and responsible for certain modifications in DNA polymerase II activity is realized. This causes DNA degeneration and increases radiosensitivity of cells.

Bacterial Proteins↗