ABSTRACT
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.
Subject(s)
Antigens/analysis , Carcinoma, Ehrlich Tumor/immunology , Chromosomal Proteins, Non-Histone/immunology , DNA-Binding Proteins/immunology , Neoplasm Proteins/immunology , Ribonucleoproteins/immunology , Animals , DNA, Single-Stranded/metabolism , Mice , Protein BindingABSTRACT
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.
Subject(s)
Chromatin , DNA, Neoplasm/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Animals , Carcinoma, Ehrlich Tumor , Electrophoresis, Disc , Mice , Phosphorylation , Protein Kinase C/metabolism , Protein Kinases/metabolismABSTRACT
Quick test for estimation of DNA-bound proteins (PBD) in mammalian blood serum involved filtration of blood serum through Wathman filters containing DNA. DNA was fixed in the Wathman filters by means of UV irradiation. Some physicochemical and chromatographic properties of PBD from mice blood serum were studied. The data obtained suggest that blood serum PBD constituted one of gamma-globulin fractions. Applicability of the PBD test for studies of tumoral diseases was shown using mice with Ehrlich ascites carcinoma.
Subject(s)
Carcinoma, Ehrlich Tumor/blood , DNA-Binding Proteins/blood , DNA/blood , Animals , Ascitic Fluid/chemistry , Chromatography, DEAE-Cellulose , Filtration , MiceABSTRACT
A number of physicochemical properties of blood plasma proteins of mice and piglets which display their primary affinity to single-stranded DNA are studied. These proteins exert an activating effect on DNA-polymerase alpha from the Ehrlich ascites carcinoma and an inhibitory effect on the venom phosphodiesterase. A comparison of properties of the proteins under study with those of antibodies to the single-stranded DNA described in the literature permits supposing an identity of these proteins.
Subject(s)
Antibodies, Antinuclear/metabolism , Blood Proteins/metabolism , DNA, Single-Stranded/metabolism , Animals , Antibodies, Antinuclear/blood , Carcinoma, Ehrlich Tumor/enzymology , DNA Polymerase II/metabolism , DNA, Single-Stranded/blood , DNA, Single-Stranded/immunology , Enzyme Activation , L-Lactate Dehydrogenase/metabolism , Mice , Phosphoric Diester Hydrolases/metabolism , SwineABSTRACT
Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.
Subject(s)
Carcinoma, Ehrlich Tumor/metabolism , DNA Replication , DNA, Neoplasm/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Animals , Chromatin/metabolism , Mice , Tumor Cells, Cultured/metabolismABSTRACT
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.
Subject(s)
DNA Repair/physiology , DNA, Neoplasm/radiation effects , DNA-Binding Proteins/physiology , Neoplasm Proteins/physiology , Animals , Carcinoma, Ehrlich Tumor/pathology , In Vitro Techniques , Mice , Neoplasm Transplantation , Ultraviolet RaysABSTRACT
Survival of Ehrlich ascites tumor cells, SSB content of the chromatin, and repair DNA synthesis rate were investigated after gamma-irradiation, the rate of repair synthesis was shown to depend on the SSB-protein content of the chromatin. Changes in the amount of SSB-protein in the chromatin were not connected with the postirradiation accumulation of single-strand sites in DNA.
Subject(s)
DNA Repair/physiology , DNA, Neoplasm/radiation effects , DNA-Binding Proteins/physiology , Neoplasm Proteins/physiology , Animals , Carcinoma, Ehrlich Tumor/pathology , Cesium Radioisotopes , Gamma Rays , In Vitro Techniques , Mice , Neoplasm TransplantationABSTRACT
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.
Subject(s)
Carcinoma, Ehrlich Tumor/metabolism , DNA Repair , DNA Replication , DNA-Binding Proteins/physiology , Neoplasm Proteins/physiology , Animals , Carcinoma, Ehrlich Tumor/enzymology , DNA Polymerase I/antagonists & inhibitors , DNA Polymerase I/metabolism , DNA Polymerase II/metabolism , DNA-Binding Proteins/isolation & purification , Enzyme Activation , T-Phages/enzymology , Tumor Cells, Cultured/enzymology , Tumor Cells, Cultured/metabolismABSTRACT
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.