[Putrescine as a oxidative stress protecting factor in Escherichia coli]. / Putrestsin kak faktor zashchity Escherichia coli ot okislitel'nogo stressa.

The level of expression of oxyR, the gene that protects Escherichia coli against oxidative stress, was enhanced by physiological concentrations of the biogenic amine putrescine. This level was directly proportional to the degree of negative DNA supercoiling. 1,4-Diamino-2-butanone (DAB), a specific inhibitor of ornithine decarboxylase, the key enzyme of polyamine synthesis, produced an inhibitory effect on the level of oxyR expression under oxidative stress, which was relieved by the addition of putrescine. The direct relationship between putrescine concentration and the degree of negative DNA supercoiling suggests that the mechanism of action of putrescine as the modulator of oxyR transcription activity is based on both its direct influence on the gene expression level and its indirect effect mediated by topological DNA changes. Putrescine was shown to produce a protective effect if the DNA is damaged by reactive oxygen species.

[Topological changes in DNA as a reflection of adaptation of Escherichia coli to oxidative stress under glucose starvation and transition to growth]. / Topologicheskie izmeneniia DNK kak otrazhenie adaptatsii Escherichia coli k okislitel'nomu stressu v usloviiakh golodaniia po gliukoze i pri perekhode k rostu.

Changes in the topological state of DNA occur in a starving Escherichia coli culture under oxidative stress caused by the addition of hydrogen peroxide. The addition of a carbon and energy source to this culture results in a second stress reaction. This supports previous data indicating that different mechanisms are responsible for the cell defense against oxidative stress in exponential and starving E. coli cultures. Polyamine synthesis is involved in the cell adaptation to the stress. Putrescine binding to DNA and its dissociation seem to modulate the DNA topological state, which regulates the expression of the adaptive genes. An increase in the activity of the polyamine-synthesizing system in response to oxidative stress leads to a putrescine flux across the cytoplasmic membrane, due to which the antioxidant activity of putrescine protects the membrane phospholipids and contributes to the restoration of the cell energy-generating function.

[The role of putrescine and the energy state of Escherichia coli in regulation of DNA topology during adaptation to oxidative stress]. / Rol' putresysina i énergeticheskogo sostoianiia Escherichia coli v re guliatsii topologii DNK pri adaptatsii k okislitel'nomu stressu.

An exponential-phase culture of E. coli responded to the addition of H2O2 by a decrease in DNA supercoiling induced by the lowering of the energy status of cells, potassium leakage, and breaking of polynucleotide chains. Extending the time of exposure of E. coli cells to hydrogen peroxide led to an increase in the intracellular pools of putrescine and potassium, promotion of cellular energy status, and the restoration of DNA supercoiling to values much in excess of the prestress level. The subsequent stabilization of the intracellular putrescine pool was accompanied by a release of this polyamine from the cell. Based on these results and those available in the literature, a mechanism of E. coli adaptation to oxidative stress is suggested that assigns roles to putrescine, potassium, and cellular energy status.

[Role of putrescine and potassium transport in regulating the topological state of DNA during adaptation of Escherichia coli to temperature stress]. / Rol' transporta putrestsina i kaliia v reguliatsii topologicheskogo sostoianiia aDNK v protsesse adaptatsii Escherichia coli k temperaturnomu stressu.

The effect of a temperature increase to 52 degrees C or the addition of ethanol (6%) to an exponential Escherichia coli culture on putrescine and potassium transport was studied. The first stage of heat shock was accompanied by a decrease in the extent of DNA supercoiling, due to the dissociation of the putrescine-DNA complex. The loss of potassium ions at this phase produced a synergistic effect. The second phase of the heat shock was characterized by a reversal in the direction of putrescine and potassium transport, which was accompanied by restoration of the prestress extent of DNA supercoiling. An increase in the ATP pool and cell energy charge resulting from the uncoupling of the energy metabolism and synthetic processes also played an important role in the restoration of the DNA initial topology at the second phase of the heat shock via the activation of the energy-dependent gyrase or the heat shock protein DnaK. A mechanism is suggested that explains the involvement of putrescine in the regulation of DNA topology, which is a universal regulator of gene expression under stress, heat shock in particular.

[Exchange of putrescine and potassium between cells and media as a factor in the adaptation of Escherichia coli to hyperosmotic shock]. / Obmen putrestsina i kaliia mezhdu kletkoi i sredoi kak faktor adaptatsii Escherichia coli k giperosmoticheskomu shoku.

Putrescine/potassium exchange in response to hyperosmotic stress was studied. The addition of 0.3 M NaCl or 0.44 M sucrose to an exponentially growing E. coli culture induced potassium uptake and putrescine release from the cell. Potassium added to an osmotically stressed potassium-deficient culture was readily absorbed by cells; this was accompanied by the loss of intracellular putrescine, both free and bound. Since DNA is the main binding site of putrescine, the loss of bound putrescine caused a relaxation of DNA supercoiling. The increase in the intracellular content of potassium not only restored but also enhanced DNA supercoiling as compared to the initial level. In vitro experiments showed the degree of plasmid DNA supercoiling to rise drastically at potassium concentrations of 300-500 mM, while different putrescine concentrations affected this parameter differently. Thus, the physiological concentrations of putrescine (below 1 mM) greatly augmented DNA supercoiling, whereas higher concentrations (5-10 mM) exerted a relaxing effect. A change in DNA supercoiling in vivo in response to osmotic stress is the result of competition between biogenic and abiogenic cations for the sites of binding to polyanionic DNA structures. A change in DNA topology serves as the regulatory factor controlling the expression of genes responsible for cell adaptation to osmotic stress.

[Role of putrescine and potassium transport in the adaptation of Escherichia coli to ammonium starvation]. / Rol' transporta putrestsina i kaliia v adaptatsii Escherichia coli k golodaniiu po ammoniiu.

Ammonium depletion in the nutrient medium induced an active transport of putrescine into the cell, which was not associated with its utilization as a nitrogen source. The uptake of putrescine was accompanied by a proportional release of potassium from the cells. Quantitative analysis of free as well as weakly and tightly bound pools of putrescine showed that this diamine was bound mainly to DNA. Topological studies by the plasmid method indicated an increase in the DNA supercoiling in response to the putrescine transport. In vitro experiments made it possible to establish an ambiguous dependence of DNA topology on putrescine content--its physiological concentrations (0.5-1.0 mM) enhanced DNA supercoiling, while higher concentrations (2-10 mM) caused gradual relaxation of DNA. A possible physiological significance of these effects in adaptive response of cells to ammonium deficiency is discussed.

Base analog 6-N-hydroxylaminopurine mutagenesis in the yeast Saccharomyces cerevisiae is controlled by replicative DNA polymerases.

Genetic control of mutagenesis by the base analog 6-N-hydroxylaminopurine (HAP) was studied in a set of isogenic yeast strains carrying null or point mutations in DNA repair and replication genes. Null alleles of the PMS1, RAD6, REV3 and RAD52 genes did not affect HAP mutagenesis. Defects in 3'- > 5' exonucleases associated with DNA polymerases epsilon and delta led to 2- to 3-fold increases in HAP-induced forward Can(r) mutant frequency. A similar increase was observed for FOAr mutants but only in the strain with a defective exonuclease of the polymerase epsilon (mutation pol2-4). The polymerase epsilon mutations, pol2-9 and pol2-18, which lead to temperature-sensitivity, and pol2-1 (insertion of URA3 at the position coding for amino acid 1134 in the POL2 gene) substantially reduced HAP mutagenesis. The polymerase delta mutation, cdc2-2, slightly reduced HAP mutagenesis. Enhanced proofreading was not the cause of the antimutator effect in the pol2-18 bearing strain, inasmuch as antimutator effect was observed in the pol2-4,18 mutant strain lacking proofreading. From the data obtained, we conclude that both DNA polymerase epsilon and delta participate in mutation generation by HAP.

The genetic activity of N6-hydroxyadenine and 2-amino-N6-hydroxyadenine in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae.

The genetic activity of 2-amino-N6-hydroxyadenine or 2-amino-N-hydroxylaminopurine (AHA) and N6-hydroxyadenine or 6-N-hydroxylaminopurine (HAP) was studied in S. typhimurium, E. coli and Saccharomyces cerevisiae strains. AHA was a more potent mutagen for bacteria and a less potent mutagen for yeast than HAP. The mutagenic activity of analogs was not influenced by excision, mutagenic or double-strand DNA repair mutations. On the other hand, the uvrBdel mutation has a drastic effect on the mutagenicity and toxicity of both analogs in the Salmonella strains studied. HAP was a very potent mutagen in yeast with a low capability of inducing mitotic recombination contrary to common mutagens, possessed unique intergenic specificity and was able to induce mutations in diploids at rather high frequency.

[Genetic activity of aminofluorene and 2-acetylaminofluorene in strains of Saccharomycetes under conditions of in vitro metabolic activation: influence of rad 1-5 mutation]. / Geneticheskaia aktivnost' 2-aminofliuorena i 2-atsetilaminofliuorena u shtammov drozhzhei sakharomitsetov v usloviiakh metabolicheskoi aktivatsii i vitro: vliianie mutatsii rad 1-5.

It has been shown that the rad1-5 mutation which alters excision repair in Saccharomyces cerevisiae yeast increased reversion frequency of the ochre mutation his7-1 and the frequency of intragenic mitotic recombination in the LYS2 gene induced by 2-aminofluorene and 2-acetylaminofluorene, as compared with the wild type strains activated in vitro by 39 mix from chicken liver.