Influence of polyamines on the activity of DNA polymerase I from Escherichia coli.

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A--T)n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA)n . (dT)10 and (rA)n . (dT)10 . (20:1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA)n . (dT)10 and somewhat less for (dA)n . (dT)10. The apparent Km for the primer (rA)n . (dT)10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent Km for dTTP. The influence of polyamines on both the 5' leads to 3' and 3' leads to 5' nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A--T)n and T7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of the activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12.

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.

Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.

Purification and characterization of the 17 K protein, a DNA-binding protein from Escherichia coli.

A basic protein of molecular mass 17 kDa (protein 17 K) which binds to relaxed DNA has been isolated and purified to homogeneity from Escherichia coli cells. The protein behaves as a tetramer in solution and there are 4800 monomers per cell in exponentially growing cells. The amino-acid composition and N-terminal sequence were determined. No effect of the protein on in vitro transcription was observed. The protein was shown to be different from the Ssb protein (Sigal, N. et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3537-3541), protein H1 (Cukier-Kahn et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3643-3647) and the HLP-1 protein (Lathe, R. et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3548-3552).

DNA- and RNA-binding proteins of chromatin from Escherichia coli.

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sjåstad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.

Cloning and sequencing of the gene for the DNA-binding 17K protein of Escherichia coli.

The skp gene encoding the 17K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The Mr of the mature polypeptide was 15,674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.

Affinity of protein HU for different nucleic acids.

The binding of protein HU from Escherichia coli to nucleic acids was investigated by affinity chromatography under various conditions, by a nitrocellulose retention assay and by isopycnic centrifugations in metrizamide gradients. The results indicate that HU has a preference for binding to RNA and single-stranded DNA over double-stranded DNA. The affinity of HU for supercoiled DNA was also less than that of the corresponding relaxed DNA.

Mechanism of caffeine-induced inhibition of DNA synthesis in escherichia coli.

Caffeine inhibited DNA synthesis in toluene-treated Escherichia coli K12 strains to the same extent as in intact cells using the incorporation of [3H]thymidine as a measure of DNA synthesis. The inhibition was found to be competitive with ATP, and it was not influenced by the concentrations of deoxynucleoside triphosphates to any extent. When caffeine was added together with other DNA synthesis inhibitors such as novobiocin, nalidixic acid or actinomycin D, the inhibition in all cases was non-additive. It is suggested that caffeine inhibits one of the ATP-requiring enzymes in the DNA replication machinery, possibly DNA polymerase III or one of the DNA helicases.

Separation of damage specific DNA endonuclease activities present in calf thymus.

A DNA endonuclease activity present in calf thymus specific for incision on DNA damaged by ultraviolet light, osmium tetroxide, potassium permanganate, hydrogen peroxide and acid has been purified from whole cell extracts. The enzymatic activity was heterogeneous both with regard to molecular mass and charge. The molecular mass of the enzyme varied from 25 to 35 kDa, but the different enzymatic species appeared to possess similar activities. The enzymes acted equally well on damage in supercoiled and relaxed forms of DNA. It further had a narrow optimum with regard to salt concentrations, the optimum activity being observed at a concentration of KCl from 40 to 65 mM.

The nuclear-associated endoplasmic reticulum is an early target for the action of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in C3H/10T1/2 fibroblasts.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) had a dual effect on the cellular membranes of C3H/10T1/2 cells in that it caused both a stimulation of [3H]choline incorporation and an enhancement of the solubilization of choline from prelabelled cells. Subfractionation studies showed that the release of [3H]choline occurred almost exclusively from nuclear-associated endoplasmic reticulum. The release was dependent on the presence of Mg2+ and Ca2+, indicating an enzyme-mediated reaction. In vivo, TPA stimulated the incorporation of [3H]choline into all subcellular fractions. The data indicate that the nuclear-associated endoplasmic reticulum represents an early target for TPA action.

Effect of caffeine on nucleotide pools in Escherichia coli.

The influence of caffeine on the intracellular concentration of various nucleoside triphosphates in addition do dTMP and dTDP, in Escherichia coli has been investigated. For most of the nucleoside triphosphates the presence of 10 mM caffeine in the medium resulted in a small increase in pool size 5 min after addition, followed by a slow decrease to the initial concentration. In the case of dTTP, however, the pool size reached a maximum, 2-fold higher than the initial value, 30 min after caffeine addition. This increase in dTTP level is probably due to an effect of caffeine on the DNA synthesis process and synthesis of dTDP-sugars.

The effect of caffeine on cell growth and metabolism of thymidine in Escherichia coli.

(1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in various E. coli strains. Caffeine caused filamentous growth in all strains investigated. The caffeine effect was reversible. (2) The incorporation of thymidine into DNA was inhibited by caffeine, and the inhibition was most pronounced with bacterial cultures grown overnight in the presence of caffeine before the addition of thymidine. For cells not pretreated with caffeine the inhibitory effect of caffeine decreased with increasing concentrations of thymidine up to about 1 microM whereafter it remained constant. The effect of thymidine concentration on the inhibition was less for bacteria that had grown overnight in the presence of caffeine than for bacteria not pretreated with caffeine. (3) Caffeine inhibited thymidine kinase, but it had no effect on thymidine phosphorylase or thymidine nucleotide kinases. (4) It is suggested that caffeine interferes with uptake of thymidine, conversion of thymidine to dTTP and the DNA synthesis process itself. Filamentous growth could be the result of the inhibition of DNA synthesis.

Effect of caffeine on DNA polymerase I from Escherichia coli: studies in vitro and in vivo.

The influence of caffeine on the activity of DNA polymerase I from E. coli was investigated. Caffeine had no effect on the polymerizing activity but did inhibit both 5' leads to 3' and 3' leads to 5' nuclease activities. The highest inhibition was observed with d(A--T)n as substrate: at a concentration of caffeine of 10 mM, inhibition was about 50%. In studies in vivo with 3 isogenic strains of E. coli, carrying different mutations in the DNA polymerase I gene, the effect of caffeine on survival after ultraviolet irradiation was most marked for the wild-type, pol+, followed by those mutants defective in 3' leads to 5', polA1, and 5' leads to 3' nuclease activities, polA107. Caffeine had little influence on survival of the resA1 mutant which lacks both 5' leads to 3' and 3' leads to 5' nuclease activities. These results support the idea that the influence of caffeine on dark repair may be explained in part by its effect on the nuclease activities of DNA polymerase I.

Mitomycin-C-induced changes in the nucleoid of Escherichia coli K12.

The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data.